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Posts Tagged ‘Apoptosis’


Embryonic Stem Cell differentiation

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1

Yan Tang1, Ya-Zhen Hong1, Hua-Jun Bai1, Qiang Wu1, Charlie Degui Chen2, Jing-Yu Lang1, Kenneth R. Boheler3 and Huang-Tian Yang1,4,*

STEM CELLS: 18 APR 2016     http://dx.doi.org:/10.1002/stem.2333

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8-/Y) model, we found that deletion ofphf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8+/Y) and ph8-/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown ofpmaip1 mimicked the phenotype of ph8-/Y by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8-/Y ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells2016

 

Significance Statement

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Thus, ESCs provide a unique model for the study of early embryonic development. We report here previously unrecognized effects of histone demethylase plant homeo domain finger protein 8 (PHF8) on mesodermal and early cardiac differentiation. This effect is resulted from the regulation of PHF8 on apoptosis through activating the transcription of pro-apoptotic gene pmaip1. These findings extend the knowledge in understanding of the epigenetic modification in apoptosis during ESC differentiation and of the link between apoptosis and cell lineage decision as well as cardiogenesis.

 

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Due to this plasticity, mechanisms controlling cell autonomous and regulatory events critical to in vivo mammalian development have benefitted from the in vitro study of differentiating ESCs [1, 2]. Early embryogenesis and cavity formation as well as early ESC differentiation, for example, are accompanied by a reduction in proliferation and increased apoptosis [3-5]. Withdrawal of leukemia inhibitory factor (LIF) from mouse embryonic stem cells (mESCs) cultivated in vitro causes approximately 20%-30% of the cells to die by spontaneous (constitutive) apoptosis [4, 5]. This occurs secondary to the induction of cleaved caspase 3 [3] and apoptosis-inducing factor (AIF)-complex proteins [6]. Blockade of spontaneous apoptosis in vitro by a p38 mitogen-activated protein kinase (MAPK) inhibitor alters the differentiation markers and increases the abundance of both antiapoptotic proteins (Bcl-2, Bcl-XL) and Ca2+-binding proteins [4, 7]. In addition, Ca2+ released from type 3 inositol 1, 4, 5-trisphosphate receptors (IP3R3) negatively regulates this apoptotic response, which in turn modulates the mesodermal lineage commitment of early differentiating mESCs [5]. These findings explain, in part, how apoptosis contributes to specific lineage commitment during early development. However, in contrast to the relatively advanced knowledge of signaling pathways [8], little is known about the contribution of epigenetic regulators, especially, histone lysine demethylases (KDMs), in the regulation of apoptosis during ESC differentiation and how the affected programmed cell death by KDMs contributes to the lineage commitment.

Epigenetic regulators and dynamic histone modifications by KDMs are emerging as important players in ESC fate decisions [9]. Histone modifications coordinate transient changes in gene transcription and help maintaining differential patterns of gene expression during differentiation [10-13]. The molecular and biological functions of many KDMs, however, remain enigmatic during ESC differentiation. PHF8, an X-linked gene encoding an evolutionarily conserved histone demethylase harboring an N-terminal plant homeo domain (PHD) and an active jumonji-C domain (JmjC), is able to catalyze demethylation from histones [14, 15]. It is actively recruited to and enriched in the promoters of transcriptionally active genes [14], and it functions to maintain the active state of rRNA through the removal of the repressive H3K9me2 methylation mark at the active rRNA promoters. Mutation of PHF8 is associated with X-linked mental retardation with cleft lip/cleft palate in human [16-18]. Knockdown of phf8 in mouse embryonic carcinoma P19 cells impairs neuronal differentiation [19] and leads to brain defects in zebrafish by directly regulating the expression of the homeo domain transcription factor MSX1/MSXB [20]. However, the precise function of PHF8 in the regulation of lineage differentiation derived from other germ layers remains to be identified.

Here, we report previously unrecognized effects of the PHF8 histone demethylase on germ layer commitment and differentiation of mESCs. The results are based on an assessment of early steps of differentiation to mesodermal lineages and cardiomyocytes using phf8 knockout (phf8-/Y) and wild-type (phf8+/Y) mESCs. The data show that PHF8 regulates gene transcription of a proapoptotic gene pmaip1 (also named Noxa) [21]. Activation or repression of pmaip1 controlled by PHF8 ultimately determines mESC lineage commitment through the regulation of caspase 3-independent apoptosis during mesodermal and cardiac differentiation. Our data reveal that PHF8-mediated the demethylation of histone proteins coordinates ESC lineage commitment through the regulation of apoptosis in early differentiating ESCs.

 

Deletion of phf8 Promotes Mesodermal and Cardiac Lineage Commitment

The PHF8 protein was detectable in undifferentiated ESCs, but its abundance significantly increased within one day of LIF withdrawal. Then it gradually decreased to a level at day 5 lower than that observed in the undifferentiated ESCs (Fig. 1A).    http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0001-t.gif

 

Figure 1. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and early cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Western blot analysis of PHF8 expression in undifferentiated and differentiating ESCs. n = 3. (B): quantitative RT-PCR (qRT-PCR) analysis of pluripotency markers nanog, rex1, sox2, and oct4. n = 8. (C): qRT-PCR analysis of gene expression of pluripotency marker oct4; early mesodermal markers brachyury (T), gsc, eomes, and mesp1; cardiovascular progenitor markers flk-1 and nrp1; and the cardiac transcription factors hand1, tbx5, and mef2c during ESC differentiation. n = 5. (D): qRT-PCR analysis of the early ectodermal markersnestin and fgf5 during ESC differentiation. n = 3. (E): qRT-PCR analysis of early endodermal markers afp, foxa2, sox17, and gata4 during ESC differentiation. n = 3. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.

To determine the significance of phf8 gene expression on ESC fate decision, we knocked out the X-chromosome-encoded phf8 gene in one allele of male SCR012 ESCs by deletion of exons 7 and 8 through Cre-mediated recombination (Supporting Information Fig. S1A). Gene inactivation was confirmed by the lack of Phf8 mRNA and PHF8 protein expression in these targeted ESCs (Supporting Information Fig. S1B). Transcripts for pluripotency marker genes nanog, rex1 (zfp42), sox2, and oct4 (pou5f1) were not significantly different between phf8+/Y and phf8-/Y ESCs (Fig. 1B). No significant difference was observed in cell morphology (Supporting Information Fig. S1C) of undifferentiated phf8+/Y and phf8-/Y ESCs or in alkaline phosphatase activity (Supporting Information Fig. S1D). Immunofluorescence staining confirmed that the expression of pluripotency marker SOX2 and SSEA-1 did not differ between the phf8+/Y and phf8-/Y ESCs (Supporting Information Fig. S1E). These results indicate that phf8 may be dispensable for normal growth and maintenance of mESCs.

We then analyzed the role of PHF8 in the mesodermal and cardiac lineage commitment. By microarray analysis of differentiating phf8+/Y and phf8-/Y cells from days 0, 1, to 3.5, we found a significant decrease in transcripts for pluripotency markers, accompanied by a significant increase in transcripts for ectoderm, mesoderm and endoderm, while in phf8-/Y cells some transcripts for mesodermal and cardiac lineage commitment were significantly enhanced compared with those in phf8+/Y cells (Supporting Information Fig. S2A). These differentiation-dependent changes in transcript abundance were confirmed by qRT-PCR for early mesodermal markers brachyury (T) [28], goosecoid (gsc),eomes[29], and mesp1[30], cardiovascular progenitor marker flk-1[31, 32] and neuropilin 1 (nrp1) [33]. Early cardiac transcription factors, including myocyte enhancer factor 2C (mef2c) [34], hand1[35], and tbx5[36, 37] were also up-regulated in phf8-/Y cells at differentiation day 5, while no difference in the expression levels of pluripotent markersoct4 (Fig. 1C), rex1, and nanog (Supporting Information Fig. S2B) were detected between phf8+/Y and phf8-/Y cells at the time points examined.

Because mESCs can differentiate into all three germ layers, we also examined whether phf8 affected ectodermal and endodermal differentiation. qRT-PCR analysis did not show any significantly difference in the expression of early ectodermal markers nestin and fgf5 between the phf8+/Y and phf8-/Y cells (Fig. 1D). Moreover, in the induced early ectodermal differentiation system [23], the expression of ectodermal markers nestin, fgf5, and pax6 were comparable between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3A). Besides, the expression of endodermal markers afp, foxa2, sox17, and gata4 were not significantly different between the phf8+/Y and phf8-/Y cells (Fig.1E). Consistently, the expression of endodermal markers foxa2, sox17, and gata4 were comparable during induced endodermal differentiation [24] between the phf8+/Y andphf8-/Y cells (Supporting Information Fig. S3B). Thus, phf8 appears not to affect early ectodermal and endodermal differentiation.

The increased mesodermal and cardiac marker expressions were associated with a significant increase in the total number of cardiac progenitors and cardiomyocytes in differentiating phf8-/Y cells. By flow cytometry analysis, marked increases in the population of FLK-1 positive (FLK-1+) cells were detected in phf8-/Y cells at differentiation day 3 and day 4 (Fig. 2A). Consistently, the percentage of contracting EBs was higher in phf8-/Y cells than in phf8+/Y cells (Fig. 2B). The transcripts for progenitor marker nrp1, early cardiac transcription factor tbx5, and cardiac specific genes tnnt2, myh6, myl2, and gja1 were higher in phf8-/Y EBs than those in phf8+/Y ones (Fig. 2C). The areas of immunostained EBs positive for the cardiac cytoskeletal and myofilamental proteins α-actinin and TNNT2 were also greater in phf8-/Y than in phf8+/Y EBs (Fig. 2D). Flow cytometry analysis of MYH6+ (Fig. 2E) and TNNT2+ (Fig. 2F) cells at differentiation day 9 further confirmed the increase of cardiomyocytes in phf8-/Y cells. Taken together, these data indicate that the phf8 deletion facilitates the differentiation of mesodermal and cardiac linage commitment.

Figure 2. phf8 deletion promotes cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Left, representative flow cytometry plots showing FLK-1 expression at differentiation day 3 (n = 6), day 4 and day 5 (n = 3 each). Right, the quantification of flow cytometry data. (B): Differentiation profile of cardiomyocytes during embryoid bodies (EB) outgrowth. n = 6. (C): qRT-PCR analysis of ESCs for the expression of cardiac markers at differentiation day 14. n = 3. (D): Immunofluorescence analysis of TNNT2 and α-actinin in day 14 EBs. Scale bars = 400 μm. (E) Flow cytometry analysis of MYH6 positive cells and (F) TNNT2 positive cells in day 9 EBs. n = 3 each. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.   http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0002-t.gif

PHF8 Inactivation Increases Cell Viability but not Proliferation of the Differentiating ESCs

Differentiation of both phf8+/Y and phf8-/Y ESCs via EB formation produced normal round shaped EBs but, by day 3, phf8-/Y EBs were larger than those generated from phf8+/YESCs, and the size differences were visibly obvious at differentiation days 5 and 7 (Fig. 3A). Although no significant differences in cell viability could be demonstrated between undifferentiated phf8+/Y and phf8-/Y ESCs (Fig. 3B), the viability of phf8-/Y cells was significantly greater than that in phf8+/Y cells at differentiation days 3 to 7 (Fig. 3C). However, no significant change in BrdU staining was detected by flow cytometry between phf8+/Y and phf8-/Y ESCs at differentiation days 0, 3, or 5 (Fig. 3D). Moreover, no significant difference in the cell cycle distribution between the differentiating Phf8+/Y and Phf8-/Y ESCs was detected, although the percentage of cells in S phase gradually decreased while those in G1 phase increased upon differentiation (Fig. 3E). Knockout of phf8 thus increases cell numbers in the early differentiating ESCs through the improvement of cell viability without changes in cell proliferation or cell cycle progression.

Figure 3. phf8 deletion increases cell viability in differentiating mouse embryonic stem cells (mESCs) without affecting cell proliferation. (A): Left, phase-contrast images of embryoid bodies (EB) morphology during EB formation from ESCs. Scale bar = 200 μm. Right, the diameter of EB formed from ESCs. (B): Cell viability of undifferentiated and (C): differentiating ESCs analyzed by MTT assay for seven consecutive days. n = 3. (D): Flow cytometry analysis of BrdU positive proportion of undifferentiated (n = 4) and differentiating ESCs at day 3 and day 5.n = 5 each. (E): Flow cytometry analysis of cell cycle distribution by propidium iodide (PI) staining at differentiation day 3 (n = 6) and day 5 (n = 3). Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Y value.   http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0003-t.gif

PHF8 Regulates Apoptosis During the Early Stage of Cardiac Lineage Commitment

We then examined whether cell death might account for the differences in the cell viability observed between the differentiating phf8+/Y and phf8-/Y ESCs. In undifferentiated ESCs, no significant difference was demonstrated with Annexin V (an early apoptosis marker) staining, TUNEL assay, total DNA fragmentation or caspase 3 protein cleavage between phf8+/Y and phf8-/Y cells (Fig. 4A–4C, 4E). In contrast, Annexin V staining (Fig. 4A) and TUNEL assay (Fig. 4B) showed significant decreases in the number of apoptotic cells in phf8-/Y ESCs at differentiation days 3 and 5 compared with those in phf8+/Y cells. Genomic DNA fragmentation with a pattern typical for apoptosis was detected in phf8+/Y cells at differentiation days 3 and 5, but it was reduced in phf8-/Y cells at the same time points (Fig. 4C). Moreover, approximately 35% of Annexin V+ cells were present in FLK-1+/phf8+/Y cells at differentiation day 4, whereas only 9% of the cells were Annexin V+ in FLK-1+/phf8-/Y cells (Fig. 4D). The ratio of TUNEL+ to either NESTIN+ (ectoderm) or SOX17+ (endoderm) cells did not differ between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3C, S3D). In addition, phf8+/Y ESCs at differentiation days 3 and 5 increased the caspase 3 cleavage (Fig. 4E, upper and lower left panels) and the ratio of cleaved caspase 3 to total caspase 3 protein (Fig. 4E, lower right panel). Unexpectedly, the ratio of cleaved caspase 3 to total caspase 3 in phf8-/Y ESCs did not significantly differ from that observed in phf8+/Y ones. Consistently, a significant enhancement of the downstream target PARP1 cleavage [38, 39] was observed at differentiation days 3 and 5, but it was comparable between the phf8+/Y andphf8-/Y cells (Fig. 4F). These data suggest that the cell death associated with phf8 function does not operate through the conventional caspase 3-mediated apoptosis.

Figure 4. Plant homeo domain finger protein 8 (PHF8) regulates apoptosis during the early mouse embryonic stem cells (mESC) differentiation. (A): Left, representative flow cytometry plots showing Annexin V (x-axis), and PI (y-axis) staining in ECSs at differentiation day 0 (n = 4), day 3 (n = 3) and day 5 (n = 7). Right, the quantification of flow cytometry data. (B): Flow cytometry detection of apoptotic responses of TUNEL positive cells at differentiation day 0 (n = 3), day 3 (n = 4), and day 5 (n = 4). (C): DNA laddering analysis at differentiation days 0, 3, and 5. n = 6 each. (D): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 3. (E): Western blot analysis of caspase 3 during the mESC differentiation. β-actin was used as a loading control. n = 4. (F): Western blot analysis of PARP1 expression during the differentiation. β-actin was used as a loading control. n = 4. Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Yvalue; #, p < .05; ##, p < .01 compared with the corresponding d0 value.  http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0004-t.gif

pmaip1 is a Direct Target Gene of PHF8 in the Early Differentiating ESCs

To understand how PHF8 might regulate apoptosis during early ESC differentiation, we compared the profiles of apoptosis-related gene transcripts in undifferentiated and early differentiating phf8+/Y and phf8-/Y ESCs using gene expression microarrays. Among the apoptosis-related genes, the transcript to pmaip1, a proapoptotic Bcl-2 family member crucial in fine-turning the cell death decision [21, 40-42], was markedly increased during early differentiation of phf8+/Y cells but it was reduced in phf8-/Y cells at differentiation days 1 and 3.5 (Fig. 5A). These expression patterns were confirmed by qRT-PCR during cardiac differentiation (Fig. 5B), and the results were consistent with the apoptotic pattern observed during the early ESC differentiation (Fig. 4A–4C). In addition, qRT-PCR analysis showed that the expression of pmaip1 did not show any significant difference between the phf8+/Y and phf8-/Y cells during the induced ectodermal (Supporting Information Fig. S3E) or endodermal (Supporting Information Fig. S3F) differentiation.

Figure 5. pmaip1 is a direct target gene of plant homeo domain finger protein 8 (PHF8) in mouse embryonic stem cells (mESCs). (A): Microarray gene expression heat map depicting the expression of apoptosis-related genes at differentiation days 0, 1 and 3.5 in phf8+/Y and phf8-/Y ESCs. The expression values in log2 scale were calculated and presented on the heat map with red representing highly abundant transcripts and green representing poorly abundant transcripts. n = 3. (B): qRT-PCR analysis of pmaip1 during the ESC differentiation. (C): ChIP assay of PHF8 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation days 0 and 3. n = 4 each. (D): Western blot analysis of H3K9me2 and H3 in phf8+/Y and phf8-/Y ESCs during the differentiation. H3 was used as a loading control. n = 9. (E): H3K9me2 staining inphf8+/Y and phf8-/Y embryoid bodies (EBs) at differentiation day 1. Scale bars = 25 μm. (F): ChIP assay of H3K9me2 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation day 3. n = 4. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value or d0.  http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0005-t.gif

A direct link between the PHF8 and pmaip1 was then confirmed by ChIP analysis. We detected the endogenous binding of PHF8 at the transcription start site (TSS, from −45 bp to 104 bp) of pmaip1 in phf8+/Y ESCs and determined that binding was enhanced at differentiation day 3. The binding of PHF8 was not detectable above the IgG control levels in phf8-/Y cells (Fig. 5C). Global methylation (H3K9me2 normalized to H3) was unchanged at differentiation days 3 and 5, but it was significantly enhanced at differentiation day 1 in phf8-/Y cells (Fig. 5D). The augmentation of H3K9me2 methylation in phf8-/Y ESCs was then confirmed by immunostaining at differentiation day 1 (Fig.5E). An increase in the repressive mark of H3K9me2 was also observed at the TSS of pmaip1 in the early differentiating phf8-/Y ESCs (Fig. 5F), indicating that the PHF8 demethylase activity is actively involved in the regulation of pmaip1 gene.

Transient Knockdown of pmaip1 Decreases Apoptosis and Promotes Mesodermal and Cardiac Differentiation

To clarify the role of pmaip1 in mESC differentiation, we transfected specific siRNAs against pmaip1 (si-Pmaip1) into phf8+/Y ESCs followed by EB formation. The negative control siRNA (si-NC) did not alter pmaip1 transcript levels compared with untreated (NT) cells, while si-Pmaip1 significantly inhibited pmaip1 transcripts by 74%-76% at differentiation days 0 and 1 (Fig. 6A-a). si-Pmaip1 cells had fewer TUNEL+ cells compared with the NT and si-NC cells at differentiation day 3 in both phf8+/Y and phf8-/Y cells (Fig. 6A-b). We then examined whether the pmaip1 knockdown influences mesodermal and early cardiac differentiation. As shown in Figure 6B, the apoptosis of FLK-1+ cells was significantly decreased in si-Pmaip1 mESCs (Fig. 6B). The expression of T and gsc as well as nrp1 and flk-1 were increased in si-Pmaip1 cells compared with those in NT and si-NC cells at differentiation day 3. In addition, the expression of cardiac transcript factors mef2c and tbx5 was up-regulated at differentiation day 5, and myh6 andtnnt2 were up-regulated at differentiation day 9 (Fig. 6C). We also transfected si-Pmaip1 into phf8-/Y ESCs. The expression of pmaip1 was downregulated at differentiation day 0 and day 1 in phf8-/Y ESCs with si-Pmaip1 (Supporting Information as Fig. S4A-a), accompanied by a decrease in TUNEL+ cells compared with NT and si-NC (Fig. 6A-b), while Annexin V remained unchanged (Supporting Information Fig. S4A-b). The expression of nrp1 and flk1 did not significantly change in phf8-/Y ESCs with si-Pmaip1 at differentiation day 3, while mef2c was upregulated at differentiation day 5, and myh6 was upregulated at differentiation day 9 (Supporting Information as Fig. S4B). These results suggest that downregulation of pmaip1 in phf8-/Y ESCs may not lead to as robust of a phenotype as it did in phf8+/Y ESCs. This difference is likely due to the level ofpmaip1 during early differentiation of phf8-/Y ESCs was already decreased to a low level similar to that observed in the undifferentiated cells (Fig. 5B). Taken together, these data demonstrate that the decreased apoptosis via down-regulation of pmaip1 contributes, at least partially, to the phf8-/Y-facilitated mesodermal and cardiomyocyte commitment.

Figure 6. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and cardiac differentiation through pmaip1. (A-a): qRT-PCR analysis of thepmaip1 expression in phf8+/Y ESCs after being transiently transfected with si-NC or si-Pmaip1. n = 4. (A-b): Apoptosis cells were quantified by flow cytometry analysis of TUNEL assay at differentiation day 3 in phf8+/Y and phf8-/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 3. (B): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 4. (C): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8+/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 5. (D): Flow cytometry detection of TUNEL positive cells at differentiation day 3, Annexin V positive cells and double stained FLK-1 and Annexin V at differentiation day 4 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mouse embryonic stem cells (mESCs). n = 4. (E): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mESCs. n = 3. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y or phf8-/Y value.
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Overexpression of pmaip1 or hPHF8 in phf8-/Y ESCs Increases Apoptosis and Weakens Mesodermal and Cardiac Differentiation

To further determine whether PHF8 contributes to mesoderm and cardiac cell commitment through the regulation of apoptosis via targeting pmaip1, we rescued the expression of pmaip1 and phf8 in phf8-/Y ESCs by generating pmaip1-ovexpressing phf8-/Y mESCs (phf8-pmaip1-/+ mESCs) and hPHF8-overexpressing phf8-/Y mESCs (phf8-hPHF8-/+ mESCs). The qRT-PCR analysis confirmed that the expression of hPHF8 or pmaip1 was significantly upregulated in the respective overexpressing cell lines (Supporting Information Fig. S4C). The expression of pmaip1 in undifferentiated phf8-/Y mESCs was not affected by hPHF8 overexpression. However, Pmaip1 transcripts increased by differentiation day 3 in overexpressing cells (Supporting Information Fig. S4D), indicating that PHF8 does regulate the expression of pmaip1 during differentiation. Both TUNEL and Annexin V analysis revealed significant increases of apoptosis in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs compared with the phf8-/Y andphf8-NC-/+ mESCs at differentiation day 3 or day 4, accompanied by a higher apoptosis ratio in FLK-1+ cells (Fig. 6D). Moreover, the expression of T and gsc as well as nrp1and flk-1 were significantly decreased in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs at differentiation day 3, followed by a down-regulation of mef2c and tbx5 at differentiation day 5, and myh6 and tnnt2 at differentiation day 9 (Fig. 6E). In addition, TUNEL analysis showed no changes in the apoptotic responses either through knockout or overexpression of phf8 compared with the corresponding wild-type cells or phf8+/Y cells during induced ectodermal differentiation (Supporting information Fig. S4E). These data are consistent with a regulatory role of phf8 on mesodermal and cardiac differentiation through targeting of pmaip1

Discussion

This is the first study to unravel a regulatory role of histone demethylase in the differentiation of ESCs through the control of apoptosis and subsequent effects on cell lineage commitment. The role of PHF8 in the regulation of ESC differentiation to the mesodermal lineage and cardiac differentiation is supported by selective changes in RNA markers for mesodermal lineages, and an increase in cardiomyocyte progenitors and cardiomyocytes (Figs. 1C, 2C). Moreover, deletion of phf8 specifically inhibits apoptosis of Flk-1+ mesodermal cells with a concomitant reduction in Annexin V+ staining (Fig. 4D) and cardiac differentiation (Fig. 2B–E), while the ratio of TUNEL+ to either NESTIN+(ectodermal cells) or SOX17+ (endodermal cells) cells does not differ between the phf8+/Y and phf8-/Y lines (Supporting Information Fig. S3C, S3D). Consistently, the proportion of early apoptotic cells (Annexin V+) in pmaip1-knockdown (Fig. 6B) is also decreased, while pmaip1-overexpression or hPHF8-overexpression in phf8-/Y cells increase the proportion of TUNEL+ and Annexin V+ cells simultaneously with a reduction in mesodermal and cardiac differentiation (Fig. 6D, 6E). These findings indicate that the PHF8 functions, at least partially, through regulation of apoptosis.

It is well known that the regulation of apoptosis is of critical importance for proper ESC differentiation and embryo development [8, 43]. ESC differentiation is regulated by apoptosis induced by MAPK activation [7] and IP3R3-regulated Ca2+ release [5]. Previously only histone 3 lysine 4 methyltransferase MLL2 had been shown to activate the antiapoptotic gene bcl2 to inhibit apoptosis during ESC differentiation [44]. The data presented, here, extends and reveals the importance of epigenetic controls in the activation of proapoptotic gene associated with ESC differentiation.

Mesodermal and cardiac differentiation have been shown to be regulated by the histone demethylase ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX)[13, 45] and jumonji domain–containing protein 3 (JMJD3) [12] through transcriptional activation of mesodermal and cardiac genes. These findings together with those presented in this paper support the critical role of histone demethylases in lineage commitment through regulatory mechanisms that control the expression of core lineage specific transcription factors and apoptotic genes. The decrease in apoptosis through deletion of phf8 can be attributed to the maintenance of repressive H3K9me2 mark on the TSS of pmaip1 after phf8 deletion, resulting in a ∼70% downregulation of pmaip1 at differentiation day 3 in the phf8-/Y cells (Fig. 5B). The pro-apoptotic gene pmaip1 is, therefore, epigenetically regulated by the histone demethylase, which subsequently affects the mesodermal and cardiac differentiation.

PMAIP1 is a Bcl2 homology domain 3 (BH3)-only protein that acts as an important mediator of apoptosis [46]. Its expression is regulated transcriptionally by various transcription factors and, when present, it acts to promote cell death in a variety of ways [21] including caspase 3 dependent [47] and independent apoptosis [48] and autophagy [40]. Here, we find that PHF8 and its regulation on the pmaip1 promote DNA fragmentation and cell death most likely through a caspase 3-independent pathway. This conclusion is based on the observation that neither the ratio of cleaved caspase 3 to total caspase 3 [49, 50] nor PARP1, a downstream target of caspase 3, is significantly affected. While this may be explained as the inhibitor of apoptosis proteins can counteract the function of caspase 3 [51, 52], the exact mechanisms we observed here need to be further explored.

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Biology, Physiology and Pathophysiology of Heat Shock Proteins

Curation: Larry H. Bernstein, MD, FCAP

 

 

Heat Shock Proteins (HSP)

  1. Exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes

Hsp70 chaperones: Cellular functions and molecular mechanism

M. P. MayerB. Bukau
Cell and Molec Life Sci  Mar 2005; 62:670  http://dx.doi.org:/10.1007/s00018-004-4464-6

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [17]. Hsp70s have thus housekeeping functions in the cell in which they are built-in components of folding and signal transduction pathways, and quality control functions in which they proofread the structure of proteins and repair misfolded conformers. All of these activities appear to be based on the property of Hsp70 to interact with hydrophobic peptide segments of proteins in an ATP-controlled fashion. The broad spectrum of cellular functions of Hsp70 proteins is achieved through

  • the amplification and diversification of hsp70genes in evolution, which has generated specialized Hsp70 chaperones,
  • co-chaperones which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and
  • cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines.

Protein folding processes assisted by Hsp70

The role of Hsp70s in the folding of non-native proteins can be divided into three related activities: prevention of aggregation, promotion of folding to the native state, and solubilization and refolding of aggregated proteins. In the cellular milieu, Hsp70s exert these activities in the quality control of misfolded proteins and the co- and posttranslational folding of newly synthesized proteins. Mechanistically related but less understood is the role of Hsp70s in the disassembly of protein complexes such as clathrin coats, viral capsids and the nucleoprotein complex, which initiates the replication of bacteriophage λ DNA. A more complex folding situation exists for the Hsp70-dependent control of regulatory proteins since several steps in the folding and activation process of these substrates are assisted by multiple chaperones.

Hsp70 proteins together with their co-chaperones of the J-domain protein (JDP) family prevent the aggregation of non-native proteins through association with hydrophobic patches of substrate molecules, which shields them from intermolecular interactions (‘holder’ activity). Some JDPs such as Escherichia coli DnaJ and Saccharomyces cerevisiae Ydj1 can prevent aggregation by themselves through ATP-independent transient and rapid association with the substrates. Only members of the Hsp70 family with general chaperone functions have such general holder activity.

Hsp70 chaperone systems assist non-native folding intermediates to fold to the native state (‘folder’ activity). The mechanism by which Hsp70-chaperones assist the folding of non-native substrates is still unclear. Hsp70-dependent protein folding in vitro occurs typically on the time scale of minutes or longer. Substrates cycle between chaperone-bound and free states until the ensemble of molecules has reached the native state. There are at least two alternative modes of action. In the first mechanism Hsp70s play a rather passive role. Through repetitive substrate binding and release cycles they keep the free concentration of the substrate sufficiently low to prevent aggregation, while allowing free molecules to fold to the native state (‘kinetic partitioning’). In the second mechanism, the binding and release cycles induce local unfolding in the substrate, e.g. the untangling of a misfolded β-sheet, which helps to overcome kinetic barriers for folding to the native state (‘local unfolding’) [8–11]. The energy of ATP may be used to induce such conformational changes or alternatively to drive the ATPase cycle in the right direction.

Hsp70 in cellular physiology and pathophysiology

Two Hsp70 functions are especially interesting, de novo folding of nascent polypeptides and interaction with signal transduction proteins, and therefore some aspects of these functions shall be discussed below in more detail. Hsp70 chaperones were estimated to assist the de novo folding of 10–20% of all bacterial proteins whereby the dependence on Hsp70 for efficient folding correlated with the size of the protein [12]. Since the average protein size in eukaryotic cells is increased (52 kDa in humans) as compared to bacteria (35 kDa in E. coli) [25], it is to be expected that an even larger percentage of eukaryotic proteins will be in need of Hsp70 during de novo folding. This reliance on Hsp70 chaperones increases even more under stress conditions. Interestingly, mutated proteins [for example mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxid dismutase (SOD) 1] seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein [2629]. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations [30], a function demonstrated earlier for Hsp90 [3132]. Such mutations are only uncovered when the overall need for Hsp70 action exceeds the chaperone capacity of the Hsp70 proteins, for example during stress conditions [30], at certain stages in development or during aging, when the magnitude of stress-induced increase in Hsp70 levels declines [3334]. Alternatively, the mutant protein can be targeted by Hsp70 and its co-chaperones to degradation as shown e.g. for CFTRΔF508 and some of the SOD1 mutant proteins [35,36]. Deleterious mutant proteins may then only accumulate when Hsp70 proteins are overwhelmed by other, stress-denatured proteins. Both mechanisms may contribute to pathological processes such as oncogenesis (mutant p53) and neurodegenerative diseases, including amyotrophic, lateral sclerosis (SOD1 mutations), Parkinsonism (α-synuclein mutations), Huntington’s chorea (huntingtin with polyglutamin expansions) and spinocerebellar ataxias (proteins with polyglutamin expansions).

De novo folding is not necessarily accelerated by Hsp70 chaperones. In some cases folding is delayed for different reasons. First, folding of certain proteins can only proceed productively after synthesis of the polypeptide is completed as shown, e.g. for the reovirus lollipop-shaped protein sigma 1 [37]. Second, proteins destined for posttranslational insertion into organellar membranes are prevented from aggregation and transported to the translocation pore [38]. Third, in the case of the caspase-activated DNase (CAD), the active protein is dangerous for the cell and therefore can only complete folding in the presence of its specific inhibitor (ICAD). Hsp70 binds CAD cotranslationally and mediates folding only to an intermediate state. Folding is completed after addition of ICAD, which is assembled into a complex with CAD in an Hsp70-dependent manner [39]. Similar folding pathways may exist also for other potentially dangerous proteins.

As mentioned above Hsp70 interacts with key regulators of many signal transduction pathways controlling cell homeostasis, proliferation, differentiation and cell death. The interaction of Hsp70 with these regulatory proteins continues in activation cycles that also involve Hsp90 and a number of co-chaperones. The regulatory proteins, called clients, are thereby kept in an inactive state from which they are rapidly activated by the appropriate signals. Hsp70 and Hsp90 thus repress regulators in the absence of the upstream signal and guarantee full activation after the signal transduction pathway is switched on [6]. Hsp70 can be titrated away from these clients by other misfolded proteins that may arise from internal or external stresses. Consequently, through Hsp70 disturbances of the cellular system induced by environmental, developmental or pathological processes act on these signal transduction pathways.

In this way stress response and apoptosis are linked to each other. Hsp70 inhibits apoptosis acting on the caspase-dependent pathway at several steps both upstream and downstream of caspase activation and on the caspase-independent pathway. Overproduction of Hsp70 leads to increased resistance against apoptosis-inducing agents such as tumor necrosis factor-α(TNFα), staurosporin and doxorubicin, while downregulation of Hsp70 levels by antisense technology leads to increased sensitivity towards these agents [1840]. This observation relates to many pathological processes, such as oncogenesis, neurodegeneration and senescence. In many tumor cells increased Hsp70 levels are observed and correlate with increased malignancy and resistance to therapy. Downregulation of the Hsp70 levels in cancer cells induce differentiation and cell death [41]. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s corea and spinocerebellar ataxias are characterized by excessive apoptosis. In several different model systems overexpression of Hsp70 or one of its co-chaperones could overcome the neurodegenerative symptoms induced by expression of a disease-related gene (huntingtin, α-synuclein or ataxin) [20,42]. Senescence in cell culture as well as aging in vivo is correlated with a continuous decline in the ability to mount a stress response [3443]. Age-related symptoms and diseases reflect this decreased ability to cope with cellular stresses. Interestingly, centenarians seem to be an exception to the rule, as they show a significant induction of Hsp70 production after heat shock challenge [44].

ATPase domain and ATPase cycle

Substrate binding

The coupling mechanism: nucleotide-controlled opening and closing of the substrate binding cavity

The targeting activity of co-chaperones

J-domain proteins

Bag proteins

Hip, Hop and CHIP

Perspectives

The Hsp70 protein family and their co-chaperones constitute a complex network of folding machines which is utilized by cells in many ways. Despite considerable progress in the elucidation of the mechanistic basis of these folding machines, important aspects remain to be solved. With respect to the Hsp70 proteins it is still unclear whether their activity to assist protein folding relies on the ability to induce conformational changes in the bound substrates, how the coupling mechanism allows ATP to control substrate binding and to what extent sequence variations within the family translate into variations of the mechanism. With respect to the action of co-chaperones we lack a molecular understanding of the coupling function of JDPs and of how co-chaperones target their Hsp70 partner proteins to substrates. Furthermore, it can be expected that more cellular processes will be discovered that depend on the chaperone activity of Hsp70 chaperones.

 

  1. The biochemistry and ultrastructure of molecular chaperones

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Laurence H. Pearl and Chrisostomos Prodromou
Ann Rev of Biochem July 2006;75:271-294
http://dx.doi.org:/10.1146/annurev.biochem.75.103004.142738

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90’s diverse “clientele.” Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

 

  1. Properties of heat shock proteins (HSPs) and heat shock factor (HSF)

Heat shock factors: integrators of cell stress, development and lifespan

Malin Åkerfelt,*‡ Richard I. Morimoto,§ and Lea Sistonen*‡
Nat Rev Mol Cell Biol. 2010 Aug; 11(8): 545–555.  doi:  10.1038/nrm2938

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

In the early 1960s, Ritossa made the seminal discovery of temperature-induced puffs in polytene chromosomes of Drosophila melanogaster larvae salivary glands1. A decade later, it was shown that the puffing pattern corresponded to a robust activation of genes encoding the heat shock proteins (HSPs), which function as molecular chaperones2. The heat shock response is a highly conserved mechanism in all organisms from yeast to humans that is induced by extreme proteotoxic insults such as heat, oxidative stress, heavy metals, toxins and bacterial infections. The conservation among different eukaryotes suggests that the heat shock response is essential for survival in a stressful environment.

The heat shock response is mediated at the transcriptional level by cis-acting sequences called heat shock elements (HSEs; BOX 1) that are present in multiple copies upstream of the HSP genes3. The first evidence for a specific transcriptional regulator, the heat shock factor (HSF) that can bind to the HSEs and induce HSP gene expression, was obtained through DNA–protein interaction studies on nuclei isolated from D. melanogaster cells4,5. Subsequent studies showed that, in contrast to a single HSF in invertebrates, multiple HSFs are expressed in plants and vertebrates68. The mammalian HSF family consists of four members: HSF1,HSF2, HSF3 and HSF4. Distinct HSFs possess unique and overlapping functions (FIG. 1), exhibit tissue-specific patterns of expression and have multiple post-translational modifications (PTMs) and interacting protein partners7,9,10. Functional crosstalk between HSF family members and PTMs facilitates the fine-tuning of HSF-mediated gene regulation. The identification of many targets has further extended the impact of HSFs beyond the heat shock response. Here, we present the recent discoveries of novel target genes and physiological functions of HSFs, which have changed the view that HSFs act solely in the heat shock response. Based on the current knowledge of small-molecule activators and inhibitors of HSFs, we also highlight the potential for pharmacologic modulation of HSF-mediated gene regulation.

Box 1

The heat shock element

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u1.jpg

Heat shock factors (HSFs) act through a regulatory upstream promoter element, called the heat shock element (HSE). In the DNA-bound form of a HSF, each DNA-binding domain (DBD) recognizes the HSE in the major groove of the double helix6. The HSE was originally identified using S1 mapping of transcripts of the Drosophila melanogaster heat shock protein (HSP) genes3 (see the figure; part a). Residues –47 to –66 are necessary for heat inducibility. HSEs in HSP gene promoters are highly conserved and consist of inverted repeats of the pentameric sequence nGAAn132. The type of HSEs that can be found in the proximal promoter regions of HSP genes is composed of at least three contiguous inverted repeats: nTTCnnGAAnnTTCn132134. The promoters of HSF target genes can also contain more than one HSE, thereby allowing the simultaneous binding of multiple HSFs. The binding of an HSF to an HSE occurs in a cooperative manner, whereby binding of an HSF trimer facilitates binding of the next one135. More recently, Trinklein and colleagues used chromatin immunoprecipitation to enrich sequences bound by HSF1 in heat-shocked human cells to define the HSE consensus sequence. They confirmed the original finding of Xiao and Lis, who identified guanines as the most conserved nucleotides in HSEs87,133 (see the figure; part b). Moreover, in a pair of inverted repeats, a TTC triplet 5′ of a GAA triplet is separated by a pyrimidine–purine dinucleotide, whereas the two nucleotides separating a GAA triplet 5′ from a TTC triplet is unconstrained87. The discovery of novel HSF target genes that are not involved in the heat shock response has rendered it possible that there may be HSEs in many genes other than the HSP genes. Although there are variations in these HSEs, the spacing and position of the guanines are invariable7. Therefore, both the nucleotides and the exact spacing of the repeated units are considered as key determinants for recognition by HSFs and transcriptional activation. Part b of the figure is modified, with permission, from REF. 87 © (2004) The American Society for Cell Biology.

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f1.gif

The mammalian HSF machinery

HSFs as stress integrators

A hallmark of stressed cells and organisms is the increased synthesis of HSPs, which function as molecular chaperones to prevent protein misfolding and aggregation to maintain protein homeostasis, also called proteostasis11. The transcriptional activation of HSP genes is mediated by HSFs (FIG. 2a), of which HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance1215. HSF1 is constitutively expressed in most tissues and cell types16, where it is kept inactive in the absence of stress stimuli. Thus, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple PTMs, protein–protein interactions and subcellular localization. HSF1 also has an intrinsic stress-sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a homotrimer in vitro in response to thermal or oxidative stress1719.

Figure 2    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f2.gif

Members of the mammalian HSF family

Functional domains

HSFs, like other transcription factors, are composed of functional domains. These have been most thoroughly characterized for HSF1 and are schematically presented in FIG. 2b. The DNA-binding domain (DBD) is the best preserved domain in evolution and belongs to the family of winged helix-turn-helix DBDs2022. The DBD forms a compact globular structure, except for a flexible wing or loop that is located between β-strands 3 and 4 (REF. 6). This loop generates a protein– protein interface between adjacent subunits of the HSF trimer that enhances high-affinity binding to DNA by cooperativity between different HSFs23. The DBD can also mediate interactions with other factors to modulate the transactivating capacity of HSFs24. Consequently, the DBD is considered as the signature domain of HSFs for target-gene recognition.

The trimerization of HSFs is mediated by arrays of hydrophobic heptad repeats (HR-A and HR-B) that form a coiled coil, which is characteristic for many Leu zippers6,25 (FIG. 2b). The trimeric assembly is unusual, as Leu zippers typically facilitate the formation of homodimers or heterodimers. Suppression of spontaneous HSF trimerization is mediated by yet another hydrophobic repeat, HR-C2628. Human HSF4 lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity29. Positioned at the extreme carboxyl terminus of HSFs is the transactivation domain, which is shared among all HSFs6except for yeast Hsf, which has transactivation domains in both the amino and C termini, and HSF4A, which completely lacks a transactivation domain2931. In HSF1, the transactivation domain is composed of two modules — AD1 and AD2, which are rich in hydrophobic and acidic residues (FIG. 3a) — that together ensures a rapid and prolonged response to stress32,33. The transactivation domain was originally proposed to provide stress inducibility to HSF1 (REFS 34,35), but it soon became evident that an intact regulatory domain, located between the HR-A and HR-B and the transactivation domain, is essential for the responsiveness to stress stimuli32,33,36,37. Because several amino acids that are known targets for different PTMs reside in the regulatory domain33,3842, the structure and function of this domain are under intensive investigation.

Figure 3    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f3.gif

HSF1 undergoes multiple PTMs on activation

Regulation of the HSF1 activation–attenuation cycle

The conversion of the inactive monomeric HSF1 to high-affinity DNA-binding trimers is the initial step in the multistep activation process and is a common feature of all eukaryotic HSFs43,44 (FIG. 3b). There is compelling evidence for HSF1 interacting with multiple HSPs at different phases of its activation cycle. For example, monomeric HSF1 interacts weakly with HSP90 and, on stress, HSF1 dissociates from the complex, allowing HSF1 trimerization45,46 (FIG. 3b). Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex of HSP90, co-chaperone p23 (also known as PTGES3) and immunophilin FK506-binding protein 5 (FKBP52; also known as FKBP4)4651. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation on heat shock52. Activated HSF1 trimers also interact with HSP70 and the co-chaperone HSP40 (also known as DNAJB1), but instead of suppressing the DNA-binding activity of HSF1, this interaction inhibits its transactivation capacity5254. Although the inhibitory mechanism is still unknown, the negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides.

A ribonucleoprotein complex containing eukaryotic elongation factor 1A (eEF1A) and a non-coding RNA, heat shock RNA-1 (HSR-1), has been reported to possess a thermosensing capacity. According to the proposed model, HSR-1 undergoes a conformational change in response to heat stress and together with eEF1A facilitates trimerization of HSF1 (REF. 55). How this activation mode relates to the other regulatory mechanisms associated with HSFs remains to be elucidated.

Throughout the activation–attenuation cycle, HSF1 undergoes extensive PTMs, including acetylation, phosphorylation and sumoylation (FIG. 3). HSF1 is also a phosphoprotein under non-stress conditions, and the results from mass spectrometry (MS) analyses combined with phosphopeptide mapping experiments indicate that at least 12 Ser residues are phosphorylated41,5659. Among these sites, stress-inducible phosphorylation of Ser230 and Ser326 in the regulatory domain contributes to the transactivation function of HSF1 (REFS 38,41). Phosphorylation-mediated sumoylation on a single Lys residue in the regulatory domain occurs rapidly and transiently on exposure to heat shock; Ser303 needs to be phosphorylated before a small ubiquitin-related modifier (SUMO) can be conjugated to Lys298 (REF. 39). The extended consensus sequence ΨKxExxSP has been named the phosphorylation-dependent sumoylation motif (PDSM; FIG. 3)40. The PDSM was initially discovered in HSF1 and subsequently found in many other proteins, especially transcriptional regulators such as HSF4, GATA1, myocyte-specific enhancer factor 2A (MEF2A) and SP3, which are substrates for both SUMO conjugation and Pro-directed kinases40,6062.

Recently, Mohideen and colleagues showed that a conserved basic patch on the surface of the SUMO-conjugating enzyme ubiquitin carrier protein 9 (UBC9; also known as UBE2I) discriminates between the phosphorylated and non-phosphorylated PDSM of HSF1 (REF. 63). Future studies will be directed at elucidating the molecular mechanisms for dynamic phosphorylation and UBC9-dependent SUMO conjugation in response to stress stimuli and establishing the roles of kinases, phosphatases and desumoylating enzymes in the heat shock response. The kinetics of phosphorylation-dependent sumoylation of HSF1 correlates inversely with the severity of heat stress, and, as the transactivation capacity of HSF1 is impaired by sumoylation and this PTM is removed when maximal HSF1 activity is required40, sumoylation could modulate HSF1 activity under moderate stress conditions. The mechanisms by which SUMO modification represses the transactivating capacity of HSF1, and the functional relationship of this PTM with other modifications that HSF1 is subjected to, will be investigated with endogenous substrate proteins.

Phosphorylation and sumoylation of HSF1 occur rapidly on heat shock, whereas the kinetics of acetylation are delayed and coincide with the attenuation phase of the HSF1 activation cycle. Stress-inducible acetylation of HSF1 is regulated by the balance of acetylation by p300–CBP (CREB-binding protein) and deacetylation by the NAD+-dependent sirtuin, SIRT1. Increased expression and activity of SIRT1 enhances and prolongs the DNA-binding activity of HSF1 at the human HSP70.1promoter, whereas downregulation of SIRT1 enhances the acetylation of HSF1 and the attenuation of DNA-binding without affecting the formation of HSF1 trimers42. This finding led to the discovery of a novel regulatory mechanism of HSF1 activity, whereby SIRT1 maintains HSF1 in a state that is competent for DNA binding by counteracting acetylation (FIG. 3). In the light of current knowledge, the attenuation phase of the HSF1 cycle is regulated by a dual mechanism: a dependency on the levels of HSPs that feed back directly by weak interactions with HSF1, and a parallel step that involves the SIRT1-dependent control of the DNA-binding activity of HSF1. Because SIRT1 has been implicated in caloric restriction and ageing, the age-dependent loss of SIRT1 and impaired HSF1 activity correlate with an impairment of the heat shock response and proteostasis in senescent cells, connecting the heat shock response to nutrition and ageing (see below).

HSF dynamics on the HSP70 promoter

For decades, the binding of HSF to the HSP70.1 gene has served as a model system for inducible transcription in eukaryotes. In D. melanogaster, HSF is constitutively nuclear and low levels of HSF are associated with the HSP70promoter before heat shock6466. The uninduced HSP70 promoter is primed for transcription by a transcriptionally engaged paused RNA polymerase II (RNAP II)67,68. RNAP II pausing is greatly enhanced by nucleosome formation in vitro, implying that chromatin remodelling is crucial for the release of paused RNAP II69. It has been proposed that distinct hydrophobic residues in the transactivation domain of human HSF1 can stimulate RNAP II release and directly interact withBRG1, the ATPase subunit of the chromatin remodelling complex SWI/SNF70,71. Upon heat shock, RNAP II is released from its paused state, leading to the synthesis of a full-length transcript. Rapid disruption of nucleosomes occurs across the entire HSP70 gene, at a rate that is faster than RNAP II-mediated transcription72. The nucleosome displacement occurs simultaneously with HSF recruitment to the promoter in D. melanogaster. Downregulation of HSF abrogates the loss of nucleosomes, indicating that HSF provides a signal for chromatin rearrangement, which is required for HSP70 nucleosome displacement. Within seconds of heat shock, the amount of HSF at the promoter increases drastically and HSF translocates from the nucleoplasm to several native loci, including HSP genes. Interestingly, the levels of HSF occupying the HSP70 promoter reach saturation soon after just one minute65,73.

HSF recruits the co-activating mediator complex to the heat shock loci, which acts as a bridge to transmit activating signals from transcription factors to the basal transcription machinery. The mediator complex is recruited by a direct interaction with HSF: the transactivation domain of D. melanogaster HSF binds to TRAP80(also known as MED17), a subunit of the mediator complex74. HSF probably has other macromolecular contacts with the preinitiation complex as it binds to TATA-binding protein (TBP) and the general transcription factor TFIIB in vitro75,76. In contrast to the rapid recruitment and elongation of RNAP II on heat shock, activated HSF exchanges very slowly at the HSP70 promoter. HSF stays stably bound to DNA in vivo and no turnover or disassembly of transcription activator is required for successive rounds of HSP70 transcription65,68.

Functional interplay between HSFs

Although HSF1 is the principal regulator of the heat shock response, HSF2 also binds to the promoters of HSP genes. In light of our current knowledge, HSF2 strictly depends on HSF1 for its stress-related functions as it is recruited to HSP gene promoters only in the presence of HSF1 and this cooperation requires an intact HSF1 DBD77. Nevertheless, HSF2 modulates, both positively and negatively, the HSF1-mediated inducible expression of HSP genes, indicating that HSF2 can actively participate in the transcriptional regulation of the heat shock response. Coincident with the stress-induced transcription of HSP genes, HSF1 and HSF2 colocalize and accumulate rapidly on stress into nuclear stress bodies (NSBs; BOX 2), where they bind to a subclass of satellite III repeats, predominantly in the human chromosome 9q12 (REFS 7880). Consequently, large and stable non-coding satellite III transcripts are synthesized in an HSF1-dependent manner in NSBs81,82. The function of these transcripts and their relationship with other HSF1 targets, and the heat shock response in general, remain to be elucidated.

 

Box 2

Nuclear stress bodies  

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u2.jpg

The cell nucleus is highly compartmentalized and dynamic. Many nuclear factors are diffusely distributed throughout the nucleoplasm, but they can also accumulate in distinct subnuclear compartments, such as nucleoli, speckles, Cajal bodies and promyelocytic leukaemia (PML) bodies136. Nuclear stress bodies (NSBs) are different from any other known nuclear bodies137,138. Although NSBs were initially thought to contain aggregates of denatured proteins and be markers of heat-shocked cells, their formation can be elicited by various stresses, such as heavy metals and proteasome inhibitors137. NSBs are large structures, 0.3–3 μm in diameter, and are usually located close to the nucleoli or nuclear envelope137,138. NSBs consist of two populations: small, brightly stained bodies and large, clustered and ring-like structures137.

NSBs appear transiently and are the main site of heat shock factor 1 (HSF1) and HSF2 accumulation in stressed human cells80. HSF1 and HSF2 form a physically interacting complex and colocalize into small and barely detectable NSBs after only five minutes of heat shock, but the intensity and size of NSBs increase after hours of continuous heat shock. HSF1 and HSF2 colocalize in HeLa cells that have been exposed to heat shock for one hour at 42°C (see the figure; confocal microscopy image with HSF1–green fluorescent protein in green and endogenous HSF2 in red). NSBs form on specific chromosomal loci, mainly on q12 of human chromosome 9, where HSFs bind to a subclass of satellite III repeats78,79,83. Stress-inducible and HSF1-dependent transcription of satellite III repeats has been shown to produce non-coding RNA molecules, called satellite III transcripts81,82. The 9q12 locus consists of pericentromeric heterochromatin, and the satellite III repeats provide scaffolds for docking components, such as splicing factors and other RNA-processing proteins139143.

HSF2 also modulates the heat shock response through the formation of heterotrimers with HSF1 in the NSBs when bound to the satellite III repeats83 (FIG. 4). Studies on the functional significance of heterotrimerization indicate that HSF1 depletion prevents localization of HSF2 to NSBs and abolishes the stress-induced synthesis of satellite III transcripts. By contrast, increased expression of HSF2 leads to its own activation and the localization of both HSF1 and HSF2 to NSBs, where transcription is spontaneously induced in the absence of stress stimuli. These results suggest that HSF2 can incorporate HSF1 into a transcriptionally competent heterotrimer83. It is possible that the amounts of HSF2 available for heterotrimerization with HSF1 influence stress-inducible transcription, and that HSF1–HSF2 heterotrimers regulate transcription in a temporal manner. During the acute phase of heat shock, HSF1 is activated and HSF1–HSF2 heterotrimers are formed, whereas upon prolonged exposures to heat stress the levels of HSF2 are diminished, thereby limiting heterotrimerization83. Intriguingly, in specific developmental processes such as corticogenesis and spermatogenesis, the expression of HSF2 increases spatiotemporarily, leading to its spontaneous activation. Therefore, it has been proposed that HSF-mediated transactivation can be modulated by the levels of HSF2 to provide a switch that integrates the responses to stress and developmental stimuli83 (FIG. 4). Functional relationships between different HSFs are emerging, and the synergy of DNA-binding activities among HSF family members offers an efficient way to control gene expression in a cell- and stimulus-specific manner to orchestrate the differential upstream signalling and target-gene networks.

Figure 4   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f4.gif

 

Interactions between different HSFs provide distinct functional modes in transcriptional regulation

A new member of the mammalian HSF family, mouse HSF3, was recently identified10. Avian HSF3 was shown to be activated at higher temperatures and with different kinetics than HSF1 (REF. 84), whereas in mice, heat shock induces the nuclear translocation of HSF3 and activation of stress-responsive genes other than HSP genes10. Future experiments will determine whether HSF3 is capable of interacting with other HSFs, potentially through heterocomplex formation. HSF4 has not been implicated in the heat shock response, but it competes with HSF1 for common target genes in mouse lens epithelial cells85, which will be discussed below. It is important to elucidate whether the formation of homotrimers or hetero trimers between different family members is a common theme in HSF-mediated transcriptional regulation.

 

HSFs as developmental regulators

Evidence is accumulating that HSFs are highly versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physioogical functions, especially during development. The initial observations using deletion experiments of the D. melanogaster Hsf gene revealed defective oogenesis and larvae development86. These effects were not caused by obvious changes in HSP gene expression patterns, which is consistent with the subsequent studies showing that basal expression of HSP genes during mouse embryogenesis is not affected by the lack of HSF1 (REF. 13). These results are further supported by genome-wide gene expression studies revealing that numerous genes, not classified as HSP genes or molecular chaperones, are under HSF1-dependent control87,88.

Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility13. Fertilized oocytes do not develop past the zygotic stage when HSF1-deficient female mice are mated with wild-type male mice, indicating that HSF1 is a maternal factor that is essential for early post-fertilization development89. Recently, it was shown that HSF1 is abundantly expressed in maturing oocytes, where it regulates specifically Hsp90α transcription90. The HSF1-deficient oocytes are devoid of HSP90α and exhibit a blockage of meiotic maturation, including delayed G2–M transition or germinal vesicle breakdown and defective asymmetrical division90. Moreover, intra-ovarian HSF1-depleted oocytes contain dysfunctional mitochondria and are sensitive to oxidative stress, leading to reduced survival91. The complex phenotype of Hsf1-knockout mice also demonstrates the involvement of HSF1 in placenta formation, placode development and the immune system15,85,92,93, further strengthening the evidence for a protective function of HSF1 in development and survival.

Both HSF1 and HSF2 are key regulators in the developing brain and in maintaining proteostasis in the central nervous system. Disruption of Hsf1 results in enlarged ventricles, accompanied by astrogliosis, neurodegeneration, progressive myelin loss and accumulation of ubiquitylated proteins in specific regions of the postnatal brain under non-stressed conditions94,95. The expression of HSP25 (also known as HSPB1) and α-crystallin B chain (CRYAB), which are known to protect cells against stress-induced protein damage and cell death, is dramatically decreased in brains lacking HSF1 (REF. 13). In contrast to HSF1, HSF2 is already at peak levels during early brain development in mice and is predominantly expressed in the proliferative neuronal progenitors of the ventricular zone and post-mitotic neurons of the cortical plate9699. HSF2-deficient mice have enlarged ventricles and defects in cortical lamination owing to abnormal neuronal migration9799. Incorrect positioning of superficial neurons during cortex formation in HSF2-deficient embryos is caused by decreased expression of the cyclin-dependent kinase 5 (CDK5) activator p35, which is a crucial regulator of the cortical migration signalling pathway100,101. The p35 gene was identified as the first direct target of HSF2 in cortex development99. As correct cortical migration requires the coordination of multiple signalling molecules, it is likely that HSF2, either directly or indirectly, also regulates other components of the same pathway.

 

Cooperativity of HSFs in development

In adult mice, HSF2 is most abundantly expressed in certain cell types of testes, specifically pachytene spermatocytes and round spermatids102. The cell-specific expression of HSF2 in testes is regulated by a microRNA, miR-18, that directly binds to the 3′ untranslated region (UTR) of HSF2 (J.K. Björk, A. Sandqvist, A.N. Elsing, N. Kotaja and L.S., unpublished observations). Targeting of HSF2 in spermatogenesis reveals the first physiological role for miR-18, which belongs to the oncomir-1 cluster associated mainly with tumour progression103. In accordance with the expression pattern during the maturation of male germ cells, HSF2-null male mice display several abnormal features in spermatogenesis, ranging from smaller testis size and increased apoptosis at the pachytene stage to a reduced amount of sperm and abnormal sperm head shape97,98,104. A genome-wide search for HSF2 target promoters in mouse testis revealed the occupancy of HSF2 on the sex chromosomal multi-copy genes spermiogenesis specific transcript on the Y 2 (Ssty2), Sycp3-like Y-linked (Sly) and Sycp3-like X-linked (Slx), which are important for sperm quality104. Compared with the Hsf2-knockout phenotype, disruption of both Hsf1 and Hsf2 results in a more pronounced phenotype, including larger vacuolar structures, more widely spread apoptosis and a complete lack of mature spermatozoa and male sterility105. The hypo thesis that the activities of HSF1 and HSF2 are intertwined and essential for spermatogenesis is further supported by our results that HSF1 and HSF2 synergistically regulate the sex chromosomal multi-copy genes in post-meiotic round spermatids (M.Å., A. Vihervaara, E.S. Christians, E. Henriksson and L.S., unpublished observations). Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are currently the only known transcriptional regulators during post-meiotic repression. These results, together with the earlier findings that HSF2 can also form heterotrimers with HSF1 in testes83, strongly suggest that HSF1 and HSF2 act in a heterocomplex and fine-tune transcription of their common target genes during the maturation of male germ cells.

HSF1 and HSF4 are required for the maintenance of sensory organs, especially when the organs are exposed to environmental stimuli for the first time after birth85,88. During the early postnatal period, Hsf1-knockout mice display severe atrophy of the olfactory epithelium, increased accumulation of mucus and death of olfactory sensory neurons88. Although lens development in HSF4-deficient mouse embryos is normal, severe abnormalities, including inclusion-like structures in lens fibre cells, appear soon after birth and the mice develop cataracts85,106,107. Intriguingly, inherited severe cataracts occurring in Chinese and Danish families have been associated with a mutation in the DBD of HSF4 (REF. 108). In addition to the established target genes, Hsp25Hsp70 and Hsp90, several new targets for HSF1 and HSF4, such as crystallin γF (Crygf), fibroblast growth factor 7 (Fgf7) and leukaemia inhibitory factor (Lif) have been found to be crucial for sensory organs85,88. Furthermore, binding of either HSF1 or HSF4 to the Fgf7 promoter shows opposite effects on gene expression, suggesting competitive functions between the two family members85. In addition to the proximal promoters, HSF1, HSF2 and HSF4 bind to other genomic regions (that is, introns and distal parts of protein-coding genes in mouse lens), and there is also evidence for either synergistic interplay or competition between distinct HSFs occupying the target-gene promoters109. It is possible that the different HSFs are able to compensate for each other to some extent. Thus, the identification of novel functions and target genes for HSFs has been a considerable step forward in understanding their regulatory mechanisms in development.

 

HSFs and lifespan

The lifespan of an organism is directly linked to the health of its tissues, which is a consequence of the stability of the proteome and functionality of its molecular machineries. During its lifetime, an organism constantly encounters environmental and physiological stress and requires an efficient surveillance of protein quality to prevent the accumulation of protein damage and the disruption of proteostasis. Proteotoxic insults contribute to cellular ageing, and numerous pathophysiological conditions, associated with impaired protein quality control, increase prominently with age11. From studies on the molecular basis of ageing, in which a wide range of different model systems and experimental strategies have been used, the insulin and insulin-like growth factor 1 receptor (IGF1R) signalling pathway, which involves the phosphoinositide 3-kinase (PI3K) and AKT kinases and the Forkhead box protein O (FOXO) transcription factors (such as DAF-16 in Caenorhabditis elegans), has emerged as a key process. The downregulation of HSF reduces the lifespan and accelerates the formation of protein aggregates in C. elegans carrying mutations in different components of the IGF1R-mediated pathway. Conversely, inhibition of IGF1R signalling results in HSF activation and promotes longevity by maintaining proteostasis110,111. These results have prompted many laboratories that use other model organisms to investigate the functional relationship between HSFs and the IGF1R signalling pathway.

The impact of HSFs on the lifespan of whole organisms is further emphasized by a recent study, in which proteome stability was examined during C. elegansageing112. The age-dependent misfolding and downregulation of distinct metastable proteins, which display temperature-sensitive missense mutations, was examined in different tissues. Widespread failure in proteostasis occurred rapidly at an early stage of adulthood, coinciding with the severely impaired heat shock response and unfolded protein response112. The age-dependent collapse of proteostasis could be restored by overexpression of HSF and DAF-16, strengthening the evidence for the unique roles of these stress-responsive transcription factors to prevent global instability of the proteome.

Limited food intake or caloric restriction is another process that is associated with an enhancement of lifespan. In addition to promoting longevity, caloric restriction slows down the progression of age-related diseases such as cancer, cardiovascular diseases and metabolic disorders, stimulates metabolic and motor activities, and increases resistance to environmental stress stimuli113. To this end, the dynamic regulation of HSF1 by the NAD+-dependent protein deacetylase SIRT1, a mammalian orthologue of the yeast transcriptional regulator Sir2, which is activated by caloric restriction and stress, is of particular interest. Indeed, SIRT1 directly deacetylates HSF1 and keeps it in a state that is competent for DNA binding. During ageing, the DNA-binding activity of HSF1 and the amount of SIRT1 are reduced. Consequently, a decrease in SIRT1 levels was shown to inhibit HSF1 DNA-binding activity in a cell-based model of ageing and senescence42. Furthermore, an age-related decrease in the HSF1 DNA-binding activity is reversed in cells exposed to caloric restriction114. These results indicate that HSF1 and SIRT1 function together to protect cells from stress insults, thereby promoting survival and extending lifespan. Impaired proteostasis during ageing may at least partly reflect the compromised HSF1 activity due to lowered SIRT1 expression.

 

Impact of HSFs in disease

The heat shock response is thought to be initiated by the presence of misfolded and damaged proteins, and is thus a cell-autonomous response. When exposed to heat, cells in culture, unicellular organisms, and cells in a multicellular organism can all trigger a heat shock response autonomously115117. However, it has been proposed that multicellular organisms sense stress differently to isolated cells. For example, the stress response is not properly induced even if damaged proteins are accumulated in neurodegenerative diseases like Huntington’s disease and Parkinson’s disease, suggesting that there is an additional control of the heat shock response at the organismal level118. Uncoordinated activation of the heat shock response in cells in a multicellular organism could cause severe disturbances of interactions between cells and tissues. In C. elegans, a pair of thermosensory neurons called AFDs, which sense and respond to temperature, regulate the heat shock response in somatic tissues by controlling HSF activity119,120. Moreover, the heat shock response in C. elegans is influenced by the metabolic state of the organism and is reduced under conditions that are unfavourable for growth and reproduction121. Neuronal control may therefore allow organisms to coordinate the stress response of individual cells with the varying metabolic requirements in different tissues and developmental stages. These observations are probably relevant to diseases of protein misfolding that are highly tissue-specific despite the often ubiquitous expression of damaged proteins and the heat shock response.

Elevated levels of HSF1 have been detected in several types of human cancer, such as breast cancer and prostate cancer122,123. Mice deficient in HSF1 exhibit a lower incidence of tumours and increased survival than their wild-type counterparts in a classical chemical skin carcinogenesis model and in a genetic model expressing an oncogenic mutation of p53. Similar results have been obtained in human cancer cells lines, in which HSF1 was depleted using an RNA interference strategy124. HSF1 expression is likely to be crucial for non-oncogene addiction and the stress phenotype of cancer cells, which are attributes given to many cancer cells owing to their high intrinsic level of proteotoxic and oxidative stress, frequent spontaneous DNA damage and aneuploidy125. Each of these features may disrupt proteostasis, raising the need for efficient chaperone and proteasome activities. Accordingly, HSF1 would be essential for the survival of cancer cells that experience constant stress and develop non-oncogene addiction.

 

HSFs as therapeutic targets

Given the unique role of HSF1 in stress biology and proteostasis, enhanced activity of this principal regulator during development and early adulthood is important for the stability of the proteome and the health of the cell. However, HSF1 is a potent modifier of tumorigenesis and, therefore, a potential target for cancer therapeutics125. In addition to modulating the expression of HSF1, the various PTMs of HSF1 that regulate its activity should be considered from a clinical perspective. As many human, age-related pathologies are associated with stress and misfolded proteins, several HSF-based therapeutic strategies have been proposed126. In many academic and industrial laboratories, small molecule regulators of HSF1 are actively being searched for (see Supplementary information S1 (table)). For example, celastrol, which has antioxidant properties and is a natural compound derived from the Celastreace family of plants, activates HSF1 and induces HSP expression with similar kinetics to heat shock, and could therefore be a potential candidate molecule for treating neurodegenerative diseases127,128. In a yeast-based screen, a small-molecule activator of human HSF1 was found and named HSF1A129. HSF1A, which is structurally distinct from the other known activators, activates HSF1 and enhances chaperone expression, thereby counteracting protein misfolding and cell death in polyQ-expressing neuronal precursor cells129. Triptolide, also from the Celastreace family of plants, is a potent inhibitor of the transactivating capacity of HSF1 and has been shown to have beneficial effects in treatments of pancreatic cancer xenografts130,131. These examples of small-molecule regulators of HSF1 are promising candidates for drug discovery and development. However, the existence of multiple mammalian HSFs and their functional interplay should also be taken into consideration when planning future HSF-targeted therapies.

 

Concluding remarks and future perspectives

HSFs were originally identified as specific heat shock-inducible transcriptional regulators of HSP genes, but now there is unambiguous evidence for a wide variety of HSF target genes that extends beyond the molecular chaperones. The known functions governed by HSFs span from the heat shock response to development, metabolism, lifespan and disease, thereby integrating pathways that were earlier strictly divided into either cellular stress responses or normal physiology.

Although the extensive efforts from many laboratories focusing on HSF biology have provided a richness of understanding of the complex regulatory mechanisms of the HSF family of transcription factors, several key questions remain. For example, what are the initial molecular events (that is, what is the ‘thermometer’) leading to the multistep activation of HSFs? The chromatin-based interaction between HSFs and the basic transcription machinery needs further investigation before the exact interaction partners at the chromatin level can be established. The activation and attenuation mechanisms of HSFs require additional mechanistic insights, and the roles of the multiple signal transduction pathways involved in post-translational regulation of HSFs are only now being discovered and are clearly more complex than anticipated. Although still lacking sufficient evidence, the PTMs probably serve as rheostats to allow distinct forms of HSF-mediated regulation in different tissues during development. Further emphasis should therefore be placed on understanding the PTMs of HSFs during development, ageing and different protein folding diseases. Likewise, the subcellular distribution of HSF molecules, including the mechanism by which HSFs shuttle between the cytoplasm and the nucleus, remains enigmatic, as do the movements of HSF molecules in different nuclear compartments such as NSBs.

Most studies on the impact of HSFs in lifespan and disease have been conducted with model organisms such as D. melanogaster and C. elegans, which express a single HSF. The existence of multiple members of the HSF family in mammals warrants further investigation of their specific and overlapping functions, including their extended repertoire of target genes. The existence of multiple HSFs in higher eukaryotes with different expression patterns suggests that they may have functions that are triggered by distinct stimuli, leading to activation of specific target genes. The impact of the HSF family in the adaptation to diverse biological environments is still poorly understood, and future studies are likely to broaden the prevailing view of HSFs being solely stress-inducible factors. To this end, the crosstalk between distinct HSFs that has only recently been uncovered raises obvious questions about the stoichiometry between the components in different complexes residing in different cellular compartments, and the mechanisms by which the factors interact with each other. Interaction between distinct HSF family members could generate new opportunities in designing therapeutics for protein-folding diseases, metabolic disorders and cancer.

 

  1. Role in the etiology of cancer

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Dan Tang,1 Md Abdul Khaleque,2 Ellen L. Jones,1 Jimmy R. Theriault,2 Cheng Li,3 Wing Hung Wong,3 Mary Ann Stevenson,2 and Stuart K. Calderwood1,2,4
Cell Stress Chaperones. 2005 Mar; 10(1): 46–58. doi:  10.1379/CSC-44R.1

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.

Heat shock proteins (HSPs) were first discovered as a cohort of proteins that is induced en masse by heat shock and other chemical and physical stresses in a wide range of species (Lindquist and Craig 1988Georgopolis and Welch 1993). The HSPs (Table 1) have been subsequently characterized as molecular chaperones, proteins that have in common the property of modifying the structures and interactions of other proteins (Lindquist and Craig 1988Beckmann et al 1990;Gething and Sambrook 1992Georgopolis and Welch 1993Netzer and Hartl 1998). Molecular chaperone function dictates that the HSP often interact in a stoichiometric, one-on-one manner with their substrates, necessitating high intracellular concentrations of the proteins (Lindquist and Craig 1988Georgopolis and Welch 1993). As molecules that shift the balance from denatured, aggregated protein conformation toward ordered, functional conformation, HSPs are particularly in demand when the protein structure is disrupted by heat shock, oxidative stress, or other protein-damaging events (Lindquist and Craig 1988;Gething and Sambrook 1992Georgopolis and Welch 1993). The HSP27, HSP40,HSP70, and HSP110 genes have therefore evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation (Voellmy 1994). HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress (Hickey and Weber 1982Landry et al 1982Li and Werb 1982Subjeck et al 1982Henics et al 1999) (Zhao et al 2002). Heat shock factor (HSF) proteins have been shown to interact with the promoters of many HSP genes and ensure prompt transcriptional activation in stress and equally precipitous switch off after recovery (Sorger and Pelham 1988Wu 1995). The hsf gene family includes HSF1 (hsf1), the molecular coordinator of the heat shock response, as well as 2 less well-characterized genes, hsf2 and hsf4(Rabindran et al 1991Schuetz et al 1991) (Nakai et al 1997). In addition to the class of HSPs induced by heat, cells also contain a large number of constitutively expressed HSP homologs, which are also listed in Table 1. The constitutive HSPs are found in a variety of multiprotein complexes containing both HSPs and cofactors (Buchner 1999). These include HSP10-HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with regulatory proteins within the cell (Netzer and Hartl 1998). HSP90 plays a particularly versatile role in cell regulation, forming complexes with a large number of cellular kinases, transcription factors, and other molecules (Buchner 1999Grammatikakis et al 2002).

 

Table 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1074571/bin/i1466-1268-10-1-46-t01.jpg

 

Heat shock protein family genes studied by microchip array analysis

Many tumor types contain high concentrations of HSP of the HSP28, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca et al 1993Yano et al 1999Cornford et al 2000Strik et al 2000Ricaniadis et al 2001Ciocca and Vargas-Roig 2002). We have concentrated here on HSP gene expression in prostate carcinoma. The progression of prostatic epithelial cells to the fully malignant, metastatic phenotype is a complex process and involves the expression of oncogenes as well as escape from androgen-dependent growth and survival (Cornford et al 2000). There is a molecular link between HSP expression and tumor progression in prostate cancer in that HSP56, HSP70, and HSP90 regulate the function of the androgen receptor (AR) (Froesch et al 1998Grossmann et al 2001). Escape from AR dependence during tumorigenesis may involve altered HSP-AR interactions (Grossmann et al 2001). The role of HSPs in tumor development may also be related to their function in the development of tolerance to stress (Li and Hahn 1981). Thermotolerance is induced in cells preconditioned by mild stress coordinately with the expression of high HSP levels (Landry et al 1982Li and Werb 1982Subjeck et al 1982). Elevated HSP expression appears to be a factor in tumor pathogenesis, and, among other mechanisms, this may involve the ability of individual HSPs to block the pathways of apoptosis and permit malignant cells to arise despite the triggering of apoptotic signals during transformation (Volloch and Sherman 1999). De novo HSP expression may also afford protection of cancer cells from treatments such as chemotherapy and hyperthermia by thwarting the proapoptotic influence of these modalities (Gabai et al 1998Hansen et al 1999Blagosklonny 2001Asea et al 2001Van Molle et al 2002). The mechanisms underlying HSP induction in tumor cells are not known but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment, which would be expected to lead to cellular stress.

Here, we have examined expression of hsf and HSP genes in immortalized normal human prostate epithelial cells and a range of prostate carcinoma cells obtained from human tumors at the mRNA and protein levels. Our aim was to determine whether hsf-HSP expression profiles are conserved in cells that express varying degrees of malignancy, under resting conditions and after heat and ionizing radiation. In addition, we have compared HSP expression profiles of a metastatic human prostate carcinoma cell line growing either in monolayer culture or as a tumor xenograft in nude mice. These studies were prompted by findings in our laboratory that prostate carcinoma cells are considerably more sensitive to heat-induced apoptosis in vivo growing as tumors compared with similar cells growing in tissue culture in vitro. Our studies show that, although the hsf-HSP expression profiles are similar in normal and malignant prostate-derived cells at the mRNA level, expression at the protein level was very different. HSF1 and HSP protein expression was highest in the 3 aggressively metastatic prostate cancer cell lines (PC-3, DU-145, and CA-HPV-10). Although the gene expression patterns of constitutive HSP differ enormously in PC-3 cells in vitro and in xenografts in vivo, stress induction of HSP genes is not markedly altered by exposure to the tumor microenvironment, indicating the hierarchical rank of the stress response that permits it to override other forms of regulation. ……

The experiments described here are largely supportive of the notion that HSP gene expression and HSF activity and expression are increased in more advanced stages of cancer (Fig 4). The most striking finding in the study was the elevation of HSF1 and HSP levels in aggressively malignant prostate carcinoma cell lines (Fig 4). It is significant that these changes in HSF and HSP levels would not have been predicted from microarray studies of HSF (Fig 3) and HSP (Fig 1) mRNA levels. The increased HSF levels observed in the metastatic prostate carcinoma cell lines in particular appear to be due to altered regulation of either mRNA translation or protein turnover (or both) (Figs 3 and ​and4).4). Although we do not at this stage know the mechanisms involved, 1 candidate could be differential activity of the proteosome in the metastatic cell lines: both HSF1 and HSF2 are targets for proteosomal degradation (Mathew et al 1998). Despite these differences in HSP expression between cells of varying degrees of malignancy under growth conditions, stress caused a major shift in HSP gene expression and activation of HSP40-1, HSP70-1A, HSP70-1B, HSP70-6 (HSP70B), DNA-J2–like, and HSP105 in all cells (Fig 2). Even in LnCap cells with minimal HSF1 and HSF2 expression, heat-inducible HSP70 protein expression was observed (Fig 4). Interestingly, we observed minimal induction of the HSP70B gene in LnCap cells: because the HSP70B promoter is known to be almost exclusively induced by stress through the HSE in its promoter, the findings may suggest that a mechanism for HSP70 induction alternative to HSF1 activation may be operative in LnCap cells (Schiller et al 1988). Increased HSP expression in cancer patients has been shown to signal a poor response to treatment by a number of modalities, suggesting that HSP expression is involved with development of resistance to treatment in addition to being involved in the mechanisms of malignant progression (Ciocca et al 1993Cornford et al 2000Yamamoto et al 2001Ciocca and Vargas-Roig 2002;Mese et al 2002). In addition, subpopulations of LnCap-derived cells, selected for enhanced capacity to metastasize, have been shown to express elevated levels of HSF1, HSP70, and HSP27 compared with nonselected controls (Hoang et al 2000). This may be highly significant because our studies indicate minimal levels of HSF1 and HSP in the poorly metastatic parent LnCap cells (Figs 1 and ​and4).4). Previous studies have also indicated that elevated HSP70 expression occurs at an early stage in cellular immortalization from embryonic stem cells (Ravagnan et al 2001). We had to use immortalized prostatic epithelial cells for our normal controls and may have missed a very early change in HSP expression during the immortalization process.

As indicated by the kinetic studies (Figs 5–7), HSPs are activated at a number of regulatory levels by stress in addition to transcriptional activation, and these may include stress-induced mRNA stabilization, differential translation, and protein stabilization (Hickey and Weber 1982Zhao et al 2002). HSF1 activity and HSP expression appear to be subject to differential regulation by a number of pathways at normal temperatures but are largely independent of such regulation when exposed to heat shock, which overrides constitutive regulation and permits prompt induction of this emergency response.

Growth of PC-3 cells in vivo as tumor xenografts was accompanied by a marked decrease in constitutive HSP expression (Figs 8 and ​and11).11). Decreased HSP expression was part of a global switch in gene expression that accompanies the switch of PC-3 cells from growth as monolayers in tissue culture to growth as tumors in vivo (D. Tang and S.K. Calderwood, in preparation). Many reports indicate changes in a wide range of cellular properties as cells grow as tumors, and these properties may reflect the remodeling of gene expression patterns. These changes may reflect adaptation to the chemical nature of the tumor microenvironment and the alterations in cell-cell interaction in growth as a tumor in vivo. Our studies also indicate the remarkable sturdiness of the heat shock response that remains intact in the PC-3 cells growing in vivo despite the global rearrangements in other gene expressions mentioned above (Figs 10 and ​and1111).

The elevation in HSF1 and HSP levels in cancer shown in our studies and in those of others and its association with a poor prognosis and inferior response to therapy suggests the strategy of targeting HSP in cancer therapy. Treatment with HSP70 antisense oligonucleotides, for instance, can cause tumor cell apoptosis on its own and can synergize with heat shock in cell killing (Jones et al 2004). Indeed, it has been shown that antagonizing heat-inducible HSP expression with quercitin, a bioflavonoid drug that inhibits HSF1 activation, or by using antisense oligonucleotides directed against HSP70 mRNA further sensitizes PC-3 cells to heat-induced apoptosis in vitro and leads to tumor regression in vivo (Asea et al 2001Lepchammer et al 2002Jones et al 2004) (A. Asea et al, personal communication). The strategy of targeting HSP expression or function in cancer cells may thus be indicated. Such a strategy might prove particularly effective because constitutive HSP expression is reduced in tumors, and this might be related to increased killing of PC-3 tumor cells by heat (Fig 12).

 

  1. Molecular chaperones in aging

Aging and molecular chaperones

Csaba So˝ti*, Pe´ter Csermely
Exper Geront 2003; 38:1037–1040  http://195.111.72.71/docs/pcs/03exger.pdf

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Molecular chaperones Chaperones are ubiquitous, highly conserved proteins (Hartl, 1996), either assisting in the folding of newly synthesized or damaged proteins in an ATP-dependent active process or working in an ATP-independent passive mode sequestering damaged proteins for future refolding or digestion. Environmental stress leads to proteotoxic damage. Damaged, misfolded proteins bind to chaperones, and liberate the heat shock factor (HSF) from its chaperone complexes. HSF is activated and transcription of chaperone genes takes place (Morimoto, 2002). Most chaperones, therefore, are also called stress or (after the archetype of experimental stress) heat shock proteins (Hsp-s).

Aging proteins—proteins of aging organisms During the life-span of a stable protein, various posttranslational modifications occur including backbone and side chain oxidation, glycation, etc. In aging organisms, the disturbed cellular homeostasis leads to an increased rate of protein modification: in an 80-year old human, half of all proteins may become oxidized (Stadtman and Berlett, 1998). Susceptibility to various proteotoxic damages is mainly increased due to dysfunction of mitochondrial oxidation of starving yeast cells (Aguilaniu et al., 2001). In prokaryotes, translational errors result in folding defects and subsequent protein oxidation (Dukan et al., 2000), which predominantly takes place in growth arrested cells (Ballesteros et al., 2001). Additionally, damaged signalling networks loose their original stringency, and irregular protein phosphorylation occurs (e.g.: the Parkinson disease-related a-synuclein also becomes phosphorylated, leading to misfolding and aggregation; Neumann et al., 2002).

Aging protein degradation Irreversibly damaged proteins are recognized by chaperones, and targeted for degradation. Proteasome level and function decreases with aging, and some oxidized, aggregated proteins exert a direct inhibition on proteasome activity. Chaperones also aid in lysosomal degradation. The proteolytic changes are comprehensively reviewed by Szweda et al. (2002). Due to the degradation defects, damaged proteins accumulate in the cells of aged organisms, and by aggregation may cause a variety of protein folding diseases (reviewed by So˝ti and Csermely, 2002a).

Aging chaperones I: defects in chaperone induction Damaged proteins compete with the HSF in binding to the Hsp90-based cytosolic chaperone complex, which may contribute to the generally observed constitutively elevated chaperone levels in aged organisms (Zou et al., 1998; So˝ti and Csermely, 2002b). On the contrary, the majority of the reports showed that stress-induced synthesis of chaperones is impaired in aged animals. While HSF activation does not change, DNA binding activity may be reduced during aging (Heydari et al., 2000). A number of signaling events use an overlapping network of chaperones not only to establish the activation-competent state of different transcription factors (e.g. steroid receptors), but also as important elements in the attenuation of respective responses. HSF transcriptional activity is also negatively influenced by higher levels of chaperones (Morimoto, 2002). Differential changes of these proteins in various organisms and tissues may lead to different extents of (dys)regulation. More importantly, the cross-talk between different signalling pathways through a shared pool of chaperones may have severe consequences during aging when the cellular conformational homeostasis is deranged (see below).

Aging chaperones II: defects in chaperone function   Direct studies on chaperone function in aged organisms are largely restricted to a-crystallin having a decreased activity in aged human lenses (Cherian and Abraham, 1995; Cherian-Shaw et al., 1999). In a recent study, an initial test of passive chaperone function of whole cytosols was assessed showing a decreased chaperone capacity in aged rats compared to those of young counterparts (Nardai et al., 2002). What can be the mechanism behind these deleterious changes in chaperone function? Chaperones may also be prone to oxidative damage, as GroEL is preferentially oxidized in growth-arrested E. coli (Dukan and Nystro¨m, 1999). Macario and Conway de Macario (2002) raised the idea of ‘sick chaperones’ in aged organisms in a recent review. Indeed, chaperones are interacting with a plethora of other proteins (Csermely, 2001a), which requires rather extensive binding surfaces. These exposed areas may make chaperones a preferential target for proteotoxic damage: chaperones may behave as ‘suicide proteins’ during aging, sacrificing themselves instead of ‘normal’ proteins. The high abundance of chaperones (which may constitute more than 5% of cellular proteins), and their increased constitutive expression in aged organisms makes them a good candidate for this ‘altruistic courtesy.’ It may be especially true for mitochondrial Hsp60, the role of which would deserve extensive studies.

Aging chaperones III: defects in capacity, the chaperone overload Another possible reason of decreased chaperone function is chaperone overload (Csermely, 2001b). In aging organisms, the balance between misfolded proteins and available free chaperones is grossly disturbed: increased protein damage, protein degradation defects increase the amount of misfolded proteins, while chaperone damage, inadequate synthesis of molecular chaperones and irreparable folding defects (due to posttranslational changes) decrease the amount of available free chaperones. Chaperone overload occurs, where the need for chaperones may greatly exceed the available chaperone capacity (Fig. 1). Under these conditions, the competition for available chaperones becomes fierce and the abundance of damaged proteins may disrupt the folding assistance to other chaperone targets, such as: (1) newly synthesized proteins; (2) ‘constantly damaged’ (mutant) proteins; and (3) constituents of the cytoarchitecture (Csermely, 2001a). This may cause defects in signal transduction, protein transport, immune recognition, cellular organization as well as the appearance of previously buffered, hidden mutations in the phenotype of the cell (Csermely, 2001b). Chaperone overload may significantly decrease the robustness of cellular networks, as well as shift their function towards a more stochastic behavior. As a result of this, aging cells become more disorganized, their adaptation is impaired.

Fig. 1. Chaperone overload: a shift in the balance between misfolded proteins and available free chaperones in aging organisms. The accumulation of chaperone substrates along with an impaired chaperone function may exhaust the folding assistance to specific chaperone targets and leads to deterioration in vital processes. Chaperone overload may significantly decrease the robustness of cellular networks, and compromise the adaptative responses. See text for details.

Senescent cells and chaperones The involvement of chaperones in aging at the cellular level is recently reviewed (So˝ti et al., 2003). Non-dividingsenescent-peripheral cells tend to have increased chaperone levels (Verbeke et al., 2001), and cannot preserve the induction of several chaperones (Liu et al., 1989), similarly to cells from aged animals. Activation and binding of HSF to the heat shock element is decreased in aged cells (Choi et al., 1990). Interestingly, cellular senescence seems to unmask a proteasomal activity leading to the degradation of HSF (Bonelli et al., 2001). Chaperone induction per se seems to counteract senescence. Repeated mild heat shock (a kind of hormesis) has been reported to delay fibroblast aging (Verbeke et al., 2001), though it does not seem to extend replicative lifespan. A major chaperone, Hsp90 is required for the correct function of telomerase, an important enzyme to extend the life-span of cells (Holt et al., 1999). Mortalin (mtHsp70/Grp75), a member of the Hsp70 family, produces opposing phenotypic effects related to its localization. In normal cells, it is pancytoplasmically distributed, and its expression causes senescence. Its upregulation and perinuclear distribution, however, is connected to transformation, probably via p53 inactivation. Mortalin also induces life-span extension in human fibroblasts or in C. elegans harboring extra copies of the orthologous gene (Kaul et al., 2002).

Aging organisms and chaperones: age-related diseases Unbalanced chaperone requirement and chaperone capacity in aged organisms helps the accumulation of aggregated proteins, which often cause folding diseases, mostly of the nervous system, due to the very limited proliferation potential of neurons. Over expression of chaperones often delays the onset or diminishes the symptoms of the disease (So˝ti and Csermely, 2002b). Other aging diseases, such as atherosclerosis and cancer are also related to chaperone action. Here space limitation precludes a detailed description of these rapidly developing fields, however, numerous recent reviews were published on these subjects, where the interested readers may find a good summary and several hints for further readings (Ferreira and Carlos, 2002; Neckers, 2002; Sarto et al., 2000; Wick and Xu, 1999).

 

Chaperones and Longevity

Increased chaperone induction leads to increased longevity (Tatar et al., 1997). Moreover, a close correlation exists between stress resistance and longevity in several long-lived C. elegans and Drosophila mutants (Lithgow and Kirkwood, 1996). As the other side of the same coin, damaged HSF has been found as an important gene to cause accelerated aging in C. elegans (Garigan et al., 2002). Caloric restriction, the only effective experimental manipulation known to retard aging in rodents and primates (Ramsey et al., 2000), restores age-impaired chaperone induction, while reversing the age-induced changes in constitutive Hsp levels (see So˝ti and Csermely, 2002a,b). These examples confirm the hypothesis that a better adaptation capacity to various stresses greatly increases the chances to reach longevity. 10. Conclusions and perspectives Aging can be defined as a multicausal process leading to a gradual decay of self-defensive mechanisms, and an exponential accumulation of damage at the molecular, cellular and organismal level. The protein oxidation, damage, misfolding and aggregation together with the simultaneously impaired function and induction of chaperones in aged organisms disturb the balance between chaperone requirement and availability. There are several important aspects for future investigation of this field: † the measurement of active chaperone function (i.e. chaperone-assisted refolding of damaged proteins) in cellular extracts does not have a well-established method yet; † we have no methods to measure free chaperone levels; † among the consequences of chaperone overload, changes in signal transduction, protein transport, immune recognition and cellular organization have not been systematically measured and/or related to the protein folding homeostasis of aging organisms and cells.

 

  1. Extracellular HSPs in inflammation and immunity

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1

Amir Kol,* Andrew H. Lichtman,† Robert W. Finberg,‡ Peter Libby,*† and Evelyn A. Kurt-Jones2‡
J  Immunol 2000; 164: 13–17.  https://www.researchgate.net/profile/Robert_Finberg/publication/12696457_Cutting_Edge_Heat_Shock_Protein_(HSP)_60_Activates_the_Innate_Immune_Response_CD14_Is_an_Essential_Receptor_for_HSP60_Activation_of_Mononuclear_Cells/links/53ee00460cf23733e80b21c0.pdf

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

There is increasing interest in the role of nontraditional mediators of inflammation in atherosclerosis (1). Recent studies from our laboratory have shown that chlamydial and human heat shock protein 60 (HSP60)3 colocalize in human atheroma (2), and either HSP60 induces adhesion molecule and cytokine production by human vascular cells and macrophages, in a pattern similar to that induced by Escherichia coli LPS (3, 4). These results suggested that HSP60 and LPS might share similar signaling mechanisms. CD14 is the major high-affinity receptor for bacterial LPS on the cell membrane of mononuclear cells and macrophages (5, 6). In addition to LPS, CD14 functions as a signaling receptor for other microbial products, including peptidoglycan from Gram-positive bacteria and mycobacterial lipoarabinomann (7, 8). CD14 is considered a pattern recognition receptor for microbial Ags and, with Toll-like receptor (TLR) proteins, an important mediator of innate immune responses to infection (9–14). We have examined the role of CD14 in the response of human monocytes and macrophages to HSP60.  …..

HSP may play a central role in the innate immune response to microbial infections. Because both microbes and stressed or injured host cells produce abundant HSP (36), and dying cells likely release these proteins, it is conceivable that HSP furnish signals that inform the innate immune system of the presence of infection and cell damage. The findings reported here, that human HSP60 induces IL-6 production by mononuclear cells and macrophages via the CD14, supports this hypothesis, suggesting that human HSP60 may act together with LPS or other microbial products to provoke innate immune responses.

Inflammation and immunity can contribute to the pathogenesis and complications of atherosclerosis (37). Moreover, the search for novel risk factors for atherosclerosis has revived the concept that microbial products might substantially contribute to the inflammatory reaction in the atheromatous vessel wall (38, 39). We have shown that chlamydial HSP60 colocalizes with human HSP60 in the macrophages of human atheroma (2). Therefore, bacterial and human HSP60, released from dying or injured cells during atherogenesis (40) or myocardial injury (41), may further promote local inflammation and possibly activate the innate immune system. Previous reports that immunization with mycobacterial HSP65 enhances atheroma formation in rabbits (42), have suggested an important role for HSPs in atherogenesis, particularly because the high degree of homology between HSPs of the same m.w. among different species might stimulate autoimmunity (43).

In conclusion, our findings, that CD14 mediates cellular activation induced by human HSP60 provide further insight into the molecular mechanisms by which HSP may activate the innate immune system and participate in atherogenesis and other inflammatory disorders.

DAMPs, PAMPs and alarmins: all we need to know about danger

Marco E. Bianchi1
J. Leukoc. Biol. 81: 1–5; 2007.   http://aerozon.ru/documents/publications/37_Bianche.pdf

Multicellular animals detect pathogens via a set of receptors that recognize pathogen associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term “alarmin” is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage associated molecular patterns (DAMPs).

Multicellular animals must distinguish whether their cells are alive or dead and detect when microorganisms intrude, and have evolved surveillance/defense/repair mechanisms to this end. How these mechanisms are activated and orchestrated is still incompletely understood, and I will argue that that these themes define a unitary field of investigation, of both basic and medical interest.

A complete system for the detection, containment, and repair of damage caused to cells in the organism requires warning signals, cells to respond to them via receptors and signaling pathways, and outputs in the form of physiological responses. Classically, a subset of this system has been recognized and studied in a coherent form: pathogen-associated molecular patterns (PAMPs) are a diverse set of microbial molecules which share a number of different recognizable biochemical features (entire molecules or, more often, part of molecules or polymeric assemblages) that alert the organism to intruding pathogens [1]. Such exogenous PAMPs are recognized by cells of the innate and acquired immunity system, primarily through toll-like receptors (TLRs), which activate several signaling pathways, among which NF-kB is the most distinctive. As a result, some cells are activated to destroy the pathogen and/or pathogen-infected cells, and an immunological response is triggered in order to produce and select specific T cell receptors and antibodies that are best suited to recognize the pathogen on a future occasion. Most of the responses triggered by PAMPs fall into the general categories of inflammation and immunity.

However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Tissues can be ripped, squashed, or wounded by mechanical forces, like falling rocks or simply the impact of one’s own body hitting the ground. Animals can be wounded by predators. In addition, tissues can be damaged by excessive heat (burns), cold, chemical insults (strong acids or bases, or a number of different cytotoxic poisons), radiation, or the withdrawal of oxygen and/or nutrients. Finally, humans can also be damaged by specially designed drugs, such as chemotherapeutics, that are meant to kill their tumor cells with preference over their healthy cells. Very likely, we would not be here to discuss these issues if evolution had not incorporated in our genetic program ways to deal with these damages, which are not caused by pathogens but are nonetheless real and common enough. Tellingly, inflammation is also activated by these types of insults. A frequently quoted reason for the similarity of the responses evoked by pathogens and trauma is that pathogens can easily breach wounds, and infection often follows trauma; thus, it is generally effective to respond to trauma as if pathogens were present. In my opinion, an additional reason is that pathogens and trauma both cause tissue and cell damage and thus trigger similar responses.

None of these considerations is new; however, a new awareness of the close relationship between trauma- and pathogenevoked responses emerged from the EMBO Workshop on Innate Danger Signals and HMGB1, which was held in February 2006 in Milano (Italy); many of the findings presented at the meeting are published in this issue of the Journal of Leukocyte Biology. At the end of the meeting, Joost Oppenheim proposed the term “alarmin” to differentiate the endogenous molecules that signal tissue and cell damage. Together, alarmins and PAMPs therefore constitute the larger family of damage-associated molecular patterns, or DAMPs.

Extranuclear expression of HMGB1 has been involved in a number of pathogenic conditions: sepsis [44], arthritis [45, 46], atherosclerosis [10], systemic lupus erythematosus (SLE) [47], cancer [48] and hepatitis [49, this issue]. Uric acid has been known to be the aethiologic agent for gout since the 19th century. S100s may be involved in arthritis [31, this issue] and psoriasis [50]. However, although it is clear that excessive alarmin expression might lead to acute and chronic diseases, the molecular mechanisms underlying these effects are still largely unexplored.

The short list of alarmins presented above is certainly both provisional and incomplete and serves only as an introduction to the alarmin concept and to the papers published in this issue of JLB. Other molecules may be added to the list, including cathelicidins, defensins and eosinophil-derived neurotoxin (EDN) [51], galectins [52], thymosins [53], nucleolin [54], and annexins [55; and 56, this issue]; more will emerge with time. Eventually, the concept will have to be revised and adjusted to the growing information. Indeed, I have previously argued that any misplaced protein in the cell can signal damage [57], and Polly Matzinger has proposed that any hydrophobic surface (“Hyppo”, or Hydrophobic protein part) might act as a DAMP [58]. As most concepts in biology, the alarmin category serves for our understanding and does not correspond to a blueprint or a plan in the construction of organisms. Biology proceeds via evolution, and evolution is a tinkerer or bricoleur, finding new functions for old molecules. In this, the reuse of cellular components as signals for alerting cells to respond to damage and danger, is a prime example.

 

  1. Role of heat shock and the heat shock response in immunity and cancer

 

Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

Stuart K. Calderwood,1,* Ayesha Murshid,1 and Jianlin Gong1
Front Immunol. 2012; 3: 75.  doi:  10.3389/fimmu.2012.00075

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock protein, vaccine, inflammation, antigen presentation

Heat shock proteins (HSP) were first discovered as a group of polypeptides whose level of expression increases to dominate the cellular proteome after stress (Lindquist and Craig, 1988). These increases in HSPs synthesis correlate with a marked resistance to potentially toxic stresses such as heat shock (Li and Werb,1982). The finding that such proteins have extracellular immune functions suggested that, as highly abundant intracellular proteins they could be prime candidates as danger signals to the immune response (Srivastava and Amato,2001). There are several human HSP gene families with known immune significance and their classification is reviewed in Kampinga et al. (2009). These include the HSPA (Hsp70) family, which includes the HPA1A and HSPA1B genes encoding the two major stress-inducible Hsp70s, that together are often referred to as Hsp72. When referring to Hsp70 in this chapter, we generally refer to the products of these two genes. The Hsp70 family also includes two other members with immune function – HSPA8 and HSPA5 genes, whose protein products are known as Hsc70 the major constitutive Hsp70 family member and Grp78, a key ER-resident protein. In addition two more Hsp70 related genes have immune significance and these include HSPH2 (Hsp110) and HSPH4 the ER-resident class H protein Grp170. The Hsp90 family also has major functions in tumor immunity and these include HSPC2 and HSPC3, which encode the major cytoplasmic proteins Hsp90a and Hsp90b, and HSPC4 that encodes ER chaperone Grp94. In addition, the product of the HSPD1 gene, the mitochondrial chaperone Hsp60 has some immunological functions. Mice have been shown to encode orthologs of each of these genes (Kampinga et al., 2009).

It has been suggested that many of the HSPs have the property of damage associated molecular patterns (DAMPs), inducers of sterile inflammation and innate immunity (Kono and Rock, 2008). The additional discovery that intracellular HSPs function as molecular chaperones and can bind to a wide spectrum of intracellular polypeptides further indicated that they could play a broad role in the immune response and might mediate both innate immunity due to their status as DAMPs and adaptive immunity by chaperoning antigens.

Heat shock proteins are currently employed as vaccines in cancer immunotherapy (Tamura et al., 1997; Murshid et al., 2011a). The rationale behind the approach is that if HSPs can be extracted from tumor tissue bound to the polypeptides which they chaperone during normal metabolism, they may retain antigenic peptides specific to the tumor (Noessner et al., 2002; Srivastava, 2002; Wang et al., 2003; Enomoto et al., 2006; Gong et al., 2010). Indeed, vaccines based on Hsp70, Hsp90, Grp94, Hsp110, and Grp170 polypeptide complexes have been used successfully to immunize mice to a range of tumor types and Hsp70 and Grp94 vaccines have undergone recent clinical trials (rev: Murshid et al., 2011a). These effects of the HSP vaccines on tumor immunity appear to be mediated largely to the associated, co-isolated tumor polypeptides, although in the case of Grp94 this question is still controversial and tumor regression was observed in mice treated with the chaperone devoid of its peptide binding domain (Udono and Srivastava, 1993; Srivastava, 2002; Nicchitta, 2003; Chandawarkar et al., 2004; Nicchitta et al.,2004). Use of such HSP vaccines is potentially a powerful approach to tumor immunotherapy as the majority of the antigenic repertoire of most individual tumor cells is unknown (Srivastava and Old, 1988; Srivastava, 1996). Individual cancer cells are likely to take a lone path in accumulating a spectrum of random mutations. Although some mutations are functional, permitting cells to become transformed and to progress into a highly malignant state, many such changes are likely to be passenger mutations not required to drive tumor growth (Srivastava and Old, 1988; Srivastava, 1996). Some of these individual mutant sequences will be novel antigenic epitopes and together with the few known shared tumor antigens comprise an “antigenic fingerprint” for each individual tumor (Srivastava,1996). Accumulation of mutations in cancer appears to be related to, and may drive the increases in HSPs observed in many tumors (Kamal et al., 2003; Whitesell and Lindquist, 2005; Trepel et al., 2010). As the mutant conformations of tumor proteins are “locked in” due to the covalent nature of the alterations, cancer cells appear to be under permanent proteotoxic stress and rich in HSP expression (Ciocca and Calderwood, 2005). For tumor immunology these conditions may offer a therapeutic opportunity as individual HSPs, whose expression is expanded in cancer will chaperone a cross-section of the “antigenic fingerprint” of the individual tumors (Murshid et al., 2011a). This approach was first utilized by Srivastava (20002006) and led to the development of immunotherapy using HSP–peptide complexes.

In addition to using HSP–peptide complexes extracted from tumors, in cases where tumor antigens are known, these can be directly loaded onto purified or recombinant HSPs and the complex used as a vaccine. This procedure has been carried out successfully in the case of the “large HSPs,” Hsp110 and Grp170 (Manjili et al., 20022003). A variant of this approach employs the molecular engineering of tumor antigens in order to produce molecular chaperone-fusion genes which encode products in which the HSP is fused covalently to the antigen. The fusion proteins are then employed as vaccines. This approach was pioneered by Young et al. who showed that a fusion between mycobacterial Hsp70 and ovalbumin could induced cytotoxic lymphocytes (CTL) in mice with the capacity to kill Ova-expressing cancer cells (Suzue et al., 1997). The vaccines could be used effectively without adjuvant and adjuvant properties were ascribed to the molecular chaperone component of the fusion protein. Subsequent studies have confirmed the utility of the approach in targeting common tumor antigens such as the melanoma antigen Mage3 (Wang et al., 2009).

HSPs and Immunosurveillance in Cancer

The question next arises as to the role of endogenous HSPs, with or without bound antigens in immunosurveillance of cancer cells. Although the immune system can recognize tumor antigens and generate a CTL response, most cancers evade immune cell killing by a range of strategies (van der Bruggen et al., 1991; Pardoll,2003). These include the down-regulation of surface MHC class I molecules by individual tumor cells and release of immunosuppressive IL-10 by tumors (Moller and Hammerling, 1992; Chouaib et al., 2002). Tumors in vivo also appear to attract a range of hematopoietic cells with immunosuppressive action including regulatory CD4+CD25+FoxP3+ T cells (Treg), M2 macrophages, myeloid-derived suppressor cells (MDSC) and some classes of natural killer cells (Pekarek et al.,1995; Terabe et al., 2005; Mantovani et al., 2008; Marigo et al., 2008). The tumor milieu also contain a small fraction of cells of mesenchymal origin identified by surface fibroblast activation protein-a (FAP cells) that suppress antitumor immune responses (Kraman et al., 2010). Endogenous tumor HSPs may also participate in immune suppression. Although the majority of the HSPs function as intracellular molecular chaperones, a fraction of these proteins can be released from cells even under unstressed conditions and may participate in immune functions (rev: Murshid and Calderwood, 2012). Intracellular Hsp70 can be actively secreted from tumor cells in either free form or packaged into lipid-bounded structures called exosomes (Mambula and Calderwood, 2006b; Chalmin et al., 2010). In addition Hsp70 and Hsp90 can also be found associated with the surfaces of tumor cells where they can function as molecular chaperones or as recognition structures for immune cells (Sidera et al., 2008; Qin et al., 2010; Multhoff and Hightower, 2011). As Hsp70 was shown in a number of earlier studies to be pro-inflammatory due to its interaction with pattern recognition receptors such as Toll-like receptors 2 and 4 (TLR2 and TLR4), these findings might suggest, as mentioned above, that Hsp70 released by tumors could be pro-inflammatory and possess the properties of DAMPs (Asea et al., 20002002; Vabulas et al., 2002). However, subsequent studies indicated that a portion of the TLR4 activation detected in the earlier reports, involving exposure of monocytes, macrophages, or dendritic cells (DC) to HSPs in vitro may be due to trace contamination with bacterial pathogen associated molecular patterns (PAMPs), potent TLR activators (Tsan and Gao,2004). In spite of these drawbacks, an overwhelming amount of evidence now seems to indicate the interaction of Hsp70 and other HSPs with TLRs (particularly TLR4) in vivo – in a wide range of physiological and pathological conditions, leading to acute inflammation in many conditions (Chase et al., 2007; Wheeler et al., 2009; see Appendix for a full list of references). Thus both TLR2 and TLR4 appear to be important components of inflammatory responses to Hsp70 under many pathophysiological conditions. In cancer therapy it has been shown that autoimmunity can be triggered in mice through necrotic killing of melanocytes engineered to overexpress Hsp70; such treatment led to the concomitant immune destruction of B16 melanoma tumors that share patterns of antigen expression with the killed melanocytes (Sanchez-Perez et al., 2006). Hsp70 appears to play an adjuvant role in this form of therapy through its interaction with TLR4 and induction of the cytokine TNF-a (Sanchez-Perez et al., 2006). However, despite these findings it has also been shown that depletion of Hsp70 in cancer cells can, in the absence of other treatments lead to tumor regression by inducing antitumor immunity (Rerole et al., 2011). This effect appears to be due to the secretion by cancer cells of immunosuppressive exosomes containing Hsp70 that activate MDSC and lead to local immunosuppression (Chalmin et al., 2010). Under normal circumstances therefore, release of endogenous Hsp70 into the extracellular microenvironment may be a component of the tumor defenses against immunosurveillance. Extracellular Hsp60 has also been shown be immunomodulatory and can increase levels of FoxP3 Treg in vitro and suppress T cell-mediated immunity (de Kleer et al., 2010; Aalberse et al., 2011).

The pro-inflammatory properties of extracellular HSPs may be more evident underin vivo situations particularly in the context of tissue damage (Sanchez-Perez et al.,2006). For instance when elevated temperatures were used to boost Hsp70 release from Lewis Lung carcinoma cells in vivo, antitumor immunity was activated along with release of chemokines CCL2, CCL5, and CCL10, in a TLR4-dependent manner, leading to attraction of DC and T cells into the tumor (Chen et al., 2009). Thus under resting conditions, the tumor milieu appears to be a specialized immunosuppressive environment, rich in inhibitory cells such as Treg, MDSC, and M2 macrophages and inaccessible to “exhausted” CD8+ T cells that often fail to penetrate the tumor microcirculation. However, under inflammatory conditions involving necrotic cell killing of tumor cells, extracellular HSPs may be able to amplify the anticancer immune response, intracellular HSPs may be released to further increase such a response and CTL may triggered to penetrate the tumor milieu, inducing antigen-specific cancer cell killing (Evans et al., 2001; Mambula and Calderwood, 2006a; Sanchez-Perez et al., 2006; Chen et al., 2009).

 

HSP-Based Anticancer Vaccines

It is apparent that a number of HSP types, conjugated to peptide complexes (HSP.PC) from cancer cells form effective bases for immunotherapy approaches with unique properties, as mentioned above (Calderwood et al., 2008; Murshid et al., 2011a). The immunogenicity of most HSP.PC appears to involve the ability of the HSPs to sample the tumor “antigenic fingerprint,” deliver the antigens to antigen presenting cells (APC) such as DC and stimulate activation of CTL (Tamura et al., 1997; Singh-Jasuja et al., 2000b; Wang et al., 2003; Murshid et al.,2010). A number of studies show that HSPs can chaperone tumor antigens and deliver them to the appropriate destination – MHC class I molecules on the DC surface (Singh-Jasuja et al., 2000a,b; Srivastava and Amato, 2001; Delneste et al.,2002; Enomoto et al., 2006; Gong et al., 2009). In addition, Hsp70 has been shown to chaperone viral antigenic peptides and increase cross priming of human CTL under ex vivo conditions (Tischer et al., 2011). However, it is still far from clear how the process of HSP-mediated cross priming unfolds. For instance, the CD8+ expressing DC subpopulation in lymph nodes is regarded as the primary cross-presenting APC (Heath and Carbone, 2009). It is not however currently known whether the CD8+ DC subset or other peripheral or lymph-node resident, DC interact with HSP vaccines to induce cross presentation. HSPs appear to be able to enter APC, such as mouse bone marrow derived DC (BMDC) and human DC in a receptor-mediated manner (Basu et al., 2001; Delneste et al., 2002; Gong et al.,2009; Murshid et al., 2010). However, no unique endocytosing HSP receptor has emerged and HSP–antigen complexes appear instead to be taken up by proteins with “scavenger” function such as LOX-1, SRECI, and CD91 that can each take up a wide range of extracellular ligands (Basu et al., 2001; Delneste et al., 2002; Theriault et al., 2006; Murshid et al., 2010). A pathway for Hsp90–peptide (Hsp90.PC) uptake has been characterized in mouse BMDC by scavenger receptor SRECI (Murshid et al., 2010). SRECI is able to mediate the whole process of Hsp90.PC endocytosis, trafficking through the cytoplasm to the sites of antigen processing and presentation of antigens to CD8+ T lymphocytes on MHC class I molecules (Murshid et al., 2010). This process is known as antigen cross presentation (Kurts et al., 2010). It is not currently clear what the relative contribution to antigen cross presentation of the various HSP receptors might be under in vivo conditions. It may be that each receptor class contributes to an individual aspect of CTL activation by HSP peptide complexes although a definitive understanding may await studies in mice deficient in each receptor class.

 

HSPs and CTL Programming

It is evident that that HSPs can mediate antigen cross presentation and activate CD8+ T lymphocytes. However, presentation of tumor antigens by DC is not sufficient for CTL programming and, in the absence of co-stimulatory molecules and innate immunity, the “helpless” CD8+ cells will cease to proliferate abundantly and will most likely undergo apoptosis (Schurich et al., 2009; Kurts et al., 2010). One mechanism for enhancing CTL programming involves activation of the TLR pathways that lead to synthesis of co-stimulatory molecules (Rudd et al.,2009; Yamamoto and Takeda, 2010). The co-stimulatory molecules, including CD80 and CD86 then become expressed on the DC cell surface and amplify the signals induced by binding of the T cell receptor on CD8+ T cells to MHC class I peptide complexes on the presenting DC (Parra et al., 1995; Rudd et al., 2009). This process is important in pathogen infection in which microbially derived antigens are encountered in the presence of inflammatory PAMPs that can activate innate immune transcriptional networks. Originally it had been thought that HSPs could provide analogous stimulation through their suspected activity as DAMPs and their inbuilt ability to trigger innate immunity through TLR2 and TLR4 on DC (Asea et al., 20002002; Vabulas et al., 2002). (The potential role of HSPs as DAMPs has been the subject of a recent review: van Eden et al., 2012). Subsequent studies on the capacity of HSPs to bind TLRs do not indicate avid binding of Hsp70 to either TLR2 or TLR4 when expressed in cells deficient in HSP receptors in vitro (Theriault et al., 2006). In vivo however, TLR signaling is essential for Hsp70 vaccine-induced tumor cell killing. Studies of tumor-bearing mice treated with an Hsp70 vaccine in vivo indicated that vaccine function is depleted by knockout of the TLR signaling intermediate Myd88 and completely abrogated by double knockout of TLR2 and TLR4 (Gong et al., 2009). These findings were somewhat complicated by the fact that TLR4 is involved in upstream regulation of the expression of Hsp70 receptor SRECI, but do strongly implicate a role for these receptors in amplifying immune signaling by Hsp70 vaccines and Hsp70-based immunotherapy (Sanchez-Perez et al., 2006; Gong et al., 2009). It is still not clear to what degree HSPs are capable of providing a sturdy DC maturing signal through TLR2/TLR4. The potency of HSP anticancer vaccines could potentially be improved by addition of PAMPs such as CpG DNA shown to activate TLR9, or double stranded RNA that can activate TLR3 (Murshid et al., 2011a). As mentioned, one contradictory factor in the earlier studies was that, although TLR2 and TLR4 are required for a sturdy Hsp70 vaccine-mediated immune response, direct binding of Hsp70 to these receptors was not observed (Theriault et al., 2006; Gong et al., 2009; Murshid et al., 2012). A rationale for these findings might be that HSPs can activate TLR signaling indirectly through primary binding to established HSP receptors such as LOX-1 and SRECI which secondarily recruit and activate the TLRs (Murshid et al., 2011b). Both of these scavenger receptors bind to TLR2 upon stimulation and activate TLR2-based signaling (Jeannin et al., 2005; A. Murshid and SK Calderwood, in preparation). In addition, we have found that Hsp90–SRECI complexes move to the lipid raft compartment of the cell, an environment highly enriched in TLR2 and TLR4 (Triantafilou et al., 2002; Murshid et al., 2010).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342006/bin/fimmu-03-00075-g001.jpg

Heat shock protein–peptide complexes extracted from tumor cells interact with endocytosing receptors (HSP-R) such as SRECI or signaling receptors (TLR) such as TLR4 on DC. SREC1 mediates uptake and intracellular processing of antigens and the presentation of resulting peptides on surface MHC class I and MHC class II proteins. MHC class II receptor–peptide complexes then bind to T cell receptors on CD4+ cells. One consequence of binding is interaction of CD40 ligand on the MHC class II cell with CD40 on the DC leading to the licensing interaction that results in enhanced expression of co-stimulatory proteins on the DC cell surface. The licensed DC may then interact with CD8+ T cells through T cell interaction with MHC class I peptide complexes. This effect will be enhanced by simultaneous interaction of CD80 or Cd86 co-stimulatory complexes on the DC with CD28 on the CD8+ cells, leading to effective CD8+ CTL that can lyse tumor cells. T cell programming can also be amplified by signals emanating from activated TLR that can boost levels of CD80 and CD86 as well as inflammatory cytokines (not shown).

 

Hsp70, Cell Damage, and Inflammation

The question of whether Hsp70 acts as DAMP and could by itself induce an inflammatory response in cancer patients in vivo is still open. However, some recent studies by Vile et al. using a gene therapy approach may shed some light on the inflammatory role of Hsp70 in tumor therapy. In this approach, as mentioned above, normal murine tissues were engineered to express high Hsp70 levels then subjected to treatments that lead to necrotic killing. The aim was to stimulate an autoimmune response that could lead to bystander immune killing of tumor cells that share the antigenic repertoire as the killed normal cells (Sanchez-Perez et al.,2006). In the initial studies, normal melanocytes were preloaded with Hsp70 plasmids and then necrotic cell death was triggered (Daniels et al., 2004). This treatment led to T cell-mediated immune killing of syngeneic B16 melanoma cells transplanted at a distant site in the mouse, presumably in response to antigens shared by the killed normal melanocytes and melanoma cell (Daniels et al., 2004). This effect only occurred when melanocytes were induced to undergo necrosis and Hsp70 levels were elevated, indicating a role for high levels of Hsp70 in the tumor specific immune response. Interestingly, these conditions did not lead to a prolonged autoimmune response, an effect mediated by the induction of a delayed Treg response (Srivastava, 2003; Daniels et al., 2004). It is notable that some early studies of chaperone-based tumor vaccines in animal models demonstrated a primary CTL response to tumors in response to treatment followed by delayed activation of a Treg reaction, and that chaperone levels must be carefully titrated for effective induction of tumor immunity (Udono and Srivastava, 1993; Liu et al.,2009). The role of Hsp70 in autoimmune rejection of tumors was also investigated in prostate cancer (Kottke et al., 2007). Ablation of normal prostate cells by necrotic killing with fusogenic viruses in the absence of Hsp70 elevation led to the induction of the cytokines IL-10 and TGF-b in the mouse prostate and a Treg response. However, when Hsp70 levels were elevated in these cells, IL-10, TGF-b, and IL-6 were induced simultaneously, the IL-6 component leading to further induction of IL-17, a profound Th17 response and tumor rejection (Kottke et al.,2007). Thus elevated levels of Hsp70, presumably released from cells undergoing necrosis can influence the local cytokine patterns and lead to an inflammatory statein vivo. Interestingly, these results seem to be tissue specific as inflammatory killing of pancreatic cells even in the presence of elevated Hsp70 did not provoke IL-6 release, a Th17 response or tumor rejection and the Treg response dominated under these conditions (Kottke et al., 2009). Thus the role of Hsp70 in tissue inflammation and tumor rejection seems to require elevated concentrations of extracellular chaperones, significant levels of necrotic cell killing, and tissue specific cytokine release.

Conclusion

  • Earlier studies investigating HSP vaccines considered such structures to be the “Swiss penknives” of immunology able to deliver antigens directly to APC and confer a maturing signal that could render DC able to effectively program CTL (Srivastava and Amato, 2001; Noessner et al., 2002). It is well established now that Hsp70, Hsp90, Hsp110, and GRP170 can chaperone tumor antigens and activate antigen cross presentation (Murshid et al., 2011a). In addition, HSPs were thought to be DAMPs with ability to strongly activate TLR signaling and innate immunity (Asea et al., 2000). However, although there is compelling evidence to indicate that Hsp70, for instance can interact with TLR4 under a number of pathological situations (see Appendix, Sanchez-Perez et al., 2006), it remains unclear whether free Hsp70 binds directly to the Toll-like receptor and induces innate immunity in the absence of other treatments in vitro(Tsan and Gao, 2004).
  • Elevated levels of extracellular HSPs appear to have the capacity to amplify the effects of inflammatory signals emanating from necrotic cells in vivoin a TLR4-dependent manner (Daniels et al., 2004; Sanchez-Perez et al., 2006; Kottke et al., 2007). In the presence of cell injury and death, elevated levels of Hsp70 appear to increase the production of inflammatory signals that involve cytokines such as IL-6 and IL-17 and lead to a specific T cell-mediated immune response to tumor cells sharing antigens with the dying cells (Kottke et al., 2007). The mechanisms involved in these processes are not clear although one possibility is that HSPs can induce the engulfment of necrotic cells. Hsp70 has been shown to increase bystander engulfment of a variety of structures (Wang et al., 2006a,b). In addition, tumor cells treated with elevated temperatures release inflammatory chemokines in an Hsp70 and TLR4-dependent mechanisms and this effect may be significant in CTL programming and tumor cell killing (Chen et al., 2009). Our studies indicate that CTL induction by Hsp70 vaccines in vivo has an absolute requirement for TLR2 and TLR4 suggesting that at least in vivo HSPs can trigger innate immunity through TLR signaling (Gong et al., 2009).
  • HSPs appear also to be able to direct antigen presentation through the class II pathway in DC and may stimulate T helper cells (Gong et al., 2009). It may thus be possible that HSPs participate in DC licensing and reinforce CTL programming during exposure to HSP vaccines. Future studies will address these questions.
  • A further interesting consideration is whether HSPs released from untreated tumor cells enhance or depress tumor immunity. One initial study shows that Hsp70 released from tumor cells in exosomes can strongly decrease tumor immunity through effects on MDSC (Chalmin et al., 2010). Further studies will be required to make a definitive statement on these questions.

 

  1. Protein aggregation disorders and HSP expression

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Christopher J. Cummings1,5, Michael A. Mancini3, Barbara Antalffy4, Donald B. DeFranco7, Harry T. Orr8 & Huda Y. Zoghbi1,2,6
Nature Genetics 19, 148 – 154 (1998) http://dx.doi.org:/10.1038/502

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease

Andreas Wyttenbach, Jenny Carmichael, Jina Swartz, Robert A. Furlong, Yolanda Narain, Julia Rankin, and David C. Rubinsztein*
https://www.researchgate.net/profile/David_Rubinsztein/publication/24447892_Effects_of_heat_shock_heat_shock_protein_40_(HDJ2)_and_proteasome_inhibition_on_protein_aggregation_in_cellular_models_of_Huntington’s_disease/links/00b7d528b80aab69bb000000.pdf

Huntington’s disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGypolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2yHSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2yHSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2yHSDJ in protein folding.

 

  1. Hsp70 in blood cell differentiation.

 

Apoptosis Versus Cell Differentiation -Role of Heat Shock Proteins HSP90, HSP70 and HSP27

David Lanneau, Aurelie de Thonel, Sebastien Maurel, Celine Didelot, and Carmen Garrido
Prion. 2007 Jan-Mar; 1(1): 53–60.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs

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Introduction

Stress or heat shock proteins (HSPs) were first discovered in 19621 as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.2 Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.3 Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α’s functions.4 HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.7 HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.8 This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.9

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.10 Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.11 One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.12Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases15 (Fig. 1).

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/figure/F1/

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and 

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.16 It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.

 

HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.2,1921 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.2224 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).

 

HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.25

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.26 A direct interaction between Akt and HSP90 was reported later.27 Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.26 Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.30 When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.27 Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.26 Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.31 HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.32 Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.33 The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p5334 and the transcription factors Her2 and Hif1α.35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.37

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,23 suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.38

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.

 

HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.39Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.40 In cancer cells, depletion of HSP70 results in spontaneous apoptosis.41

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation44 in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.45

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.46

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.47However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.48

HSP70 also prevents cell death in conditions in which caspase activation does not occur.49 Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.52 AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.53 HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation54 but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.55 During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.56

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).57 Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-150 and AIF binding,51 it is dispensable for JNK60 or GATA-161binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.61

 

HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.6466 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.65 In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.67

HSP27 can also interfere with caspase activation upstream of the mitochondria.66This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin68preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release66 (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,69 to decrease reactive oxygen species cell content,19 and to neutralize the toxic effects of oxidized proteins.70 These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.71

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.72 HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.73 In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).74 Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival75 while that of p27kip1facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.76

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.77 Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.78 In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary73,79 and it is its phosphorylated form that protects against neurotoxicity.80

 

HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.81 In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.82 In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.83

In leukemic cells HSP27 has been described as a pre-differentiation marker84because its induction occurs early during differentiation.8588 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes89 and for C2C12 muscle cells.90 This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G0/G1).19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells’ formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.91 HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.61 HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.18

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate92 and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.93 This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,94 RIP32 or Rb.95 Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.96 In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.97 In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.98 In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.4

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.99 In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G1 block. These G1 arrested cells do not differentiate but instead die by apoptosis.100

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.101Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.102 In breast cancer cells, 17-AAG-induced G1 block is accompanied by differentiation followed by apoptosis.103 The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.104

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.105 Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.16,17Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.61 Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.106It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.18 Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.

 

Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells’ aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells’ tumorigenicty and their depletion can induce a spontaneous regression of the tumors.24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.108 However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.

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HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death inC. elegans

A new pathway for non-apoptotic cell death

The results presented here allow us to construct a model for the initiation and execution of LCD in C. elegans (Figure 7). The logic of the LCD pathway may be similar to that of developmental apoptotic pathways. In C. elegans and Drosophila, where the control of specific cell deaths has been primarily examined, cell lineage or fate determinants control the expression of specific transcription factors that then impinge on proteins regulating caspase activation (Fuchs and Steller, 2011). Likewise, LCD is initiated by redundant determinants that require a transcription factor to activate protein degradation genes.

Figure 7.

https://elife-publishing-cdn.s3.amazonaws.com/12821/elife-12821-fig7-v3-480w.jpg

Figure 7. Model for linker cell death.

Green, upstream regulators. Orange, HSF-1. Purple, proteolytic components.    DOI: http://dx.doi.org/10.7554/eLife.12821.016

 

Our data suggest that three partially redundant signals control LCD initiation. The antagonistic Wnt pathways we describe may provide positional information to the linker cell, as the relevant ligands are expressed only near the region where the linker cell dies. The LIN-29 pathway, which controls timing decisions during the L4-adult molt, may ensure that LCD takes place only at the right time. Finally, while the TIR-1/SEK-1 pathway could act constitutively in the linker cell, it may also respond to specific cues from neighboring cells. Indeed, MAPK pathways are often induced by extracellular ligands. We propose that these three pathways, together, trigger activation of HSF-1. Our data support a model in which HSF-1 is present in two forms, HSF-1LC, promoting LCD, and HSF-1HS, protecting cells from stresses, including heat shock. We postulate that the redundant LCD initiation pathways tip the balance in favor of HSF-1LC, allowing this activity to bind to promoters and induce transcription of key LCD effectors, including LET-70/UBE2D2 and other components of the ubiquitin proteasome system (UPS), functioning through E3 ligase complexes consisting of CUL-3, RBX-1, BTBD-2, and SIAH-1.

Importantly, the molecular identification of LCD components and their interactions opens the door to testing the impact of this cell death pathway on vertebrate development. For example, monitoring UBE2D2 expression during development could reveal upregulation in dying cells. Likewise, genetic lesions in pathway components we identified may lead to a block in cell death. Double mutants in apoptotic and LCD genes would allow testing of the combined contributions of these processes.

The proteasome and LCD

As is the case with caspase proteases that mediate apoptosis (Pop and Salvesen, 2009), how the UPS induces LCD is not clear, and remains an exciting area of future work. That loss of BTBD-2, a specific E3 ligase component, causes extensive linker cell survival suggests that a limited set of targets may be required for LCD. Previous work demonstrated that BTBD2, the vertebrate homolog of BTBD-2, interacts with topoisomerase I (Khurana et al., 2010; Xu et al., 2002), raising the possibility that this enzyme may be a relevant target, although other targets may exist.

The UPS has been implicated in a number of cell death processes in which it appears to play a general role in cell dismantling, most notably, perhaps, in intersegmental muscle remodeling during metamorphosis in moths (Haas et al., 1995). However, other studies suggest that the UPS can have specific regulatory functions, as with caspase inhibition by IAP E3 ligases (Ditzel et al., 2008).

During Drosophila sperm development, caspase activity is induced by the UPS to promote sperm individualization, a process that resembles cytoplasm-specific activation of apoptosis (Arama et al., 2007). While C. elegans caspases are dispensible for LCD, it remains possible that they participate in linker cell dismantling or serve as a backup in case the LCD program fails.

Finally, the proteasome contains catalytic domains with target cleavage specificity reminiscent of caspases; however, inactivation of the caspase-like sites does not, alone, result in overt cellular defects (Britton et al., 2009), suggesting that this activity may be needed to degrade only specific substrates. Although the proteasome generally promotes proteolysis to short peptides, site-specific cleavage of proteins by the proteasome has been described (Chen et al., 1999). It is intriguing to speculate, therefore, that caspases and the proteasome may have common, and specific, targets in apoptosis and LCD.

A pro-death developmental function for HSF-1

Our discovery that C. elegans heat-shock factor, HSF-1, promotes cell death is surprising. Heat-shock factors are thought to be protective proteins, orchestrating the response to protein misfolding induced by a variety of stressors, including elevated temperature. Although a role for HSF1 has been proposed in promoting apoptosis of mouse spermatocytes following elevated temperatures (Nakai et al., 2000), it is not clear whether this function is physiological. In this context, HSF1 induces expression of the gene Tdag51 (Hayashida et al., 2006). Both pro- and anti-apoptotic activities have been attributed to Tdag51 (Toyoshima et al., 2004), and which is activated in sperm is not clear. Recently, pathological roles for HSF1 in cancer have been detailed (e.g. Mendillo et al., 2012), but in these capacities HSF1 still supports cell survival.

Developmental functions for HSF1 have been suggested in which HSF1 appears to act through transcriptional targets different from those of the heat-shock response (Jedlicka et al., 1997), although target identity remains obscure. Here, we have shown that HSF-1 has at least partially non-overlapping sets of stress-induced and developmental targets. Indeed, typical stress targets of HSF-1, such as the small heat-shock gene hsp-16.49 as well as genes encoding larger chaperones, likehsp-1, are not expressed during LCD, whereas let-70, a direct transcriptional target for LCD, is not induced by heat shock. Interestingly, the yeast let-70 homologs ubc4 and ubc5 are induced by heat shock (Seufert and Jentsch, 1990), supporting a conserved connection between HSF and UBE2D2-family proteins. However, the distinction between developmental and stress functions is clearly absent in this single-celled organism, raising the possibility that this separation of function may be a metazoan innovation.

What distinguishes the stress-related and developmental forms of HSF-1? One possibility is that whereas the stress response appears to be mediated by HSF-1 trimerization, HSF-1 monomers or dimers might promote LCD roles. Although this model would nicely account for the differential activities in stress responses and LCD of the HSF-1(R145A) transgenic protein, which would be predicted to favor inactivation of a larger proportion of higher order HSF-1 complexes, the identification of conserved tripartite HSEs in the let-70 and rpn-3 regulatory regions argues against this possibility. Alternatively, selective post-translational modification of HSF-1 could account for these differences. In mammals, HSF1 undergoes a variety of modifications including phosphorylation, acetylation, ubiquitination, and sumoylation (Xu et al., 2012), which, depending on the site and modification, stimulate or repress HSF1 activity. In this context, it is of note that p38/MAPK-mediated phosphorylation of HSF1 represses its stress-related activity (Chu et al., 1996), and the LCD regulator SEK-1 encodes a MAPKK. However, no single MAPK has been identified that promotes LCD (E.S.B., M.J.K. unpublished results), suggesting that other mechanisms may be at play.

Our finding that POP-1/TCF does not play a significant role in LCD raises the possibility that Wnt signaling exerts direct control over HSF-1 through interactions with β-catenin. However, we have not been able to demonstrate physical interactions between these proteins to date (M.J.K, unpublished results).

Finally, a recent paper (Labbadia and Morimoto, 2015) demonstrated that in young adult C. elegans, around the time of LCD, global binding of HSF-1 to its stress-induced targets is reduced through changes in chromatin modification. Remarkably, we showed that chromatin regulators play a key role in let-70 induction and LCD (J.A.M., M.J.K and S.S., manuscript in preparation), suggesting, perhaps, that differences in HSF-1 access to different loci may play a role in distinguishing its two functions.

LCD and neurodegeneration

Previous studies from our lab raised the possibility that LCD may be related to degenerative processes that promote vertebrate neuronal death. Nuclear crenellation is evident in dying linker cells and in degenerating cells in polyQ disease (Abraham et al., 2007) and the TIR-1/Sarm adapter protein promotes LCD in C. elegans as well as degeneration of distal axonal segments following axotomy in Drosophila and vertebrates (Osterloh et al., 2012). The studies we present here, implicating the UPS and heat-shock factor in LCD, also support a connection with neurodegeneration. Indeed, protein aggregates found in cells of patients with polyQ diseases are heavily ubiquitylated (Kalchman et al., 1996). Chaperones also colocalize with protein aggregates in brain slices from SCA patients, and HSF1 has been shown to alleviate polyQ aggregation and cellular demise in both polyQ-overexpressing flies and in neuronal precursor cells (Neef et al., 2010). While the failure of proteostatic mechanisms in neurodegenerative diseases is generally thought to be a secondary event in their pathogenesis, it is possible that this failure reflects the involvement of a LCD-like process, in which attempts to engage protective measures instead result in activation of a specific cell death program.

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Natural Killer Cell Response: Treatment of Cancer

Curator: Larry H. Bernstein, MD, FCAP

 

Molecular mechanisms of natural killer cell activation in response to cellular stress

C J Chan1,2,3, M J Smyth1,2,3,4,5 and L Martinet1,2,4,5        Edited by M Piacentini

Cell Death and Differentiation (2014) 21, 5–14;    http://www.nature.com/cdd/journal/v21/n1/full/cdd201326a.htm

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.

 

  • Stress induces specific intrinsic and extrinsic physiological mechanisms within cells that lead to their identification as functionally abnormal
  • Sources of cellular stress can be nutritional, physical, pathogenic, or oncogenic
  • Intrinsic responses to cellular stress include activation of the DNA-damage response, tumor-suppressor genes, and senescence
  • The extrinsic response to cellular stress is activation of the immune system, such as natural killer cells
  • Intrinsic responses to cellular stress can directly upregulate factors that can activate the immune system, and the immune system been shown to be indispensable for the efficacy of some chemotherapy

Further critical determinants of intrinsic responses to stress and cell death that can activate the immune system must be identified

  • Identification of the different cellular pathways and molecular determinants controlling the immunogenicity of different cancer therapies is required
  • How can we harness the ability of therapeutic agents to activate both the intrinsic and extrinsic responses to cellular stress to achieve more specific and safer approaches to cancer treatment?

Any insult to a cell that leads to its abnormal behavior or premature death can be defined as a source of stress. As the turnover and maintenance of cells in all multi-cellular organisms is tightly regulated, it is essential that stressed cells be rapidly identified to avoid widespread tissue damage and to maintain tissue homeostasis. Various intrinsic cellular mechanisms exist within cells that become activated when they are exposed to stress. These include activation of DNA-damage response proteins, senescence programs, and tumor-suppressor genes.1 Extrinsic mechanisms also exist that combat cellular stress, through the upregulation of mediators that can activate different components of the immune system.2 Although frequently discussed separately, much recent evidence has indicated that intrinsic and extrinsic responses to cellular stress are intimately linked.3

As the link between cell intrinsic and extrinsic responses to stress have been uncovered, these observations are now being harnessed therapeutically, particularly in the context of cancer.4 Indeed, various chemotherapeutic agents and radiotherapy are critically dependent on the immune system to elicit their full therapeutic benefit.5, 6 The mechanisms by which this occurs may be twofold: (i) the induction of intrinsic cellular stress mechanisms activates innate immunity and (ii) the release and presentation of tumor-specific antigens engages an inflammatory adaptive immune response.

NK cells are the major effector lymphocyte of innate immunity found in all the primary and secondary immune compartments as well as various mucosal tissues.7 Through their ability to induce direct cytotoxicity of target cells and produce pro-inflammatory cytokines such as interferon-gamma, NK cells are critically involved in the immune surveillance of tumors8, 9, 10 and microbial infections.11, 12 The major mechanism that regulates NK cell contact-dependent functions (such as cytotoxicity and recognition of targets) is the relative contribution of inhibitory and activating receptors that bind to cognate ligands.

Under normal physiological conditions, NK cell activity is inhibited through the interaction of their inhibitory receptors with major histocompatibility complex (MHC) class I.13, 14 However, upon instances of cellular stress that are frequently associated with viral infection and malignant transformation, ligands for activating receptors are often upregulated and MHC class I expression may be downregulated. The upregulation of these activating ligands and downregulation of MHC class I thus provides a signal for NK cells to become activated and display effector functions. Activating receptors are able to provide NK cells with a strong stimulus in the absence of co-stimulation due to the presence of adaptor molecules such as DAP10, DAP12, FcRγ, and CD3ζ that contain immunoreceptor tyrosine-based activating motifs (ITAMs).15, 16,17 By contrast, inhibitory receptors contain inhibitory motifs (ITIMs) within their cytoplasmic tails that can activate downstream targets such as SHP-1 and SHP-2 and directly antagonize those signaling pathways activated through ITAMs.18, 19, 20 The specific details of individual classes of inhibitory and activating receptors and their ligands are summarized in Figure 1 and have been extensively reviewed elsewhere.14, 21 Instead, this review will more focus on the relevant activating receptors that are primarily involved in the direct regulation of NK cell-mediated recognition of cellular stress: natural killer group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1).

Figure 1.

Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorNK cell receptors and their cognate ligands. Major inhibitory and activating receptors on NK cells and their cognate ligands on targets are depicted. BAT3, human leukocyte antigen (HLA)-B-associated transcript 3; CRTAM, class I-restricted T-cell-associated molecule; HA, hemagglutinin; HLA-E, HLA class I histocompatibility antigen, alpha chain E; IgG, immunoglobulin G; LFA-1, leukocyte function-associated antigen-1; LLT1, lectin-like transcript 1; TIGIT, T cell immunoglobulin and ITIM domain

Full figure and legend (185K)

NK Cell-Mediated Recognition of Cellular Stress by NKG2D and DNAM-1

NKG2D is a lectin-like type 2 transmembrane receptor expressed as a homodimer in both mice and humans by virtually all NK cells.22, 23 Upon interaction with its ligands, NKG2D can trigger NK cell-mediated cytotoxicity against their targets. The ligands for NKG2D are self proteins related to MHC class I molecules.24 In humans, these ligands consist of the MHC class I chain-related protein (MIC) family (e.g., MICA and MICB) and the UL16-binding protein (ULBP1-6) family.25, 26 In mice, ligands for NKG2D include the retinoic acid early inducible (Rae) gene family, the H60 family, and mouse ULBP-like transcript-1 (MULT-1).27, 28, 29 NKG2D ligands are generally absent on the cell surface of healthy cells but are frequently upregulated upon cellular stress associated with viral infection and malignant transformation.3, 30 Indeed, NKG2D ligand expression has been found on many transformed cell lines, and NKG2D-dependent elimination of tumor cells expressing NKG2D ligands has been well documented in vitro and in tumor transplant experiments.25, 30, 31, 32, 33 In humans, NKG2D ligands have been described on different primary tumors34, 35 and specific NKG2D gene polymorphisms are associated with susceptibility to cancer.36 Finally, blocking NKG2D through gene inactivation or monoclonal antibodies leads to an increased susceptibility to tumor development in mouse models,37, 38demonstrating the key role played by NKG2D in immune surveillance of tumors. NKG2D can also contribute to shape tumor immunogenicity, a process called immunoediting, as demonstrated by the frequent ability of tumor cells to avoid NKG2D-mediated recognition through NKG2D ligand shedding, as discussed later in this review.38, 39, 40

DNAM-1 is a transmembrane adhesion molecule constitutively expressed on T cells, NK cells, macrophages, and a small subset of B cells in mice and humans.41, 42, 43 DNAM-1 contains an extracellular region with two IgV-like domains, a transmembrane region and a cytoplasmic region containing tyrosine- and serine-phosphorylated sites that is able to initiate downstream activation cascades.41, 44 There is accumulating evidence showing that DNAM-1 not only promotes adhesion of NK cells and CTLs but also greatly enhances their cytotoxicity toward ligand-expressing targets.41, 45, 46, 47, 48, 49, 50 The ligands for DNAM-1 are the nectin/nectin-like family members CD155 (PVR, necl-5) and CD112 (PVRL2, nectin-2).45, 46 Like NKG2D ligands, DNAM-1 ligands are frequently expressed on virus-infected and transformed cells.51, 52DNAM-1 ligands, especially CD155, are overexpressed by many types of solid and hematological malignancies and blocking DNAM-1 interactions with its ligands reduces the ability of NK cells to kill tumor cells in vitro.41, 49, 53, 54, 55, 56, 57 Further evidence of the role of DNAM-1 in tumor immune surveillance is provided by studies using experimental and spontaneous models of cancer in vivo showing enhanced tumor spread in the absence of DNAM-1.47, 48, 49, 50, 58

As NKG2D and DNAM-1 ligands are frequently expressed on stressed cells, many studies have sought to determine the mechanisms that underpin these observations. The guiding hypothesis for these studies is that cell-intrinsic responses to stress are directly linked to cell-extrinsic responses that can trigger rapid NK cell surveillance and elimination of stressed cells. Indeed, major cell-intrinsic responses to cellular stress can directly lead to NK cell-activating ligand upregulation and are outlined in the following sections.

The DNA-Damage Response

Cellular stress caused by the activation of the DNA-damage response leads to downstream apoptosis or cell-cycle arrest. The activation of DNA-damage checkpoints occurs when there are excessive DNA strand breaks and replication errors, thereby representing an important tumorigenesis barrier that can slow or inhibit the progression of malignant transformation.59, 60 Two major transducers of the DNA-damage response are the PI3-kinase-related protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM and ATR can modulate numerous signaling pathways such as checkpoint kinases (Chk1 and Chk2, which inhibit cell-cycle progression and promote DNA repair) and p53 (which mediates cell-cycle arrest and apoptosis).61

In addition to the induction of cell-cycle arrest and apoptosis, activation of the DNA-damage response has been shown to promote the expression of several activating ligands that are specific for NK cell receptors, primarily those of the NKG2D receptor. These findings have shown a critical direct link between cellular transformation, apoptosis, and surveillance by the immune system.62 The first evidence of this link between DNA damage and immune cell activation was provided by Raulet and colleagues who showed that NKG2D ligands were upregulated by genotoxic stress and stalled DNA replication conditions known to activate either ATM or ATR.63 These observations have now been extended by several other studies that have defined further DNA-damaging conditions (e.g., genotoxic drugs/chemotherapy, deregulated proliferation, or oxidative stress) that can promote NKG2D ligand upregulation.64, 65, 66, 67

The role of the DNA-damage response in controlling NKG2D ligand expression and subsequent NK cell activation has also been demonstrated in the context of anti-viral immunity, specifically in Abelson murine leukemia virus infection.68 This pathogen was shown to induce activation-induced cytidine deaminase (AID) expression outside the germinal center, resulting in generalized hypermutation, DNA-damage checkpoint activation, and Chk1 phosphorylation. The genotoxic activity of virally induced AID not only restricted the proliferation of infected cells but also induced the expression of NKG2D ligands. More recently, another member of APOBEC-AID family of cytidine deaminases, A3G, has been shown to promote the recognition of HIV-infected cells by NK cells after DNA-damage response activation.69 In this study, viral protein Vpr-mediated repair processes, which generate nicks, gaps, and breaks of DNA, activate an ATM/ATR DNA-damage response that leads to NKG2D ligand expression.

The DNA-damage sensors ATM and ATR have also been shown to regulate other key NK cell-activating ligands such as the DNAM-1 ligand, CD155.58, 65, 70 For example, in the Eμ-myc spontaneous B-cell lymphoma model, activation of the DNA-damage response leads to the upregulation of CD155 in the early-stage transformed B cells, subsequently activating spontaneous tumor regression in an NK cell- and T-cell-dependent manner.58 The DNA-damage response can also regulate the expression of the death receptor DR5.71 The engagement of DR5 by the effector molecule TRAIL, which is expressed by NK cells and T cells, can induce apoptosis of target cells and has been shown to have a key role in immune surveillance against tumors.72 Collectively, these results suggest that the detection of DNA damage, primarily through ATM and ATR, may represent a conserved protection mechanism governing the immunogenicity of infected or transformed cells, leading to direct recognition by NK cells (Figure 2).

Figure 2.

Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorOverview of the molecular pathways leading to NK cell recognition of intrinsic cellular stress. Oncogenic transformation and viral infection can activate intrinsic cellular responses to stress. These responses include activation of the DNA-damage response, senescence, tumor suppressors, and the presentation and/or release of HSPs that, in turn, can activate NK cells through various receptor–ligand interactions. Senescent cells can also release pro-inflammatory cytokines that can recruit NK cells and other innate immunity, such as macrophages. CCL2, C-C motif chemokine ligand 2; CXCL11, C-X-C motif chemokine ligand 11; DR, death receptor 5; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

Full figure and legend (146K)

As a result of these studies, many therapeutic agents known to induce DNA damage have been evaluated for their ability to increase the immunogenicity of cancer cells for a more targeted therapeutic approach using NK cells.64, 65 For example, treatment of multiple myeloma cells with doxorubicin, melphalan, or bortezomib can lead to DNAM-1 and NKG2D ligand upregulation.65Indeed, many chemotherapeutic agents commonly used, especially in hematological malignancies, can trigger the DNA-damage pathway. Therefore, it is reasonable to speculate that there is a general role of ATM and ATR in the induction of NK cell activation as a therapeutic effect of these agents.

Senescence

Cellular senescence is generally defined as a growth-arrest program in mammalian cells that limits their lifespan.73 The major type of cellular senescence is replicative senescence that occurs due to telomere shortening. However, it is now generally accepted that premature senescence can also occur due to oncogene activation (oncogene-induced senescence) and/or the loss/gain of tumor-suppressor gene function, in the absence of telomere shortening.74 Thus, premature senescence is an important barrier against malignant transformation.59 Upon engagement of the senescence program, although cells are in growth arrest, they remain metabolically active and can produce many pro-inflammatory cytokines, as well as upregulate adhesion molecules and activating ligands to alert the immune system.75, 76, 77Activation of the immune system, in particular innate immunity, has a critical role in the clearance of senescent cells.78, 79, 80, 81More specifically, in a model of hepatocellular carcinoma, it has been shown that reactivation of p53 can induce a senescence program, resulting in tumor regression through the activation of NK cells, macrophages, and neutrophils. Of note, intercellular adhesion molecule (ICAM)-1, which can trigger both adhesion and cytotoxicity of NK cells,82 and interleukin-15, a cytokine that can promote NK cell effector function,83 were both upregulated in senescent tumors. More recently, the potential contribution of NK cells was also shown in the clearance of senescent hepatic stellate cells, a mechanism important in limiting liver fibrosis in response to a fibrogenic agent.80 ICAM-1, NKG2D ligands (MICA and ULPB2), and DNAM-1 ligands (CD155) were all upregulated on senescent hepatic stellate cells.

The specific mechanisms linking the senescence program to immune activation are not yet fully understood. However, the intracellular molecular mechanisms that govern induction of senescence may provide possible indications. Both replicative senescence and premature senescence (e.g., oncogene-induced senescence) have been shown to have common molecular determinants, such as the activation of the DNA-damage response pathway (e.g., ATM and ATR) and downstream activation of p53 and p16INK4A.1, 59, 84, 85, 86 Activation of the DNA-damage response would presumably initiate the upregulation of NK cell-activating ligands as previously discussed. However, how senescence may be linked to the induction of pro-inflammatory cytokine release is a more compelling question and requires further investigation (Figure 2). Nevertheless, induction of pro-inflammatory cytokines is an important protective mechanism in order to recruit immune cells that can rapidly recognize and remove senescent cells. Interestingly, activation of NK cells by senescent cells has been observed in a clinical context when multiple myeloma cells were treated with chemotherapy and genotoxic agents.65 In this setting, NKG2D and DNAM-1 ligands were both upregulated through a mechanism that required activation of the DNA-damage pathway initiated by ATM and ATR.65

Tumor Suppressors: p53

p53 is a potent tumor suppressor and central regulator of apoptosis, DNA repair, and cell proliferation, that is activated in response to DNA damage, oncogene activation, and other cellular stress.87 The number of identified cellular functions that p53 regulates has greatly increased over the past few years, and there is now a vast array of evidence that shows that p53 can be induced by viral infection88 to limit pathogen spread by inducing apoptosis.89, 90 Furthermore, p53 not only acts as an intrinsic barrier against tumorigenesis or pathogenic spread but can also lead to increased cellular immunogenicity. For example, p53 reactivation in a hepatocellular carcinoma can promote tumor regression mediated by innate immunity.78 A direct link between p53 expression and immune cell recognition was recently provided by Textor et al.91 where expression of p53 in lung cancer cell lines strongly upregulated the NKG2D ligands ULBP1 and 2, resulting in NK cell activation. Subsequently, p53-responsive elements were found to directly regulate ULBP1 and 2 expression, the deletion of which abolished the capacity of p53 to mediate ULBP1 and 2 upregulation. Another recent report that used a pharmacological activator of p53 confirmed the ability of p53 to directly induce ULBP2 expression that was independent of ATM/ATR.92 However, it has also been shown that miR34a and miR34C microRNAs (miRNAs) induced by p53 can target ULBP2 mRNA and reduce its cell-surface expression, suggesting that p53 may have a dual role in regulating ULBP2 expression.93 Finally, early work showed that NKG2D ligands can be upregulated by ATR/ATM in the total absence of p53 in tumor cell lines,62, 63 suggesting the existence of ATM/ATR-dependent and p53-independent pathways that regulate NKG2D ligand expression in response to cellular stress.

In addition to regulating NK cell ligand expression, genetic reactivation of p53 in tumors can also induce a wide array of pro-inflammatory mediators ranging from adhesion receptor (ICAM-1) expression to the production of various chemokines (CXCL11 and monocyte chemoattractant protein-1) and cytokines (interleukin-15).78 Furthermore, recent studies in anti-viral immunity indicate that several interferon-inducible genes and Toll-like receptor-3 expression are direct transcriptional targets of p53 and that p53 contributes to production of type I interferon by virally infected cells.94, 95, 96 All together, these studies suggest that p53 accumulation could represent a key determinant of the immunogenicity of stressed cells that are infected or undergoing malignant transformation through its ability to regulate innate immune activation.

Oncogenes

Malignant transformation is a complex process that frequently involves the activation of one or more oncogenes in addition to the inactivation or mutation of tumor-suppressor genes (e.g., p53). Oncogene activation is a powerful inducer of cellular stress that is able to activate intrinsic cellular programs that lead to cell apoptosis or senescence (e.g., activation of the DNA-damage response and p53).1 In addition, many recent reports have also shown that major oncogenes can activate extrinsic responses to cellular stress through inducing the upregulation of NK cell-activating ligands.63, 97, 98 This suggests that oncogene activation can represent a key cellular event in alerting the immune system to ongoing cellular transformation (Figure 3).

Figure 3.

Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorMolecular mechanisms that regulate the cell surface expression of NKG2D ligands. The major group of NK cell-activating ligands that are upregulated by intrinsic cellular responses to stress are those that bind the NKG2D receptor. Activation of the DNA-damage response, senescence, oncogenes, tumor suppressors, or sensing of deregulated proliferation can induce NKG2D ligand gene transcription and increase mRNA translation, leading to extracellular protein expression. MMP, matrix metalloproteases

Full figure and legend (183K)

The enhanced expression of the proto-oncogene Myc has been described as a critical event leading to cellular transformation and is a frequently found genetic alteration in cancer.99 In a recent study, again using the Eμ-myc model, Medzhitov and colleagues demonstrated the ability of c-Myc to alert NK cells to early oncogenic transformation through the upregulation of Rae-1.97 In this study, the induction of Rae-1 was dependent on the direct regulation of Rae-1 transcription by Myc through its interaction with the Raet1 epsilon gene. Collectively, these results provide a possible direct molecular mechanism to explain the increased susceptibility of NKG2D gene-targeted mice to lymphoma development in the Eμ-myc model.38

Recent evidence suggests that several oncogenic mutations of Ras (H-Ras, N-Ras, and K-Ras) can also regulate NKG2D ligand expression in both mice and humans.98 Interestingly, in this case, NKG2D ligands were regulated through MAPK/MEK and PI3K pathways downstream of oncogenic H-RasV12. The activation of PI3K pathways, and more particularly the p110α subunits by virus-encoded proteins, has also been shown to induce the Rae-1 family of ligands.100 As many viruses can manipulate the PI3K pathway101 and tumors often bear Ras and p110α oncogene mutations,102 collectively, this data suggests that there is the existence of a common molecular mechanism by which NK cells sense cellular stress mediated by PI3K-dependent regulation of NKG2D ligands.

Interestingly, whereas Myc was involved in the transcriptional regulation of NKG2D ligands, PI3K can increase NKG2D ligand expression by increasing the translation of Rae-1 mRNA.98 This involved the induction of eIF4E, a protein that enhances the translation of mRNA.103 As number of tumors and viruses can upregulate host translation initiation machinery through the overexpression of eIF4E,104, 105 this may represent an important means by which NK cells can discriminate tumor- and virus-infected cells from normal cells.

Heat-Shock Proteins (HSPs)

HSPs are highly conserved intracellular chaperone molecules that are present in most prokaryotic and eukaryotic cells that mediate protection against cellular damage under conditions of stress. HSPs are distributed in most intracellular compartments of cells where they support the correct folding of nascent polypeptides, prevent protein aggregation, and assist in protein transport across membranes.106 Many tumors display overexpression of HSPs as a response to cellular stress induced by oncogenic transformation.107, 108 HSPs can also be mobilized to the plasma membrane, or even released from cells, under conditions of stress.109

Although intracellular HSPs can promote cell survival by interfering with different apoptosis components, many studies have reported that membrane-bound or soluble HSPs can directly stimulate innate immunity.110 A major immunostimulatory function of HSPs is to promote the presentation of tumor-specific antigens by MHC class I to CD8 T cells.111, 112, 113 Soluble and membrane-bound HSPs can also induce antigen-presenting cell maturation and the resultant secretion of pro-inflammatory cytokines.114, 115, 116Finally, HSPs may directly activate NK cells as HSP70, when overexpressed on tumor cells, can induce a selective dose-dependent increase in NK cell-mediated cytotoxicity in vitro.117 NK cells may directly recognize HSP70 through a 14-amino-acid oligomer (TKD) that is localized in the C-terminal domain of the protein through CD94.118, 119 Tumor-specific HSP70 that is either presented at the cell surface or secreted on exosomes can also enhance NK cell activity against diverse types of cancer in vivo.120, 121 Most importantly, hepatocellular carcinoma cells that are treated with various chemotherapeutic agents can become more susceptible to NK cell-mediated cytotoxicity through their release of HSP-containing exosomes, giving the aforementioned findings a therapeutic context.122 Collectively, these results suggest that HSP translocation to the plasma membrane or secretion during cellular stress may represent a potent danger signal that can stimulate NK cell activity, particularly in the context of cancer.

 

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Eppendorf Award for Young European Investigators

Curator: Larry H. Bernstein, MD, FCAP

Series E. 2; 8.11

The independent Eppendorf Award Jury chaired by Prof. Reinhard Jahn selected Dr. Thomas Wollert (Research Group Leader at the Max Planck Institute of Biochemistry in Martinsried, Germany) as the 2015 winner of the Eppendorf Award for Young European Investigators.

Thomas receives the €20,000 prize for his groundbreaking work in reconstituting complex intracellular membrane events in the test tube using artificial membranes and purified components. His experiments have paved the way for understanding key steps in autophagy, a fundamental process required for the clearance of damaged cell parts in all eukaryotic cells.

Listen to a podcast with Thomas Wollert and learn more about his work, and read excerpts from the interview in a Q&A feature article.

Presented in partnership with Nature The Eppendorf Award for Young European Investigators was established in 1995 to recognize outstanding work in biomedical science. It also provides the opportunity for European researchers to showcase their work and communicate their research to a scientific audience. Nature is pleased to partner with Eppendorf to promote the award and celebrate the winner’s work in print and online. Nature’s Julie Gould talks to the 2015 winner Thomas Wollert (Max Planck Institute of Biochemistry, Germany) about his work — which looks at the complex molecular process that cells use to remove their waste — and how it felt to win the award.
To listen to the full interview, visit: go.nature.com/cszfl1

About the Award Thomas Wollert is the twentieth recipient of the Eppendorf Award for Young European Investigators, which recognizes talented young individuals working in the field of biomedical research in Europe. The Eppendorf Award is presented in partnership with Nature. The winner is selected by an independent jury of scientists under the chairmanship of Reinhard Jahn, Director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany. Nature and Eppendorf do not influence the selection. For more information see: eppendorf.com/award

http://www.nature.com/nature/awards/eppendorf/eppendorf_award_2015.pdf?WT.mc_id=EMI_NATURE_1509_YOUNGINVESTIGATOR2015&spMailingID=49568192&spUserID=MTYxNjA5NDg5ODE3S0&spJobID=762344014&spReportId=NzYyMzQ0MDE0S0

Julie Gould: Congratulations on being awarded this year’s prize. How did it feel when you found out that you had won?

Thomas Wollert: That came as a big surprise to me. It’s a great honor and it’s of course a major recognition of our work; not only my work, but also the work that my laboratory has done over the past five years. So this is very important to me.

JG: Tell us a little bit about the research you are working on.

TW: The cells in our bodies recycle almost everything — they do not waste much. The question in the past has been: how is this achieved? The process needs to be highly regulated. You don’t want to degrade something that you still need, but you do want to get rid of dangerous material that accumulates in the cell. We became interested in one pathway that is involved in transporting this sort of trash, or unwanted material, to recycling stations in the cell. We are particularly interested in how the molecular mechanism is driven.

JG: What sort of molecular trash are we talking about?

TW: Everything that needs to be degraded in a cell has to end up at a recycling station, one of which is called the lysosome. What ends up there is chemically degraded, and the building blocks are reused by the cell to build material. Proteins that become aggregated, big material or composite structures, and everything else in the cell cytoplasm (such as mitochondria) need to be transported to the lysosome. There is a specialized pathway to do that — this has been called autophagy for self-digestion. During autophagy, crescent-shaped membranes are formed, which expand and capture cytoplasmic components. These structures become autophagosomes, which are like entire organelles and are the containers that transport the trash to the lysosomes for degradation.

JG: How do these autophagosomes form in the cell?

TW: In yeast the system is fairly well understood. Small membrane vesicles are recruited and fuse to form the crescents haped autophagic precursor membrane. This membrane then surrounds and captures material, and, after sealing, the full autophagosome is formed and finally fuses with the lysosome. There are 40 different proteins in yeast that have been identified as those that have an essential function in autophagy — they are specific to the autophagy pathway. The question was, what are they doing with the membrane and what is their molecular function? And that was the major interest of my lab.

JG: What did you discover? TW:

We analysed two important steps in autophagy. The first is initiation and the second is expansion.

An autophagosome is built from small vesicles, which come together and fuse. This process is driven by one big complex called the Atg1-kinase complex. This complex is known to be involved in recruiting the donor vesicles that create the autophagosome. We recently published work on the expansion step. This is an interesting step that involves a small ubiquitin-like molecule, Atg8. The unique feature of this particular molecule is that it becomes covalently attached to autophagic precursor membranes. Many Atg8 molecules get conjugated to these membranes, so the question has been: why is there so much Atg8 on the membrane and what is its job there? To answer this, we analyzed the proteins independently of the complex cellular environment. We produced recombinant molecular machines that drive the formation of autophagosomes and analyzed their function in the test tube. The test-tube components include the protein subunits of these molecular machines and model membranes that serve as the platform for proteins to assemble into large complexes. What we realized — and what came as a surprise to us — was that the molecular machine that drives conjugation of Atg8 stays with Atg8 at the membrane, rather than leaving after conjugation. We predicted that something needs to happen, some bigger structure needs to form on the membrane to keep the conjugation machine there. Using high-resolution approaches, we observed that Atg8 forms together with its conjugation machine, a protein shell on membranes. It’s like a meshwork that sits on top of the membrane and stabilizes the forming autophagosome. Presumably.

JG: Why presumably?

TW: Because the details of how this expansion is driven by the scaffold is something that we are investigating.

JG: Will you be following this up over the next few years?

TW: Yes. This is an interesting question, but not an easy one to answer. We need to understand the direct relationship of how this really works in vivo.

JG: How does the autophagosome capture material from cells?

TW: The selection of cargo comes in two flavours. Under normal conditions, when the cell is happy, it only wants to degrade unwanted material or something damaged. It chooses these materials quite selectively. For example, it might only want to degrade dysfunctional mitochondria, the cell’s power plants. The membrane then wraps tightly around these structures. However, if a cell becomes stressed or starved, it can use autophagy to degrade anything that’s around. That means bulk cytoplasm without any selectivity. Imagine a big happy cell that is starved and goes on a low-value nutritional diet. The cell will shrink, but it survives. If nutritional conditions improve, it can grow again.

JG: What big impacts will this research have?

TW: The research focus at the moment is neurodegenerative disease and cancer. In certain neurodegenerative diseases, some proteins can accumulate in cells. There are a couple of diseases, such as Huntington’s disease, in which particular genetic modifications lead to alterations in proteins, which then tend to aggregate. In other diseases, such as Alzheimer’s disease, proteins also accumulate, and those protein oligomers, or aggregates, are toxic to the cell. In some neurodegenerative diseases, it has been observed that increasing autophagy is beneficial for cells, and thus patients, because increasing autophagy increases the removal of the toxic material. Neurodegenerative disease is usually not observed until the later stages, when this material has already accumulated. If you could remove this harmful material from cells, you could maybe rescue some neurons from dying. This is one application where you would really want to increase autophagy. In cancer, it has already been shown that combining chemotherapy with an inhibitor of autophagy is beneficial because autophagy just counteracts chemotherapy.

JG: What is it about this field that you find so interesting?

TW: What excites me the most is that you can use a minimal system, combining a few components and then trying to get them to work in a test tube. Our major goal, and our holy grail in this research, is to have the full autophagy pathway in a test tube, combining the autophagy components, step by step, to produce an autophagosome from small membranes, and to have some material wrapped in the autophagosome.

Award Winners

2015 Winner

In 2015 Eppendorf AG is presenting the Eppendorf Award for Young European Investigators for the 20th time. The independent Eppendorf Award Jury chaired by Prof. Reinhard Jahn selected Dr. Thomas Wollert (Research Group Leader Molecular Membrane and Organelle Biology at the Max Planck Institute of Biochemistry in Martinsried, Germany) as the 2015 winner of the Eppendorf Award for Young European Investigators. Thomas Wollert, born 1979, receives the €20,000 prize for his groundbreaking work in reconstituting complex intracellular membrane events in the test tube using artificial membranes and purified components. Thomas talks about his work in this Award Feature

The official prize ceremony took place at the EMBL Advanced Training Centre in Heidelberg, Germany, on June 25, 2015.

To hear an interview with prize winner Thomas, listen here.

2014 Winner

The independent Eppendorf Award Jury chaired by Prof. Reinhard Jahn selected Madeline Lancaster, Ph.D., of the Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria, as the 2014 winner of the Eppendorf Award for Young European Investigators. Madeline Lancaster, born 1982, receives the € 15,000 research prize for her work showing that complex neuronal tissues resembling early states of fetal human brain can be created in vitro from pluripotent stem cells. Madeline talks about her work in this Award Feature

To hear an interview with prize winner Madeline, listen here or watch the video from the award ceremony.

2013 Winner

The independent Eppendorf Award Jury chaired by Prof. Reinhard Jahn selected Ben Lehner, Ph.D., of the Centre de Regulació Genòmica, Barcelona, Spain, as the 2013 winner of the Eppendorf Award for Young European Investigators. Ben, born 1978, receives the € 15,000 research prize for his discoveries concerning the fundamental question why mutations in the genome result in variable phenotypes. Ben talks about his work in this Award Feature.

To hear an interview with prize winner Ben, listen here or watch the video from the award ceremony.

2012 Winner

The 2012 prize was awarded to Elizabeth Murchison, Ph.D. (Wellcome Trust Sanger Institute, Cambridge, United Kingdom) for her discoveries concerning a deadly cancer that is spreading among the endemic population of Tasmanian devils in Tasmania and threatening the survival of the species. Elizabeth talks about her work in this Award Feature.

To hear an interview with prize winner Elizabeth, listen here or watch the video from the award ceremony in Heidelberg.

2011 Winner

The 2011 Eppendorf Young European Investigator Award goes to Suzan Rooijakkers for her contribution to discovering how Staphylococcus aureus evades immune attack. Suzan talks about her work on this Award Feature.

To hear an interview with prize winner Suzan, listen here.

Listen here to the podcast from the award ceremony in Heidelberg.

2009 Winner

In 2009 the prize was awarded to Óscar Fernández-Capetillo, head of the Genomic Instability Group at the Spanish National Cancer Center. Read the highlights of his interview with Nature in this Award Feature.

Listen here to learn about the impact the Award had on his career.

2008 Winner

The 2008 prize was awarded to Dr. Simon Boulton of the London Research Institute. Read the highlights of his interview with Nature in this Award Feature.

Listen here to learn about the impact the Award had on his career.

2007 Winner

Dr Mónica Bettencourt-Dias is the 2007 winner of the Eppendorf Young European Investigator Award. Monica gives a personal account of her research and the Eppendorf Award in an Award Feature forNature.

Listen here to learn more about the impact the award had on her career.

2006 Winner

Dr Luca Scorrano won the award in 2006. Read more about his research on the Eppendorf Young Investigator website.

Listen here to learn more about Dr Scorrano’s work and the impact the award has had on his career.

http://www.nature.com/multimedia/podcast/eppendorf/eppendorf-podcast-15.mp3   2015

https://www.youtube.com/watch?feature=player_embedded&v=N3SXLURTI_w   2014

https://www.youtube.com/watch?feature=player_embedded&v=ntU_Ve3x6oI     2013

https://www.youtube.com/watch?feature=player_embedded&v=E2mhX9ccEHs    2012

http://media.nature.com/download/nature/nature/podcast/eppendorf/eppendorf_2011_winner.mp3

http://media.nature.com/download/nature/nature/podcast/eppendorf/eppendorf_2011.mp3

http://media.nature.com/download/nature/nature/podcast/eppendorf/eppendorf_2010.mp3

http://media.nature.com/download/nature/nature/podcast/eppendorf/eppendorf-2009.mp3

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Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

Writer and Curator: Larry H. Bernstein, MD, FCAP 

(Note that each portion of the discussion is followed by a reference)

It is now a time to pause after almost a century of a biological scientific discoveries that have transformed the practice of medicine and impacted the lives of several generations of young minds determined to probe the limits of our knowledge.  In the century that we have entered into the scientific framework of medicine has brought together a difficult to grasp evolution of the emergence of human existence from wars, famine, droughts, storms, infectious diseases, and insect born pestilence with betterment of human lives, only unevenly divided among societal classes that have existed since time immemorial. In this short time span there have emerged several generations of physicians who have benefited from a far better medical education that their forebears could have known. In this expansive volume on cancer, we follow an incomplete and continuing challenge to understand cancer, a disease that has become associated with longer life spans in developed nations.

While there are significant improvements in the diagnosis and treatment of cancers, there is still a personal as well as locality factor in the occurrence of this group of diseases, which has been viewed incorrectly as a “dedifferentiation” of mature tissue types and the emergence of a cell phenotype that is dependent on glucose, reverts to a cancer “stem cell type” (loss of stemness), loses cell to cell adhesion, loses orderly maturation, and metastasizes to distant sites. At the same time, physician and nurses are stressed in the care of patients by balancing their daily lives and maintaining a perspective.

The conceptual challenge of cancer diagnosis and management has seemed insurmountable, but owes much to the post World War I activities of Otto Heinrich Warburg. It was Warburg who made the observation that cancer cells metabolize glucose by fermentation in much the way Pasteur 60 years earlier observed fermentation of yeast cells. This metabolic phenomenon occurs even in the presence of an oxygen supply, which would provide a huge deficit in ATP production compared with respiration. The cancer cell is “addicted to glucose” and produced lactic acid. Warburg was awarded the Nobel Prize in Medicine for this work in 1931.

In the last 15 years there has been a resurgence of work on the Warburg effect that sheds much new light on the process that was not previously possible, with significant therapeutic implications.  In the first place, the metabolic mechanism for the Warburg effect was incomplete even at the beginning of the 21st century.  This has been partly rectified with the enlightening elucidation of genome modifications, cellular metabolic regulation, and signaling pathways.

The following developments have become central to furthering our understanding of malignant transformation.

  1. There is usually an identifiable risk factor, such as, H. pylori, or of a chronic inflammatory state, as in the case of Barrett’s esophagus.
  2. There are certain changes in glucose metabolism that have been unquestionably been found in the evolution of this disease. The changes are associated with major changes in metabolic pathways, miRN signaling, and the metabolism geared to synthesis of cells with an impairment of the cell death cycle. In these changes, mitochondrial function is central to both the impaired respiration and the autophagy geared to the synthesis of cancer cells.

The emergence of this cell prototype is characterized by the following, again related to the Warburg effect:

  1. Cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis
  2. The mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis.
  3. Cancer cells tend to express a partially inhibited splice variant of pyruvate kinase (PK-M2), leading to decreased pyruvate production.
  4. The two proteins that mediate pyruvate conversion to lactate and its export, M-type lactate dehydrogenase and the monocarboxylate transporter MCT-4, are commonly upregulated in cancer cells leading to decreased pyruvate oxidation.
  5. The enzymatic step following mitochondrial entry is the conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex. Cancer cells frequently exhibit increased expression of the PDH kinase PDK1, which phosphorylates and inactivates PDH. This PDH regulatory mechanism is required for oncogene induced transformation and reversed in oncogene-induced senescence.
  6. The PDK inhibitor dichloroacetate has shown some clinical efficacy, which correlates with increased pyruvate oxidation. One of the simplest mechanisms to explain decreased mitochondrial pyruvate oxidation in cancer cells, a loss of mitochondrial pyruvate import, has been observed repeatedly over the past 40 years. This process has been impossible to study at a molecular level until recently, however, as the identities of the protein(s) that mediate mitochondrial pyruvate uptake were unknown.
  7. The mitochondrial pyruvate carrier (MPC) as a multimeric complex that is necessary for efficient mitochondrial pyruvate uptake. The MPC contains two distinct proteins, MPC1 and MPC2; the absence of either leads to a loss of mitochondrial pyruvate uptake and utilization in yeast, flies, and mammalian cells.

A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

John C. Schell, Kristofor A. Olson, Lei Jiang, Amy J. Hawkins, et al.
Molecular Cell Nov 6, 2014; 56: 400–413.
http://dx.doi.org/10.1016/j.molcel.2014.09.026

In addition to the above, the following study has therapeutic importance:

Glycolysis has become a target of anticancer strategies. Glucose deprivation is sufficient to induce growth inhibition and cell death in cancer cells. The increased glucose transport in cancer cells has been attributed primarily to the upregulation of glucose transporter 1 (Glut1),  1 of the more than 10 glucose transporters that are responsible for basal glucose transport in almost all cell types. Glut1 has not been targeted until very recently due to the lack of potent and selective inhibitors.

First, Glut1 antibodies were shown to inhibit cancer cell growth. Other Glut1 inhibitors and glucose transport inhibitors, such as fasentin and phloretin, were also shown to be effective in reducing cancer cell growth. A group of inhibitors of glucose transporters has been recently identified with IC50 values lower than 20mmol/L for inhibiting cancer cell growth. However, no animal or detailed mechanism studies have been reported with these inhibitors.

Recently, a small molecule named STF-31 was identified that selectively targets the von Hippel-Lindau (VHL) deficient kidney cancer cells. STF-31 inhibits VHL deficient cancer cells by inhibiting Glut1. It was further shown that daily intraperitoneal injection of a soluble analogue of STF-31 effectively reduced the growth of tumors of VHL-deficient cancer cells grafted on nude mice. On the other hand, STF-31 appears to be an inhibitor with a narrow cell target spectrum.

These investigators recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like mechanism. These compounds target Glut1 and are efficacious in vivo as anticancer agents. A novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule.

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

Yi Liu, Yanyan Cao, Weihe Zhang, Stephen Bergmeier, et al.
Mol Cancer Ther Aug 2012; 11(8): 1672–82
http://dx.doi.org://10.1158/1535-7163.MCT-12-0131

Alterations in cellular metabolism are among the most consistent hallmarks of cancer. These investigators have studied the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, M-type lactate dehydrogenase levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.

The results are consistent with a functional connection between alterations in glucose metabolism and mitochondrial physiology in cancer. The data also reflect that the dependency of tumor cells on glucose metabolism is a liability for these cells under limited-oxygen conditions. Interfering with LDH-A activity as a means of blocking pyruvate to lactate conversion could be exploited therapeutically. Because individuals with complete deficiency of LDH-A do not show any symptoms under ordinary circumstances, the genetic data suggest that inhibition of LDH-A activity may represent a relatively nontoxic approach to interfere with tumor growth.

Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance

Valeria R. Fantin Julie St-Pierre and Philip Leder
Cancer Cell Jun 2006; 9: 425–434.
http://dx.doi.org:/10.1016/j.ccr.2006.04.02

The widespread clinical use of positron-emission tomography (PET) for the detection of aerobic glycolysis in tumors and recent findings have rekindled interest in Warburg’s theory. Studies on the physiological changes in malignant conversion provided a metabolic signature for the different stages of tumorigenesis; during tumorigenesis, an increase in glucose uptake and lactate production have been detected. The fully transformed state is most dependent on aerobic glycolysis and least dependent on the mitochondrial machinery for ATP synthesis.

Tumors ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumors and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumors leads to enhanced, oxygen-independent glucose usage and a lactate based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualized cancer therapy based on the determination of metabolic changes in tumors that might enable the targeted inhibition of invasion and metastasis.

Other important links between cancer-causing genes and glucose metabolism have been already identified. Activation of the oncogenic kinase Akt has been shown to stimulate glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Such tumor cells have been shown to be dependent on glucose because the ability to induce fatty acid oxidation in response to glucose deprivation is impaired by activated Akt. In addition, AMP-activated protein kinase (AMPK) has been identified as a link between glucose metabolism and the cell cycle, thereby implicating p53 as an essential component of metabolic cell-cycle control.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted

S Langbein, M Zerilli, A zur Hausen, W Staiger, et al.
British Journal of Cancer (2006) 94, 578–585.
http://dx.doi.org:/10.1038/sj.bjc.6602962

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both contributing toapoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DJm, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Cancer progression and its resistance to treatment depend, at least in part, on suppression of apoptosis. Although mitochondria are recognized as regulators of apoptosis, their importance as targets for cancer therapy has not been adequately explored or clinically exploited. In 1930, Warburg suggested that mitochondrial dysfunction in cancer results in a characteristic metabolic phenotype, that is, aerobic glycolysis (Warburg, 1930). Positron emission tomography (PET) imaging has now confirmed that most malignant tumors have increased glucose uptake and metabolism. This bioenergetic feature is a good marker of cancer but has not been therapeutically pursued..

The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolic electrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness  make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.

A Mitochondria-K+ Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth

Sebastien Bonnet, Stephen L. Archer, Joan Allalunis-Turner, et al.

Cancer Cell Jan 2007; 11: 37–51.
http://dx.doi.org:/10.1016/j.ccr.2006.10.020

Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state. Aim: To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Conclusion: Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-β, a cell transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic antileukemic drugs such as STI571 (Gleevec).  Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high efficacy nongenetic novel targets for cancer therapies.

A Metabolic Hypothesis of Cell Growth and Death in Pancreatic Cancer

Laszlo G. Boros, Wai-Nang Paul Lee, and Vay Liang W. Go
Pancreas 2002; 24(1):26–33

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets.

MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoral heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs.

MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.

Clear cell carcinoma (ccRCC) is the commonest subtype of renal cell carcinoma, accounting for 80% of cases. These tumors are highly resistant to cytotoxic chemotherapy and until recently, systemic treatment options for advanced ccRCC were limited to cytokine based therapies, such as interleukin-2 and interferon-α. Recently, anti-angiogenic drugs and mTOR inhibitors, all targeting the HIF–VEGF axis which is activated in up to 91% of ccRCCs through loss of the VHL tumor suppressor gene [1], have been shown to be effective in metastatic ccRCC [2–5]. Although these drugs increase overall survival to more than 2 years [6], resistance invariably occurs, making the identification of new molecular targets a major clinical need to improve outcomes in patients with metastatic ccRCC.

Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target

Marco Gerlinger, Claudio R Santos, Bradley Spencer-Dene, et al.
J Pathol 2012; 227: 146–156
http://dx.doi.org:/10.1002/path.4006

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.

Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and a catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of >90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.

O2 concentrations are significantly reduced in many human cancers compared with the surrounding normal tissue. The median PO2 in breast cancers is 10 mmHg, as compared with65 mmHg in normal breast tissue. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy.

HIF-1 is a transcription factor that consists of an O2 regulated HIF-1a and a constitutively expressed HIF-1b subunit. In well-oxygenated cells, HIF-1a is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and a-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts. Prolylhydroxylated HIF-1a is bound by the von Hippel–Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1a for proteasomal degradation (Figure 1a). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases.

The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 – up or down – results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity.

HIF-1: upstream and downstream of cancer metabolism

Gregg L Semenza
Current Opinion in Genetics & Development 2010, 20:51–56

This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Tony Hunter and Richard Marais

http://dx.doi.org:/10.1016/j.gde.2009.10.009

Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1a overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a  variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.

Intratumoral hypoxia The majority of locally advanced solid tumors contain regions of reduced oxygen availability. Intratumoral hypoxia results when cells are located too far from a functional blood vessel for diffusion of adequate amounts of O2 as a result of rapid cancer cell proliferation and the formation of blood vessels that are structurally and functionally abnormal. In the most extreme case, O2 concentrations are below those required for survival, resulting in cell death and establishing a selection for cancer cells in which apoptotic pathways are inactivated, anti-apoptotic pathways are activated, or invasion/metastasis pathways that promote escape from the hypoxic microenvironment are activated. This hypoxic adaptation may arise by alterations in gene expression or by mutations in the genome or both and is associated with reduced patient survival.

Hypoxia-inducible factor 1 (HIF-1) The expression of hundreds of genes is altered in each cell exposed to hypoxia. Many of these genes are regulated by HIF-1. HIF-1 is a heterodimer formed by the association of an O2-regulated HIF1a subunit with a constitutively expressed HIF-1b subunit. The structurally and functionally related HIF-2a protein also dimerizes with HIF-1b and regulates an overlapping battery of target genes. Under nonhypoxic conditions, HIF-1a (as well as HIF-2a) is subject to O2-dependent prolyl hydroxylation and this modification is required for binding of the von Hippel–Lindau tumor suppressor protein (VHL), which also binds to Elongin C and thereby recruits a ubiquitin ligase complex that targets HIF-1a for ubiquitination and proteasomal degradation. Under hypoxic conditions, the rate of hydroxylation and ubiquitination declines, resulting in accumulation of HIF-1a. Immunohistochemical analysis of tumor biopsies has revealed high levels of HIF-1a in hypoxic but viable tumor cells surrounding areas of necrosis.

Genetic alterations in cancer cells increase HIF-1 activity In the majority of clear-cell renal carcinomas, VHL function is lost, resulting in constitutive activation of HIF-1. After re-introduction of functional VHL, renal carcinoma cell lines are no longer tumorigenic, but can be made tumorigenic by expression of HIF2a in which the prolyl residues that are subject to hydroxylation have been mutated. In addition to VHL loss-of-function, many other genetic alterations that inactivate tumor suppressors

Evaluation of HIF-1 inhibitors as anticancer agents

Gregg L. Semenza
Drug Discovery Today Oct 2007; 12(19/20).
http://dx.doi.org:/10.1016/j.drudis.2007.08.006

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by upregulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.

Although many anticancer regimens have been introduced to date, their survival benefits are negligible, which is the reason that a more innovative treatment is required. Basically, the identification of the specific molecular features of tumor promotion has allowed for rational drug discovery in cancer treatment, and drugs have been screened based upon the modulation of specific molecular targets in tumor cells. Target-based drugs should satisfy the following two conditions.

First, they must act by a described mechanism.

Second, they must reduce tumor growth in vivo, associated with this mechanism.

Many key factors have been found to be involved in the multiple steps of cell growth signal-transduction pathways. Targeting these factors offers a strategy for preventing tumor growth; for example, competitors or antibodies blocking ligand–receptor interaction, and receptor tyrosine kinase inhibitors, downstream pathway inhibitors (i.e., RAS farnesyl transferase inhibitors, mitogen-activated protein kinase and mTOR inhibitors), and cell-cycle arresters (i.e., cyclin-dependent kinase inhibitors) could all be used to inhibit tumor growth.

In addition to the intracellular events, tumor environmental factors should be considered to treat solid tumors. Of these, hypoxia is an important cancer-aggravating factor because it contributes to the progression of a more malignant phenotype, and to the acquisition of resistance to radiotherapy and chemotherapy. Thus, transcription factors that regulate these hypoxic events are good targets for anticancer therapy and in particular HIF-1 is one of most compelling targets. In this paper, we introduce the roles of HIF-1 in tumor promotion and provide a summary of new anticancer strategies designed to inhibit HIF-1 activity.

New anticancer strategies targeting HIF-1

Eun-Jin Yeo, Yang-Sook Chun, Jong-Wan Park
Biochemical Pharmacology 68 (2004) 1061–1069
http://dx.doi.org:/10.1016/j.bcp.2004.02.040

Classical work in tumor cell metabolism focused on bioenergetics, particularly enhanced glycolysis and suppressed oxidative phosphorylation (the ‘Warburg effect’). But the biosynthetic activities required to create daughter cells are equally important for tumor growth, and recent studies are now bringing these pathways into focus. In this review, we discuss how tumor cells achieve high rates of nucleotide and fatty acid synthesis, how oncogenes and tumor suppressors influence these activities, and how glutamine metabolism enables macromolecular synthesis in proliferating cells.

Otto Warburg’s demonstration that tumor cells rapidly use glucose and convert the majority of it to lactate is still the most fundamental and enduring observation in tumor metabolism. His work, which ushered in an era of study on tumor metabolism focused on the relationship between glycolysis and cellular bioenergetics, has been revisited and expanded by generations of tumor biologists. It is now accepted that a high rate of glucose metabolism, exploited clinically by 18FDGPET scanning, is a metabolic hallmark of rapidly dividing cells, correlates closely with transformation, and accounts for a significant percentage of ATP generated during cell proliferation. A ‘metabolic transformation’ is required for tumorigenesis. Research over the past few years has reinforced this idea, revealing the conservation of metabolic activities among diverse tumor types, and proving that oncogenic mutations can promote metabolic autonomy by driving nutrient uptake to levels that often exceed those required for cell growth and proliferation.

In order to engage in replicative division, a cell must duplicate its genome, proteins, and lipids and assemble the components into daughter cells; in short, it must become a factory for macromolecular biosynthesis. These activities require that cells take up extracellular nutrients like glucose and glutamine and allocate them into metabolic pathways that convert them into biosynthetic precursors (Figure 1). Tumor cells can achieve this phenotype through changes in the expression of enzymes that determine metabolic flux rates, including nutrient transporters and enzymes [8– 10]. Current studies in tumor metabolism are revealing novel mechanisms for metabolic control, establishing which enzyme isoforms facilitate the tumor metabolic phenotype, and suggesting new targets for cancer therapy.

The ongoing challenge in tumor cell metabolism is to understand how individual pathways fit together into the global metabolic phenotype of cell growth. Here we discuss two biosynthetic activities required by proliferating tumor cells: production of ribose-5 phosphate for nucleotide biosynthesis and production of fatty acids for lipid biosynthesis. Nucleotide and lipid biosynthesis share three important characteristics.

  • First, both use glucose as a carbon source.
  • Second, both consume TCA cycle intermediates, imposing the need for a mechanism to replenish the cycle.
  • Third, both require reductive power in the form of NADPH.

In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1, not shown). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

The Tumor Microenvironment Selects for Altered Metabolism One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

Blood vessels recruited to the tumor microenvironment, however, are disorganized, may not deliver blood effectively, and therefore do not completely alleviate hypoxia (reviewed in Gatenby and Gillies, 2004). The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment. Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF. Other molecular mechanisms are likely to be important, such as the metabolic changes induced by oncogene activation and tumor suppressor loss.

Oncogene Activation Drives Changes in Metabolism Not only may the tumor microenvironment select for a deranged metabolism, but oncogene status can also drive metabolic changes. Since Warburg’s time, the biochemical study of cancer metabolism has been overshadowed by efforts to identify the mutations that contribute to cancer initiation and progression. Recent work, however, has demonstrated that the key components of the Warburg effect—

  • increased glucose consumption,
  • decreased oxidative phosphorylation, and
  • accompanying lactate production—
  • are also distinguishing features of oncogene activation.

The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Ramanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes.

Cancer Cell Metabolism: Warburg & Beyond

Hsu PP & Sabatini DM
Cell  Sep 5, 2008; 134, 703-705
http://dx.doi.org:/10.1016/j.cell.2008.08.021

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty  acid   synthesis   and   TCA   cycle   glucose   oxidation. This  primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment.

Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros, Daniel J. Brackett and George G. Harrigan
Current Cancer Drug Targets, 2003, 3, 447-455 447

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150 kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, while silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans

Daniel K. Bricker, Eric B. Taylor, John C. Schell, Thomas Orsak, et al.
Science Express 24 May 2012
http://dx.doi.org:/10.1126/science.1218099

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Review ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference 

Sara Tomaselli, Barbara Bonamassa, Anna Alisi, Valerio Nobili, Franco Locatelli and Angela Gallo
Int. J. Mol. Sci. 19 Nov 2013; 14, 22796-22816 http://dx.doi.org:/10.3390/ijms141122796

The fermented wheat germ extract (FWGE) nutraceutical (Avemar™), manufactured under “good manufacturing practice” conditions and, fulfilling the self-affirmed “generally recognized as safe” status in the United States, has been approved as a “dietary food for special medical purposes for cancer patients” in Europe. In this paper, we report the adjuvant use of this nutraceutical in the treatment of high-risk skin melanoma patients. Methods: In a randomized, pilot, phase II clinical trial, the efficacy of dacarbazine (DTIC)-based adjuvant chemotherapy on survival parameters of melanoma patients was compared to that of the same treatment supplemented with a 1-year long administration of FWGE. Results: At the end of an additional 7-year-long follow-up period, log-rank analyses (Kaplan-Meier estimates) showed significant differences in both progression-free (PFS) and overall survival (OS) in favor of the FWGE group. Mean PFS: 55.8 months (FWGE group) versus 29.9 months (control group), p  0.0137. Mean OS: 66.2 months (FWGE group) versus 44.7 months (control group), p < 0.0298. Conclusions: The inclusion of Avemar into the adjuvant protocols of high-risk skin melanoma patients is highly recommended.

Adjuvant Fermented Wheat Germ Extract (Avemar™) Nutraceutical Improves Survival of High-Risk Skin Melanoma Patients: A Randomized, Pilot, Phase II Clinical Study with a 7-Year Follow-Up

LV Demidov, LV Manziuk, GY Kharkevitch, NA Pirogova, and EV Artamonova
Cancer Biotherapy & Radiopharmaceuticals 2008; 23(4)
http://dx.doi.org:/10.1089/cbr.2008.0486

Cancer cells possess unique metabolic signatures compared to normal cells, including shifts in aerobic glycolysis, glutaminolysis, and de novo biosynthesis of macromolecules. Targeting these changes with agents (drugs and dietary components) has been employed as strategies to reduce the complications associated with tumorigenesis. This paper highlights the ability of several food components to suppress tumor-specific metabolic pathways, including increased expression of glucose transporters, oncogenic tyrosine kinase, tumor-specific M2-type pyruvate kinase, and fatty acid synthase, and the detection of such effects using various metabonomic technologies, including liquid chromatography/mass spectrometry (LC/MS) and stable isotope-labeled MS. Stable isotope-mediated tracing technologies offer exciting opportunities for defining specific target(s) for food components. Exposures, especially during the early transition phase from normal to cancer, are critical for the translation of knowledge about food components into effective prevention strategies. Although appropriate dietary exposures needed to alter cellular metabolism remain inconsistent and/or ill-defined, validated metabonomic biomarkers for dietary components hold promise for establishing effective strategies for cancer prevention.

Bioactive Food Components and Cancer-Specific Metabonomic Profiles

Young S. Kim and John A. Milner
Journal of Biomedicine and Biotechnology 2011, Art ID 721213, 9 pages
http://dx.doi.org:/10.1155/2011/721213

This reviewer poses the following observation.  The importance of the pyridine nucleotide reduced/oxidized ratio has not been alluded to here, but the importance cannot be understated. It has relevance to the metabolic functions of anabolism and catabolism of the visceral organs.  The importance of this has ties to the pentose monophosphate pathway. The importance of the pyridine nucleotide transhydrogenase reaction remains largely unexplored.  In reference to the NAD-redox state, the observation was made by Nathan O. Kaplan that the organs may be viewed with respect to their primary functions in anabolic or high energy catabolic activities. Thus we find that the endocrine organs are largely tied to anabolic functioning, and to NADP, whereas cardiac and skeletal muscle are highly dependent on NAD. The consequence of this observed phenomenon appears to be related to a difference in the susceptibility to malignant transformation.  In the case of the gastrointestinal tract, the rate of turnover of the epithelium is very high. However, with the exception of the liver, there is no major activity other than cell turnover. In the case of the liver, there is a major commitment to synthesis of lipids, storage of fuel, and synthesis of proteins, which is largely anabolic, but there is also a major activity in detoxification, which is not.  In addition, the liver has a double circulation. As a result, a Zahn infarct is uncommon.  Now we might also consider the heart.  The heart is a muscle syncytium with a high need for oxygen.  Cutting of the oxygen supply makes the myocytes vulnerable to ischemic insult and abberant rhythm abnormalities.  In addition, the cardiomyocyte can take up lactic acid from the circulation for fuel, which is tied to the utilization of lactate from vigorous skeletal muscle activity.  The skeletal muscle is tied to glycolysis in normal function, which has a poor generation of ATP, so that the recycling of excess lactic acid is required by cardiac muscle and hepatocytes.  This has not been a part of the discussion, but this reviewer considers it important to remember in considering the organ-specific tendencies to malignant transformation.

Comment (Aurelian Udristioiu):

Otto Warburg observed that many cancers lose their capacity for mitochondrial respiration, limiting ATP production to anaerobic glycolytic pathways. The phenomenon is particularly prevalent in aggressive malignancies, most of which are also hypoxic [1].
Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines [2-3]. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines [4].
Hypoxia reduces the ability of cells to maintain their energy levels, because less ATP is obtained from glycolysis than from oxidative phosphorylation. Cells adapt to hypoxia by activating the expression of mutant genes in glycolysis.
-Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.
The most direct induction of apoptosis caused by hypoxia is determined by the inhibition of the electron carrier chain from the inner membrane of the mitochondria. The lack of oxygen inhibits the transport of protons and thereby causes a decrease in membrane potential. Cell survival under conditions of mild hypoxia is mediated by phosphoinositide-3 kinase (PIK3) using severe hypoxia or anoxia, and then cells initiate a cascade of events that lead to apoptosis [5].
After DNA damage, a very important regulator of apoptosis is the p53 protein. This tumor suppressor gene has mutations in over 60% of human tumors and acts as a suppressor of cell division. The growth-suppressive effects of p53 are considered to be mediated through the transcriptional trans-activation activity of the protein. In addition to the maturational state of the clonal tumor, the prognosis of patients with CLL is dependent of genetic changes within the neoplastic cell population.

1.Warburg O. On the origin of cancer cells. Science 1956; 123 (3191):309-314
PubMed Abstract ; Publisher Full Text

2.Giardina TM, Steer JH, Lo SZ, Joyce DA. Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages. Biochim Biophys Acta 2008, 1777(2):118-129. PubMed Abstract | Publisher Full Text

3. Horimoto M, Resnick MB, Konkin TA, Routhier J, Wands JR, Baffy G. Expression of uncoupling protein-2 in human colon cancer. Clin Cancer Res 2004; 10 (18 Pt1):6203-6207. PubMed Abstract | Publisher Full Text

4. Randle PJ, England PJ, Denton RM. Control of the tricarboxylate cycle and it interactions with glycolysis during acetate utilization in rat heart. Biochem J 1970; 117(4):677-695. PubMed Abstract | PubMed Central Full Text

5. Gillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008; 49(Suppl 2):24S-42S. PubMed Abstract | Publisher Full Text

Shortened version of Comment –

Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines.
Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.

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Manipulate Signaling Pathways

Writer and Curator: Larry H Bernstein, MD, FCAP 

 

7.6  Manipulate Signaling Pathways

7.6.1 The Dynamics of Signaling as a Pharmacological Target

7.6.2 A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

7.6.3 IQGAPs choreograph cellular signaling from the membrane to the nucleus

7.6.4 Signaling cell death from the endoplasmic reticulum stress response

7.6.5 An Enzyme that Regulates Ether Lipid Signaling Pathways in Cancer Annotated by Multidimensional Profiling

7.6.6 Peroxisomes – A Nexus for Lipid Metabolism and Cellular Signaling

7.6.7 A nexus for cellular homeostasis- the interplay between metabolic and signal transduction pathways

7.6.8 Mechanisms-of-intercellular-signaling

7.6.9 Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

 

 

7.6.1 The Dynamics of Signaling as a Pharmacological Target

Marcelo Behar, Derren Barken, Shannon L. Werner, Alexander Hoffmann
Cell  10 Oct 2013; 155(2):448–461
http://dx.doi.org/10.1016/j.cell.2013.09.018

Highlights

  • Drugs targeting signaling hubs may block specific dynamic features of the signal
  • Specific inhibition of dynamic features may introduce pathway selectivity
  • Phase space analysis reveals principles for drug targeting signaling dynamics
  • Based on these principles, NFκB dynamics can be manipulated with specificity

Summary

Highly networked signaling hubs are often associated with disease, but targeting them pharmacologically has largely been unsuccessful in the clinic because of their functional pleiotropy. Motivated by the hypothesis that a dynamic signaling code confers functional specificity, we investigated whether dynamic features may be targeted pharmacologically to achieve therapeutic specificity. With a virtual screen, we identified combinations of signaling hub topologies and dynamic signal profiles that are amenable to selective inhibition. Mathematical analysis revealed principles that may guide stimulus-specific inhibition of signaling hubs, even in the absence of detailed mathematical models. Using the NFκB signaling module as a test bed, we identified perturbations that selectively affect the response to cytokines or pathogen components. Together, our results demonstrate that the dynamics of signaling may serve as a pharmacological target, and we reveal principles that delineate the opportunities and constraints of developing stimulus-specific therapeutic agents aimed at pleiotropic signaling hubs.

http://www.cell.com/cms/attachment/2021777732/2041663648/fx1.jpg

Intracellular signals link the cell’s genome to the environment. Misregulation of such signals often cause or exacerbate disease (Lin and Karin, 2007 and Weinberg, 2007) (so-called “signaling diseases”), and their rectification has been a major focus of biomedical and pharmaceutical research (Cohen, 2002Frelin et al., 2005 and Ghoreschi et al., 2009). For the identification of therapeutic targets, the concept of discrete signaling pathways that transmit intracellular signals to connect cellular sensor/receptors with cellular core machineries has been influential. In this framework, molecular specificity of therapeutic agents correlates well with their functional or phenotypic specificity. However, in practice, clinical outcomes for many drugs with high molecular specificity has been disappointing (e.g., inhibitors of IKK, MAPK, and JNK; Berger and Iyengar, 2011DiDonato et al., 2012Röring and Brummer, 2012 and Seki et al., 2012).

Many prominent signaling mediators are functionally pleiotropic, playing roles in multiple physiological functions (Chavali et al., 2010 and Gandhi et al., 2006). Indeed, signals triggered by different stimuli often travel through shared network segments that operate as hubs before reaching the effectors of the cellular response (Bitterman and Polunovsky, 2012 and Gao and Chen, 2010). Hubs’ inherent pleiotropy means that their inhibition may have broad and likely undesired effects (Karin, 2008Berger and Iyengar, 2011,Force et al., 2007Oda and Kitano, 2006 and Zhang et al., 2008); this is a major obstacle for the efficacy of drugs targeting prominent signaling hubs such as p53, MAPK, or IKK.

Recent studies have begun to address how signaling networks generate stimulus-specific responses (Bardwell, 2006Haney et al., 2010Hao et al., 2008 and Zalatan et al., 2012). For example, the activity of some pleiotropic kinases may be steered to particular targets by scaffold proteins (Park et al., 2003,Schröfelbauer et al., 2012 and Zalatan et al., 2012). Alternatively, or in addition, some signaling hubs may rely on stimulus-specific signal dynamics to activate selective downstream branches in a stimulus-specific manner in a process known as temporal or dynamic coding or multiplexing (Behar and Hoffmann, 2010,Chalmers et al., 2007Hoffmann et al., 2002Kubota et al., 2012Marshall, 1995 and Purvis et al., 2012;Purvis and Lahav, 2013Schneider et al., 2012 and Werner et al., 2005).

Although the importance of signaling scaffolds and their pharmacological promise is widely appreciated (Klussmann et al., 2008 and Zalatan et al., 2012) and isolated studies have altered the stimulus-responsive signal dynamics (Purvis et al., 2012Park et al., 2003Sung et al., 2008 and Sung and Simon, 2004), the capacity for modulating signal dynamics for pharmacological gain has not been addressed in a systematic manner. In this work, we demonstrate by theoretical means that, when signal dynamics are targeted, pharmacological perturbations can produce stimulus-selective results. Specifically, we identify combinations of signaling hub topology and input-signal dynamics that allow for pharmacological perturbations with dynamic feature-specific or input-specific effects. Then, we investigate stimulus-specific drug targeting in the IKK-NFκB signaling hub both in silico and in vivo. Together, our work begins to define the opportunities for pharmacological targeting of signaling dynamics to achieve therapeutic specificity.

Dynamic Signaling Hubs May Be Manipulated to Mute Specific Signals

Previous work has shown how stimulus-specific signal dynamics may allow a signaling hub to selectively route effector functions to different downstream branches (Behar et al., 2007). Here, we investigated the capacity of simple perturbations to kinetic parameters (caused for example by drug treatments) to produce stimulus-specific effects. For this, we examined a simple model of an idealized signaling hub (Figure 1A), reminiscent of the NFκB p53 or of MAPK signaling modules. The hub X reacts with strong but transient activity to stimulus S1 and sustained, slowly rising activity to stimulus S2. These stimulus-specific signaling dynamics are decoded by two effector modules, regulating transcription factors TF1 and TF2. TF1, regulated by a strongly adaptive negative feedback, is sensitive only to fast-changing signals, whereas TF2, regulated by a slowly activating two-state switch, requires sustained signals for activation (Figure 1B). We found it useful to characterize the X, TF1, and TF2 responses in terms of two dynamic features, namely the maximum early amplitude (“E,” time < 15′) and the average late amplitude (“L,” 15′ < t < 6 hr). These features, calculated using a mathematical model of the network (see Experimental Procedures) show good fidelity and specificity (Komarova et al., 2005) (Figure 1C), as S1 causes strong activation of TF1 with minimal crosstalk to TF2, and vice versa for S2.

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Figure 1. Pharmacologic Perturbations with Stimulus-Specific Effects

(A) A negative-feedback module transduces input signals S1 and S2, producing outputs that are decoded by downstream effectors circuits that may distinguish between different dynamics.

(B) Unperturbed dynamics of X, TF1, and TF2 in response to S1 (red) and S2 (blue). Definition of early (E) and late (L) parts of the signal is indicated.

(C) Specificity and fidelity of E and L for TF1 and TF2, as defined in Komarova et al., 2005).

(D) Partial inhibition of X activation (A) abolishes the response to S1, but not S2, whereas a perturbation targeting the feedback regulator (FBR) suppresses the response to S2, but not S1.

(E) Perturbation phenotypes defined as difference between unperturbed and perturbed values of the indicated quantities (arbitrary scales for X, TF1, and TF2). Perturbation A inhibits E and TF1, but not TF2; perturbation FBR inhibits L and TF2, but not TF1.

(F) Virtual screening pipeline showing the experimental design and the two analysis branches for characterizing feature- and input-specific effects.

See also in Experimental Procedures and Table S1.

Seeking simple (affecting a single reaction) perturbations that selectively inhibit signaling by S1 or S2, we found that perturbation A, partially inhibiting the activation of X, was capable of suppressing hub activity in response to a range of S1 amplitudes while still allowing for activity in response to S2 (Figure 1D). Consequently, this perturbation significantly reduced TF1 activity in response to S1 but had little effect on TF2 activity elicited by S2. We also found that the most effective way to inhibit S2 signaling was by targeting the deactivation of negative feedback regulator Y (FBR). This perturbation caused almost complete abrogation of late X activity yet allows for significant levels of early activity. As a result, TF2 was nearly completely abrogated in response to S2, but stimulus S1 still produced a solid TF1 response. The early (E) and late (L) amplitudes could be used to quantify the input-signal-specific effects of these perturbations (Figure 1E).

This numerical experiment showed that it is possible to selectively suppress transient or sustained dynamic signals transduced through a common negative-feedback-containing signaling hub. Moreover, the dynamic features E and L could be independently inhibited. To study how prevalent such opportunities for selective inhibition are, we established a computational pipeline for screening reaction perturbations within multiple network topologies and in response to multiple dynamic input signals; the simulation results were analyzed to identify cases of either “input-signal-specific” inhibition or “dynamic feature-specific” inhibition (Figure 1F).

A Computational Screen to Identify Opportunities for Input-Signal-Specific Inhibition

The computational screen involved small libraries of one- and two-component regulatory modules and temporal profiles of input signals (Figure 2A), both commonly found in intracellular signaling networks. All modules (M1–M7, column on left) contained a species X that, upon stimulation by an input signal, is converted into an active form X (the output) that propagates the signal to downstream effectors. One-component modules included a reversible two-state switch (M1) and a three-state cycle with a refractory state (M2). Two-component modules contained a species Y that, upon activation via a feedback (M3 and M5) or feedforward (M4 and M6) loop, either deactivates X (M3 and M4) or inhibits (M5 and M6) its activation. We also included the afore-described topology that mimics the IκB-NFκB or the Mdm2-p53 modules (M7). Mathematical descriptions may be found in the Experimental Procedures. Although many biological signaling networks may conform to one of these simple topologies, others may be abstracted to one that recapitulates the physiologically relevant emergent properties

Figure 2. A Virtual Screen for Stimulus Specificity in Pharmacologic Perturbations

(A) Signaling modules (left) and input library (top) used in the screen. Dotted lines indicate enzymatic reactions (perturbation names indicated in letter code). Time courses of hub activity for each module/input combination for the unperturbed (black) and perturbed cases (blue indicates a decrease, red an increase in parameter value).

(B) Relative sensitivity of the stimulus response to the indicated perturbation (defined as the perturbation’s effect on the area under the curve), normalized per row.

See also Experimental ProceduresFigure S1, and Tables S2 and S3.

The library of stimuli (S1–S10; Figure 2A, top row) comprises ten input functions with different combinations of “fast” and “slow” initiation and decay phases (see Experimental Procedures). The virtual screen was performed by varying the kinetic parameter for each reaction over a range of values, thereby modeling simple perturbations of different strengths and recording the temporal profile of X abundance. To quantify stimulus-specific inhibition, we measured the area under the normalized dose-response curves (time average of X versus perturbation dose) for each module-input combination (Experimental ProceduresFigure 2B, and Figure S1 available online).

Phase Space Analysis Reveals Underlying Regulatory Principles

To understand the origin of dynamic feature-specific inhibition, we investigated the perturbation effects analytically on each module’s phase space, i.e., the space defined by X∗ and Y∗ quasi-equilibrium surfaces (Figures 4 and S4). These surfaces (“q.e. surfaces”) represent the dose response of X∗ as a function of Y∗ and a stationary input signal S (“X surface”) and the dose response of Y∗ as a function of X∗ and S (“Y surface”) (Figure 4A). The points at which the surfaces intersect correspond to the concentrations of X∗ and Y∗ in equilibrium for a given value of S. In the basal state, when S is low, the system is resting at an equilibrium point close to the origin of coordinates. When S increases, the concentrations of X∗ and Y∗ adjust until the signal settles at some stationary value (Figure 4A). Gradually, changing input signals cause the concentrations to follow trajectories close to the q.e. surfaces (quasi-equilibrium dynamics), following the line defined by the intersection of the surfaces (“q.e. line”) in the extreme of infinitely slow inputs. Fast-changing stimuli drive the system out of equilibrium, causing the trajectories to deviate markedly from the q.e. surfaces.

Two main principles emerged: (1) perturbations that primarily affect the shape of a q.e. surface tend to affect steady-state levels or responses that evolve close to quasi-equilibrium, and (2) perturbations that primarily affect the balance of timescales (X, Y activation, and S) tend to affect transient out-of-equilibrium parts of the response. These principles reflect the fact that out-of-equilibrium parts of a signal are largely insensitive to the precise shape of the underlying dose-response surfaces (they may still be bounded by them) but depend on the balance between the timescales of the biochemical processes involved. Perturbation of these balances affects how a system approaches steady state (thus affecting out-of-equilibrium and quasi-equilibrium dynamics), but not steady-state levels. To illustrate these principles, we present selected results for modules M3 and M4 and discuss additional cases in the supplement (Figure S3).

Detailed Analysis of Modules M3 and M4, Related to Figure 4

Time courses and projections of the phase space for modules M3 and M4. Color coding similar to Figure 4.

In the feedback-based modules (M3 and M5), the early peak of activity in response to rapidly changing signals is an out-of-equilibrium feature that occurs when the timescale of Y activation is significantly slower than that of X. Under these conditions, the concentration of X increases rapidly (out of equilibrium) before decaying along the X surface (in quasi-equilibrium) as more Y gets activated (Figure 4A, parameters modified to better illustrate the effects being discussed; see Table S2). For input signals that settle at some stationary level of S, Y activation eventually catches up and the concentration of X settles at the equilibrium point where the X and Y curves intersect. Gradually changing signals allow X and Yactivation to continuously adapt, and the system evolves closer to the q.e. line.

In such modules, perturbation A (X activation) changes both the shape of the q.e. surface for X and the kinetics of activation. When in the unperturbed system Y saturates, perturbation A primarily reduces Xsteady-state level (Figures 4B and 4C, left and center). When Y does not saturate in the unperturbed system, the primary effect is the reduced activation kinetics. Thus the perturbation affects the out-of-equilibrium peak (Figures 4B and 4C, center and right), with only minor reduction of steady-state levels (especially when Y’s dose response respect to X is steep). The transition from saturated to not-saturated feedback (as well as the perturbation strength) underlies the dose-dependent switch from L to E observed in the screen. In both saturated and unsaturated regimes, the shift in the shape of the surfaces does change the q.e. line and thus affects responses occurring in quasi-equilibrium. In contrast, perturbation of the feedback recovery (FBR) shifts the Y surface vertically (Figure 4D), specifically affecting the steady-state levels and late signaling; the effect on Y kinetics is limited because the reaction is relatively slow. Perturbation FBA also shifts the Y surface, but the net effect is less specific because the associated increase in the rate of Y activation tends to equalize X and Y kinetics affecting also the out-of-equilibrium peak.

In resting cells, NFκB is held inactive through its association with inhibitors IκBα, β, and ε. Upon stimulation, these proteins are phosphorylated by the kinase IKK triggering their degradation. Free nuclear NFκB activates the expression of target genes, including IκB-encoding genes, which thereby provide negative feedback (Figure 5A). The IκB-NFκB-signaling module is a complex dynamic system; however, by abstracting the control mechanism to its essentials, we show below that the above-described principles can be applied profitably.

IκB-NFκB signaling module

IκB-NFκB signaling module

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Figure 5. Modulating NFκB Signaling Dynamics

(A) The IκB-NFκB signaling module.

(B) Equilibrium dose-response relationship for NFκB versus IKK.

(C) Three IKK curves representative of three stimulation regimes; TNFc (red), TNFp (green), and LPS (blue) function as inputs into the model, which computes the corresponding NFκB activity dynamics (bottom). The quasi-equilibrium line (black) was obtained by transforming the IKK temporal profiles by the dose response in (B). Deviation from the quasi-equilibrium line for the TNF response indicates out-of-equilibrium dynamics.

(D) Coarse-grained model of the IκB-NFκB module and predicted effects of perturbations.

(E) Selected perturbations with specific effects on out-of-equilibrium (top three) or steady state (bottom two). (Left to right) Feature maps in the E-L space (E: t < 60 ′, L: 120′ < t < 300′), tangent angle at the unperturbed point (θ > 0 indicates L is more suppressed than E and vice versa), and time courses (green, TNF chronic; red, TNF pulse; blue, LPS). Only inhibitory perturbations are shown. Additional perturbations are shown in Figure S4.

See also Experimental Procedures and Table S7.

Here, we delineate the potential of achieving stimulus-specific inhibition when targeting molecular reactions within pleiotropic signaling hubs. We found that it is theoretically possible to design perturbations that (1) selectively attenuate signaling in response to one stimulus but not another, (2) selectively attenuate undesirable features of dynamic signals or enhance desirable ones, or (3) remodulate output signals to fit a dynamic profile normally associated with a different stimulus.

These opportunities—not all of them possible for every signaling module topology or biological scenario—are governed by two general principles based on timescale and dose-response relationships between upstream signal dynamics and intramodule reaction kinetics (Figure 4 and Table S4). In short, a steady-state or quasi-equilibrium part of a response may be selectively affected by perturbations that introduce changes in the relevant dose-response surfaces. Out-of-equilibrium responses that are not sensitive to the precise shape of a dose-response curve may be selectively attenuated by perturbations that modify the relative timescales. Dose responses and timescales cannot, in general, be modified independently by simple perturbations (combination treatments are required), but as we show, in some cases, one effect dominates resulting in feature or stimulus specificity.

The degree to which specific dynamic features of a signaling profile or the dynamic responses to specific stimuli can be selectively inhibited depends on how distinctly they rely on quasi-equilibrium and out-of-equilibrium control. Signals that contain both features may be partially inhibited by both types of perturbation, limiting the specific inhibition achievable by simple perturbations. In practice, this limited the degree to which NFκB signaling could be inhibited in a stimulus-specific manner (Figure 5) and the associated therapeutic dose window (Figure 6). The most selective stimulus-specific effects can be introduced when a signal is heavily dependent on a particular dynamic feature; for example, suppression of out-of-equilibrium transients will abrogate the response to stimuli that produce such transients. For a selected group of target genes, this specificity at the signal level translated directly to expression patterns (Figure 6B, middle). More generally, selective inhibition of early or late phases of a signal may allow for specific control of early and late response genes (Figure 6C), a concept that remains to be studied at genomic scales. Though the principles are general, how they apply to specific signaling pathways depends not only on the regulatory topology, but also on the dynamic regime determined by the parameters. As demonstrated with the IκB-NFκB module, analysis of a coarse-grained topology in terms of the principles may allow the prediction of perturbations with a desired specificity.

 

7.6.2 A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

Marvin E. Tanenbaum, Luke A. Gilbert, Lei S. Qi, Jonathan S. Weissman, Ronald D. Vale
Cell 23 Oct 2014; 159(3): 635–646
http://dx.doi.org/10.1016/j.cell.2014.09.039

Highlights

  • SunTag allows controlled protein multimerization on a protein scaffold
  • SunTag enables long-term single-molecule imaging in living cells
  • SunTag greatly improves CRISPR-based activation of gene expression

Summary

Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

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SunTag, which can recruit multiple copies of an antibody-fusion protein
Development of the SunTag, a System for Recruiting Multiple Protein Copies to a Polypeptide Scaffold Protein multimerization on a single RNA or DNA template is made possible by identifying protein domains that bind with high affinity to a relatively short nucleic acid motif. We therefore sought a protein-based system with similar properties, specifically a protein that can bind tightly to a short peptide sequence (Figures 1A and1B).Antibodies arecapable ofbindingto short,unstructured peptide sequences with high affinity and specificity, and, importantly, peptide epitopes can be designed that differ from naturally occurring sequences in the genome. Furthermore, whereas antibodies generally do not fold properly in the cytoplasm, single-chain variable fragment (scFv) antibodies, in which the epitope-binding regions of the light and heavy chains of the antibody are fused to forma single polypeptide, have been successfully expressed in soluble form in cells (Colby et al., 2004; Lecerf et al., 2001; Wo ¨rn et al., 2000).
We expressed three previously developed single-chain antibodies (Colby et al., 2004; Lecerf et al., 2001; Wo ¨rn et al., 2000) fused to EGFP in U2OS cells and coexpressed their cognate peptides (multimerized in four tandem copies) fused to the cytoplasmic side of the mitochondrial protein mitoNEET (Colca et al., 2004) (referred to here as Mito, Figure S1A). We then assayed whether the antibody-GFP fusion proteins would be recruited to the mitochondria by fluorescence microscopy, which would indicate binding between antibody and peptide (Figure 1B). Of the three antibody-peptide pairs tested, only the GCN4 antibody-peptide pair showed robust and specific binding while not disrupting normal mitochondrial morphology (Figures 1C and S1B). Thus, we focused our further efforts on the GCN4 antibody-peptide pair. The GCN4 antibody was optimized to allow intracellular expression in yeast (Wo ¨rn et al., 2000). In human cells, however, we still observed some protein aggregates of scFv-GCN4-GFP at high expression levels (Figure S2A). To improve scFv-GCN4 stability, we added a variety of N- and C-terminal fusion proteins known to enhance protein solubility and found that fusion of superfolder-GFP (sfGFP) alone
(Pe’delacq et al., 2006) or along with the small solubility tag GB1 (Gronenborn et al., 1991) to the C terminus of the GCN4 antibody almost completely eliminated protein aggregation, even at high expression levels (Figure S2A). Thus, we performed all further experiments with scFv-GCN4-sfGFP-GB1 (hereafter referred to as scFvGCN4-GFP). Very tight binding of the antibody-peptide pair in vivo is critical fortheformation ofmultimersonaproteinscaffoldbackbone.To determine the dissociation rate of the GCN4 antibody-peptide interaction, we performed fluorescence recovery after photobleaching (FRAP) experiments on scFv-GCN4-GFP bound to the mitochondrial-localized mito-mCherry-4xGCN4pep. After photobleaching, very slow GFP recovery was observed (halflife of 5–10 min [Figures 2A and 2B]), indicating that the antibody bound very tightly to the peptide. It is also important to optimize the spacing of the scFv-GCN4 binding sites within the protein scaffold so that they could be saturated by scFvGCN4 because steric hindrance of neighboring peptide binding sites is a concern. We varied the spacing between neighboring GCN4 peptides and quantified the antibody occupancy on the peptide array.

Figure 1. Identification of an Antibody-Peptide Pair that Binds Tightly In Vivo (A) Schematic of the antibody-peptide labeling strategy. (B) Schematic of the experiment described in (C) in which the mitochondrial targeting domain of mitoNEET (yellow box, mito) fused to mCherry and four tandem copies of a peptide recruits a GFP-tagged intracellular antibody to mitochondria. (C) ScFv-GCN4-GFP was coexpressed with either mito-mCherry-4xGCN4peptide (bottom) or mito-mCherry-FKBP as a control (top) in U2OS cells, and cells were imaged using spinning-disk confocal microscopy. Scale bars, 10 mm. See also Figure S1.

Figure 2. Characterization of the Off Rate and Stoichiometry of the Binding Interaction between the scFv-GCN4 Antibody and the GCN4 Peptide Array In Vivo (A) Mito-mCherry-24xGCN4pep was cotransfected with scFv-GCN4-GFP in HEK293 cells, and their colocalization on mitochondria in a single cell is shown (10 s). At 0 s, the mitochondria-localized GFP signal was photobleached in a single z plane using a 472 nm laser, and fluorescence recovery was followed by time-lapse microscopy. Scale bar, 5 mm. (B) The FRAP was quantified for 20 cells. (C–E) Indicated constructs were transfected in HEK293 cells, and images were acquired 24 hr after transfection with identical image acquisition settings. Representative images are shown in (C). Note that the GFP signal intensity in the mito-mCherry-24xGCN4pep + scFv-GCN4-GFP is highly saturated when the same scaling is used as in the other panels. Bottom row shows a zoom of a region of interest: dynamic scaling was different for the GFP and mCherry signals, so that both could be observed. Scale bars, 10 mm. (D and E) Quantifications of the GFP:mCherry fluorescence intensity ratio on mitochondria after normalization. Eachdot represents a single cell, and dashed lines indicates the average value. See also Figure S2.

Figure 3. The SunTag Allows Long-Term Single-Molecule Fluorescence Imaging in the Cytoplasm (A–H) U2OS cells were transfected with indicated SunTag24x constructs together with the scFv-GCN4-GFP-NLS and were imaged by spinning-disk confocal microscopy 24 hr after transfection. (A) A representative image of SunTag24x-CAAX-GFP is shown (left), as well as the fluorescence intensities quantification of the foci (right, blue bars). As a control, U2OS were transfected with sfGFP-CAAX and fluorescence intensities of single sfGFP-CAAX molecules were also quantified (red bars). The average fluorescence intensity of the single sfGFP-CAAX was set to 1. Dotted line marks the outline of the cell (left). Scale bar, 10 mm. (B) Cells expressing K560-SunTag24x-GFP were imaged by spinning disk confocal microscopy (image acquisition every 200 ms). Movement is revealed by a maximum intensity projection of 50 time points (left) and a kymograph (right). Scale bar, 10 mm. (C and D) Cells expressing both EB3-tdTomato and K560-SunTag24x-GFP were imaged, and moving particles were tracked manually. Red and blue tracks (bottom) indicate movement toward the cell interior and periphery, respectively (C). The duration of the movie was 20 s. Scale bar, 5 mm. Dots in (D) represent individual cells with between 5 and 20 moving particles scored per cell. The mean and SD are indicated. (E and F) Cells expressing Kif18b-SunTag24x-GFP were imaged with a 250 ms time interval. Images in (E) show a maximum intensity projection (50 time- points, left) and a kymograph (right). Speeds of moving molecules were quantified from ten different cells (F). Scale bar, 10 mm. (G and H) Cells expressing both mCherry-a-tubulin and K560rig-SunTag24x-GFP were imaged with a 600 ms time interval.The entire cell is shown in (G), whereas H shows zoomed-instills of atime series from the same cell. Open circlestrack two foci on the same microtubule,which is indicated bythe dashed line. Asterisks indicate stationary foci. Scale bars, 10 and 2 mm (G and H), respectively. See also Figure S3 and Movies S1, S2, S3, S4, S5, and S6.
The GCN4 peptide contains many hydrophobic residues (Figure 4B) and is largely unstructured in solution (Berger et al., 1999); thus, the poor expression of the peptide array could be due to its unstructured and hydrophobic nature. To test this idea, we designed several modified peptide sequence that were predicted to increase a-helical propensity and reduce hydrophobicity. One of these optimized peptides (v4, Figure 4B) was expressed moderately well as a 243 peptide array, and even higher expression was achieved with a 103 peptide array (Figure 4C). Importantly, fluorescence imaging revealed that thescFv-GCN4antibody robustlyboundto theGCN4v4peptide array in vivo and FRAP analysis suggests that the scFv-GCN4 antibody dissociates with a similar slow off rate from the GCN4
v4 peptide array as the original peptide (Figures 4D and 4E). Furthermore, K560 motility could be observed when it was tagged with the optimized v4 243 peptide array, indicating that the optimized v4 peptide array did not interfere with protein function (Movie S7). Together, these results identify a second version of the peptide array that can be used for applications requiring higher expression.
Activation of Gene Transcription Using Cas9-SunTag Because the SunTag system could be used for amplification of a fluorescence signal, we tested whether it also could be used to amplify regulatory signals involved in gene expression. Transcription of a gene is strongly enhanced by recruiting multiple copies of transcriptional activators to endogenous or artificial gene promoters (Anderson and Freytag, 1991; Chen et al., 1992; Pettersson and Schaffner, 1990). Thus, we thought that robust, artificial activation of gene transcription might also be achieved by recruiting multiple copies of a synthetic transcriptional activator to a gene using the SunTag.

Figure 4. An Optimized Peptide Array for High Expression (A) Indicated constructs were transfected in HEK293 cells and imaged 24 hr after transfection using wide-field microscopy. All images were acquired using identical acquisition parameters. Representative images are shown (left), and fluorescence intensities were quantified (n = 3) (right). (B) Sequence of the first and second generation GCN4 peptide (modified or added residues are colored blue, hydrophobic residues are red, and linker residues are yellow). (C–E) Indicated constructs were transfected in HEK293 cells and imaged 24 hr after transfection using wide-field (C) or spinning-disk confocal (D and E) microscopy. (C) Representative images are shown (left), and fluorescence intensities were quantified (n = 3) (right). (D and E) GFP signal on mitochondria was photobleached, and fluorescence recovery was determined over time. The graph (E) represents an average of six cells per condition. (E) shows an image of a representative cell before photobleaching. Scale bars in (A) and (C), 50 mm; scale bars in (D) and (E), 10 mm. Error bars in (A) and (C) represent SDs. See also Movie S7.

Figure 5. dCas9-SunTag Allows Genetic Rewiring of Cells through Activation of Endogenous Genes (A) Schematic of gene activation by dCas9-VP64 and dCas9-SunTag-VP64. dCas9 binds to a gene promoter through its sequence-specific sgRNA (red line). Direct fusion of VP64 to dCas9 (top) results in a single VP64 domain at the promoter, which poorly activates transcription of the downstream gene. In contrast, recruitment of many VP64 domains using the SunTag potently activates transcription of the gene (bottom). (B–D) K562 cells stably expressing dCas9-VP64 or dCas9-SunTag-VP64 were infected with lentiviral particles encoding indicated sgRNAs, as well as BFP and a puromycin resistance gene and selected with 0.7 mg/ml puromycin for 3 days to kill uninfected cells. (B and C) Cells were stained for CXCR4 using adirectlylabeleda-CXCR4 antibody, and fluorescence was analyzed by FACS. (D) Trans-well migration assays (see Experimental Procedures) were performed with indicated sgRNAs. Results are displayed as the fold change in directional migrating cells over control cell migration. (E) dCas9-VP64 or dCas9-SunTag-VP64 induced transcription of CDKN1B with several sgRNAs. mRNA levels were quantified by qPCR. (F) Doubling timeofcontrolcells orcells expressing indicated sgRNAs was determined (see Experimental Procedures section). Graphs in (C), (D), and (F) are averages of three independent experiments. Graph in (E) is average of two biological replicates, each with two or three technical replicates. All error bars indicate SEM. See also Figure S4

 

7.6.3 IQGAPs choreograph cellular signaling from the membrane to the nucleus

Jessica M. Smith, Andrew C. Hedman, David B. Sacks
Trends Cell Biol Mar 2015; 25(3): 171–184
http://dx.doi.org/10.1016/j.tcb.2014.12.005

Highlights

  • IQGAP proteins scaffold diverse signaling molecules.
  • IQGAPs mediate crosstalk between signaling pathways.
  • IQGAP1 regulates nuclear processes, including transcription.

Since its discovery in 1994, recognized cellular functions for the scaffold protein IQGAP1 have expanded immensely. Over 100 unique IQGAP1-interacting proteins have been identified, implicating IQGAP1 as a critical integrator of cellular signaling pathways. Initial research established functions for IQGAP1 in cell–cell adhesion, cell migration, and cell signaling. Recent studies have revealed additional IQGAP1 binding partners, expanding the biological roles of IQGAP1. These include crosstalk between signaling cascades, regulation of nuclear function, and Wnt pathway potentiation. Investigation of the IQGAP2 and IQGAP3 homologs demonstrates unique functions, some of which differ from those of IQGAP1. Summarized here are recent observations that enhance our understanding of IQGAP proteins in the integration of diverse signaling pathways.

http://ars.els-cdn.com/content/image/1-s2.0-S0962892414002153-gr2.sml

 

7.6.4 Signaling cell death from the endoplasmic reticulum stress response

Shore GC1, Papa FR, Oakes SA
Curr Opin Cell Biol. 2011 Apr; 23(2):143-9
http://dx.doi.org/10.1016%2Fj.ceb.2010.11.003

Inability to meet protein folding demands within the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), a signaling pathway with both adaptive and apoptotic outputs. While some secretory cell types have a remarkable ability to increase protein folding capacity, their upper limits can be reached when pathological conditions overwhelm the fidelity and/or output of the secretory pathway.

The lumen of the ER is a unique cellular environment optimized to carry out the three primary tasks of this organelle:

  1. calcium storage and release,
  2. protein folding and secretion, and
  3. lipid biogenesis [1].

A range of cellular disturbances lead to accumulation of misfolded proteins in the ER, including

  • point mutations in secreted proteins that disrupt their proper folding,
  • sustained secretory demands on endocrine cells,
  • viral infection with ER overload of virus-encoding protein, and
  • loss of calcium homeostasis with detrimental effects on ER-resident calcium-dependent chaperones [24].

 

The tripartite UPR consists of three ER transmembrane proteins (IRE1α, PERK, ATF6) that

  • alert the cell to the presence of misfolded proteins in the ER and
  • attempt to restore homeostasis in this organelle through increasing ER biogenesis,
  1. decreasing the influx of new proteins into the ER,
  2. promoting the transport of damaged proteins from the ER to the cytosol for degradation, and
  3. upregulating protein folding chaperones [5].

The adaptive responses of the UPR can markedly expand the protein folding capacity of the cell and restore ER homeostasis [6]. However, if these adaptive outputs fail to compensate because ER stress is excessive or prolonged, the UPR induces cell death.

The cell death pathways collectively triggered by the UPR include both caspase-dependent apoptosis and caspase-independent necrosis. While many details remain unknown, we are beginning to understand how cells determine when ER stress is beyond repair and communicate this information to the cell death machinery. For the purposes of this review, we focus on the apoptotic outputs triggered by the UPR under irremediable ER stress.

Connections from the UPR to the Mitochondrial Apoptotic Pathway

Connections from the UPR to the Mitochondrial Apoptotic Pathway

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078187/bin/nihms256554f1.jpg

Figure 1 Connections from the UPR to the Mitochondrial Apoptotic Pathway

Under excessive ER stress, the ER transmembrane sensors IRE1α and PERK send signals through the BCL-2 family of proteins to activate the mitochondrial apoptotic pathway. In response to unfolded proteins, IRE1α oligomerizes and induces endonucleolytic decay of hundreds of ER-localized mRNAs, depleting ER protein folding components and leading to worsening ER stress. Phosphorylated IRE1α also recruits TNF receptor-associated factor 2 (TRAF2) and activates apoptosis signaling kinase 1 (ASK1) and its downstream target c-Jun NH2-terminal kinase (JNK). JNK then activates pro-apoptotic BIM and inhibits anti-apoptotic BCL-2. These conditions result in dimerization of PERK and activation of its kinase domain to phosphorylate eukaryotic translation initiation factor 2α (eIF2α), which causes selective translation of activating transcription factor-4 (ATF4). ATF4 upregulates expression of the CHOP/GADD153 transcription factor, which inhibits the gene encoding anti-apoptotic BCL-2 while inducing expression of pro-apoptotic BIM. ER stress also promotes p53-dependent transcriptional upregulation of Noxa and Puma, two additional pro-apoptotic BH3-only proteins. Furthermore, high levels of UPR signaling induce initiator caspase-2 to proteolytically cleave and activate pro-apoptotic BID upstream of the mitochondrion. In addition to antagonizing pro-survival BCL-2 members, cleaved BID, BIM and PUMA activate Bax and/or Bak. Hence, in response to excessive UPR signaling, the balance of BCL-2 family proteins shifts in the direction of apoptosis and leads to the oligomerization of BAX and BAK, two multi-domain pro-apoptotic BCL-2 family proteins that then drive the permeabilization of the outer mitochondrial membrane, apoptosome formation and activation of executioner caspases such as Caspase-3. Figure adapted with permission from the Journal of Cell Science [58].

The proximal unfolded protein response sensors

UPR signaling is initiated by three ER transmembrane proteins:

  1. IRE1α,
  2. PERK, and

The most ancient ER stress sensor, IRE1α, contains

  1. an ER lumenal domain,
  2. a cytosolic kinase domain and
  3. a cytosolic RNase domain [9,10].

In the presence of unfolded proteins, IRE1α’s ER lumenal domains homo-oligomerize, leading

  • first to kinase trans-autophosphorylation and
  • subsequent RNase activation.

Dissociation of the ER chaperone BiP from IRE1α’s lumenal domain in order to engage unfolded proteins may facilitate IRE1α oligomerization [11]; alternatively, the lumenal domain may bind unfolded proteins directly [12]. PERK’s ER lumenal domain is thought to be activated similarly [13,14]. The ATF6 activation mechanism is less clear. Under ER stress, ATF6 translocates to the Golgi and is cleaved by Site-1 and Site-2 proteases to generate the ATF6(N) transcription factor [15].

All three UPR sensors have outputs that attempt to tilt protein folding demand and capacity back into homeostasis. PERK contains a cytosolic kinase that phosphorylates eukaryotic translation initiation factor 2α (eIF2α), which impedes translation initiation to reduce the protein load on the ER [16]. IRE1α splices XBP1mRNA, to produce the homeostatic transcription factor XBP1s [17,18]. Together with ATF6(N), XBP1s increases transcription of genes that augment ER size and function[19]. When eIF2α is phosphorylated, the translation of the activating transcription factor-4 (ATF4) is actively promoted and leads to the transcription of many pro-survival genes [20]. Together, these transcriptional events act as homeostatic feedback loops to reduce ER stress. If successful in reducing the amount of unfolded proteins, the UPR attenuates.

However, when these adaptive responses prove insufficient, the UPR switches into an alternate mode that promotes apoptosis. Under irremediable ER stress, PERK signaling can induce ATF-4-dependent upregulation of the CHOP/GADD153 transcription factor, which inhibits expression of the gene encoding anti-apoptotic BCL-2 while upregulating the expression of oxidase ERO1α to induce damaging ER oxidation [21,22]. Sustained IRE1α oligomerization leads to activation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream target c-Jun NH2-terminal kinase (JNK) [23,24]. Phosphorylation by JNK has been reported to both activate pro-apoptotic BIM and inhibit anti-apoptotic BCL-2 (see below). Small molecule modulators of ASK1 have been shown to protect cultured cells against ER stress-induced apoptosis, emphasizing the importance of the IRE1α-ASK1-JNK output as a death signal in this pathway [25]. In response to sustained oligomerization, the IRE1α RNase also causes endonucleolytic decay of hundreds of ER-localized mRNAs [26]. By depleting ER cargo and protein folding components, IRE1α-mediated mRNA decay may worsen ER stress, and could be a key aspect of IRE1α’s pro-apoptotic program [27]. Recently, inhibitors of IRE1α’s kinase pocket have been shown to conformationally activate its adjacent RNase domain in a manner that enforces homeostatic XBP1s without causing destructive mRNA decay [27], a potentially exciting strategy for preventing ER stress-induced cell loss.

The BCL-2 family and the Mitochondrial Apoptotic Pathway

A wealth of genetic and biochemical data argues that the intrinsic (mitochondrial) apoptotic pathway is the major cell death pathway induced by the UPR, at least in most cell types. This apoptotic pathway is set in motion when several toxic proteins (e.g., cytochrome c, Smac/Diablo) are released from mitochondria into the cytosol where they lead to activation of downstream effector caspases (e.g., Caspase-3) [30]. The BCL-2 family, a large class of both pro- and anti- survival proteins, tightly regulates the intrinsic apoptotic pathway by controlling the integrity of the outer mitochondrial membrane [31]. This pathway is set in motion when cell injury leads to the transcriptional and/or post-translational activation of one or more BH3-only proteins that share sequence similarity in a short alpha helix (~9–12 a.a.) known as the Bcl-2 homology 3 (BH3) domain [32]. Once activated, BH3-only proteins lead to loss of mitochondrial integrity by disabling mitochondrial protecting proteins that drive the permeabilization of the outer mitochondrial membrane.

ER stress has been reported to activate at least four distinct BH3-only proteins (BID, BIM, NOXA, PUMA) that then signal the mitochondrial apoptotic machinery (i.e., BAX/BAK) [3335]. Each of these BH3-only proteins is activated by ER stress in a unique way. Cells individually deficient in any of these BH3-only proteins are modestly protected against ER stress-inducing agents, but not nearly as resistant as cells null for their common downstream targets BAX and BAK [36]—the essential gatekeepers to the mitochondrial apoptotic pathway. Moreover, cells genetically deficient in both Bim andPuma are more protected against ER stress-induced apoptosis than Bim or Puma single knockout cells [37].

The ER stress sensor that signals these BH3-only proteins is known in a few cases (i.e., BIM is downstream of PERK); however, we do not yet understand how the UPR communicates with most of the BH3-only proteins. Moreover, it is not known if all of the above BH3-only proteins are simultaneously set in motion by all forms of ER stress or if a subset is activated under specific pathological stimuli that injure this organelle. Understanding the molecular details of how ER damage is communicated to the mitochondrial apoptotic machinery is critical if we want to target disease specific apoptotic signals sent from the ER.

Initiator and Executor Caspases

Caspases, or cysteine-dependent aspartate-directed proteases, play essential roles in both initiating apoptotic signaling (initiator caspases- 2, 4, 8, 12) and executing the final stages of cell demise (executioner caspases- 3, 7, 9) [38]. It is not surprising that the executioner caspases (casp-3,7,9) are critical for cell death resulting from damage to this organelle. Caspase 12 was the first caspase reported to localize to the ER downstream of BAX/BAK-dependent mitochondrial permeabilization becomes activated by UPR signaling in murine cells [39],but humans fail to express a functional Caspase 12 [41. Genetic knockdown or pharmacological inhibition of caspase-2 confers resistance to ER stress-induced apoptosis [42]. How the UPR activates caspase-2 and whether other initiator caspasesare also involved remains to be determined.

Calcium and Cell Death

Although an extreme depletion of ER luminal Ca2+ concentrations is a well-documented initiator of the UPR and ER stress-induced apoptosis or necrosis, it represents a relatively non-physiological stimulus. Ca2+ signaling from the ER is likely coupled to most pathways leading to apoptosis. UPR-induced activation of ERO1-α via CHOP in macrophages results in stimulation of inositol 1,4,5-triphosphate receptor (IP3R) [43]. All three sub-groups of the Bcl-2 family at the ER regulate IP3R activity. A significant fraction of IP3R is a constituent of highly specialized tethers that physically attach ER cisternae to mitochondria (mitochondrial-associated membrane) and regulate local Ca2+ dynamics at the ER-mitochondrion interface [4546]. This results in propagation of privileged IP3R-mediated Ca2+ oscillations into mitochondria. In an extreme scenario, massive transmission of Ca2+ into mitochondria results in Ca2+ overload and cell death by caspase-dependent and –independent means [46,47]. More refined transmission regulated by the Bcl-2 axis at the ER can influence cristae junctions and the availability of cytochrome c for its release across the outer mitochondrial membrane [48]. Finally, such regulated Ca2+transmission to mitochondria is a key determinant of mitochondrial bioenergetics [49].

ER Stress-Induced Cell Loss and Disease

Mounting evidence suggests that ER stress-induced apoptosis contributes to a range of human diseases of cell loss, including diabetes, neurodegeneration, stroke, and heart disease, to name a few (reviewed in REF [50]). The cause of ER stress in these distinct diseases varies depending on the cell type affected and the intracellular and/or extracellular conditions that disrupt proteostasis. Both mutant SOD1 and mutant huntingtin proteins aggregate, exhaust proteasome activity, and result in secondary accumulations of misfolded proteins in the ER [5152].

In the case of IRE1α, it may be possible to use kinase inhibitors to activate its cytoprotective signaling and shut down its apoptotic outputs [27]. Whether similar strategies will work for PERK and/or ATF6 remains to be seen. Alternatively, blocking the specific apoptotic signals that emerge from the UPR is perhaps a more straightforward strategy to prevent ER stress-induced cell loss. To this end, small molecular inhibitors of ASK and JNK are currently being tested in a variety preclinical models of ER stress [5253,5657]. This is just the beginning, and much work needs to be done to validate the best drugs targets in the ER stress pathway.

Conclusions

The UPR is a highly complex signaling pathway activated by ER stress that sends out both adaptive and apoptotic signals. All three transmembrane ER stress sensors (IRE1α, PERK, AFT6) have outputs that initially decrease the load and increase capacity of the ER secretory pathway in an effort to restore ER homeostasis. However, under extreme ER stress, continuous engagement of IRE1α and PERK results in events that simultaneously exacerbate protein misfolding and signal death, the latter involving caspase-dependent apoptosis and caspase-independent necrosis. Advances in our molecular understanding of how these stress sensors switch from life to death signaling will hopefully lead to new strategies to prevent diseases caused by ER stress-induced cell loss.

7.6.5 An Enzyme that Regulates Ether Lipid Signaling Pathways in Cancer Annotated by Multidimensional Profiling

Chiang KP, Niessen S, Saghatelian A, Cravatt BF.
Chem Biol. 2006 Oct; 13(10):1041-50.
http://dx.doi.org/10.1016/j.chembiol.2006.08.008

Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.

Elucidation of the metabolic and signaling networks that regulate health and disease stands as a principal goal of postgenomic research. The remarkable complexity of these molecular pathways has inspired the advancement of “systems biology” methods for their characterization [1]. Toward this end, global profiling technologies, such as DNA microarrays 2 and 3 and mass spectrometry (MS)-based proteomics 4 and 5, have succeeded in generating gene and protein signatures that depict key features of many human diseases. However, extricating from these associative relationships the roles that specific biomolecules play in cell physiology and pathology remains problematic, especially for proteins of unknown biochemical or cellular function.

The functions of certain proteins, such as adaptor or scaffolding proteins, can be gleaned from large-scale protein-interaction maps generated by technologies like yeast two-hybrid 6 and 7, protein microarrays [8], and MS analysis of immunoprecipitated protein complexes 9 and 10. In contrast, enzymes contribute to biological processes principally through catalysis. Thus, elucidation of the activities of the many thousands of enzymes encoded by eukaryotic and prokaryotic genomes requires knowledge of their endogenous substrates and products. The functional annotation of enzymes in prokaryotic systems has been facilitated by the clever analysis of gene clusters or operons 11 and 12, which correspond to sets of genes adjacently located in the genome that encode for enzymes participating in the same metabolic cascade. The assembly of eukaryotic enzymes into metabolic pathways is more problematic, however, as their corresponding genes are not, in general, physically organized into operons, but rather are scattered randomly throughout the genome.

We hypothesized that the determination of endogenous catalytic activities for uncharacterized enzymes could be accomplished directly in living systems by the integrated application of global profiling technologies that survey both the enzymatic proteome and its primary biochemical output (i.e., the metabolome). Here, we have tested this premise by utilizing multidimensional profiling to characterize an integral membrane enzyme of unknown function that is highly elevated in human cancer.

Development of a Selective Inhibitor for the Uncharacterized Enzyme KIAA1363

Previous studies using the chemical proteomic technology activity-based protein profiling (ABPP) 15, 16 and 17 have identified enzyme activity signatures that distinguish human cancer cells based on their biological properties, including tumor of origin and state of invasiveness [18]. A primary component of these signatures was the protein KIAA1363, an uncharacterized integral membrane hydrolase found to be upregulated in aggressive cancer cells from multiple tissues of origin. To investigate the role that KIAA1363 plays in cancer cell metabolism and signaling, a selective inhibitor of this enzyme was generated by competitive ABPP 20 and 21.

Previous competitive ABPP screens that target the serine hydrolase superfamily identified a set of trifluoromethyl ketone (TFMK) inhibitors that showed activity in mouse brain extracts [20]. These TFMK inhibitors showed only limited activity in living human cells (data not shown). We postulated that the activity of KIAA1363 inhibitors could be enhanced by replacing the TFMK group with a carbamate, which inactivates serine hydrolases via a covalent mechanism (Figure S1; see the Supplemental Data available with this article online). Carbamate AS115 (Figure 1A) was synthesized and tested for its effects on the invasive ovarian cancer cell line SKOV-3 by competitive ABPP (Figure 1B). AS115 was found to potently and selectively inactivate KIAA1363, displaying an IC50 value of 150 nM, while other serine hydrolase activities were not affected by this agent (IC50 values > 10 μM) (Figures 1B and 1C). AS115 also selectively inhibited KIAA1363 in other aggressive cancer cell lines that possess high levels of this enzyme, including the melanoma lines C8161 and MUM-2B (Figure S2B).

Figure 1. Characterization of AS115, a Selective Inhibitor of the Cancer-Related Enzyme KIAA1363

Profiling the Metabolic Effects of KIAA1363 Inactivation in Cancer Cells

We next compared the global metabolite profiles of SKOV-3 cells treated with AS115 to identify endogenous small molecules regulated by KIAA1363, using a recently described, untargeted liquid chromatography-mass spectrometry (LC-MS) platform for comparative metabolomics [22]. AS115 (10 μM, 4 hr) was found to cause a dramatic reduction in the levels of a specific set of lipophilic metabolites (m/z 317, 343, and 345) in SKOV-3 cells ( Figure 2A). These metabolites did not correspond to any of the typical lipid species found in cells, none of which were significantly altered by AS115 treatment ( Table S1). High-resolution MS of the m/z 317 metabolite provided a molecular formula of C19H40O3 ( Figure 2B), which suggests that this compound might represent a monoalkylglycerol ether bearing a C16:0 alkyl chain (C16:0 MAGE).  This structure assignment was corroborated by tandem MS and LC analysis, in which the endogenous m/z 317 product and synthetic C16:0 MAGE displayed equivalent fragmentation and migration patterns, respectively ( Figure S3). By extension, the m/z 343 and 345 metabolites were interpreted to represent the C18:1 and C18:0 MAGEs, respectively. A control carbamate inhibitor, URB597, which targets other hydrolytic enzymes [23], but not KIAA1363, did not affect MAGE levels in cancer cells ( Figure S4).

Pharmacological Inhibition of KIAA1363 Reduces Monoalkylglycerol Ether, MAGE, Levels in Human Cancer Cells

Pharmacological Inhibition of KIAA1363 Reduces Monoalkylglycerol Ether, MAGE, Levels in Human Cancer Cells

http://ars.els-cdn.com/content/image/1-s2.0-S1074552106003000-gr2.jpg

Figure 2. Pharmacological Inhibition of KIAA1363 Reduces Monoalkylglycerol Ether, MAGE, Levels in Human Cancer Cells

(A) Global metabolite profiling of AS115-treated SKOV-3 cells (10 μM AS115, 4 hr) with untargeted LC-MS methods [22]revealed a specific reduction in a set of structurally related metabolites with m/z values of 317, 343, and 345 (p < 0.001 for AS115- versus DMSO-treated SKOV-3 cells). Results represent the average fold change for three independent experiments. See Table S1for a more complete list of metabolite levels.

(B) High-resolution MS analysis of the sodium adduct of the purified m/z 317 metabolite provided a molecular formula of C19H40O3, which, in combination with tandem MS and LC analysis ( Figure S3), led to the determination of the structure of this small molecule as C16:0 monoalkylglycerol ether (C16:0 MAGE).

Biochemical Characterization of KIAA1363 as a 2-Acetyl MAGE Hydrolase

The correlation between KIAA1363 inactivation and reduced MAGE levels suggests that these lipids are products of a KIAA1363-catalyzed reaction. A primary route for the biosynthesis of MAGEs has been proposed to occur via the enzymatic hydrolysis of their 2-acetyl precursors 24 and 25. This 2-acetyl MAGE hydrolysis activity was first detected in cancer cell extracts over a decade ago [25], but, to date, it has eluded molecular characterization. To test whether KIAA1363 functions as a 2-acetyl MAGE hydrolase, this enzyme was transiently transfected into COS7 cells. KIAA1363-transfected cells possessed significantly higher 2-acetyl MAGE hydrolase activity compared to mock-transfected cells, and this elevated activity was blocked by treatment with AS115 (Figure 3A). In contrast, KIAA1363- and mock-transfected cells showed no differences in their respective hydrolytic activity for 2-oleoyl MAGE, monoacylglycerols, or phospholipids (e.g., platelet-activating factor [PAF], phosphatidylcholine) (Figure S5A). These data indicate that KIAA1363 selectively catalyzes the hydrolysis of 2-acetyl MAGEs to MAGEs.

KIAA1363 Regulates an Ether Lipid Signaling Network that Bridges Platelet-Activating Factor and the Lysophospholipids

Examination of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [26] suggests that the KIAA1363-MAGE pathway might serve as a unique metabolic node linking the PAF [27] and lysophospholipid [28] signaling systems in cancer cells (Figure 4A). Consistent with a direct pathway leading from MAGEs to these lysophospholipids, addition of 13C-MAGE to SKOV-3 cells resulted in the formation of 13C-labeled alkyl-LPC and alkyl-LPA (Figure 4C).
Conversely, the levels of 2-acetyl MAGE in SKOV-3 cells, as judged by metabolic labeling experiments, were significantly stabilized by treatment with AS115, which, in turn, led to an accumulation of PAF (Figure 4D).  A comparison of the metabolite profiles of SKOV-3 and OVCAR-3 cells revealed significantly higher levels of MAGE, alkyl-LPC, and alkyl-LPA in the former line (Figure 4E). These data indicate that the lysophospholipid branch of the MAGE network is elevated in aggressive cancer cells, and that this metabolic shift is regulated by KIAA1363.

Figure 4. KIAA1363 Serves as a Key Enzymatic Node in a Metabolic Network that Connects the PAF and Lysophospholipid Families of Signaling Lipids

Stable Knockdown of KIAA1363 Impairs Tumor Growth In Vivo

Figure 6. KIAA1363 Contributes to Ovarian Tumor Growth and Cancer Cell Migration

The decrease in tumorigenic potential of shKIAA1363 cells was not associated with a change in proliferation potential in vitro (Figure S8). shKIAA1363 cells were, however, impaired in their in vitro migration capacity compared to control cells (Figure 6B). Neither MAGE nor alkyl-LPC impacted cancer cell migration at concentrations up to 1 μM (Figure 6B). In contrast, alkyl-LPA (10 nM) completely rescued the reduced migratory activity of shKIAA1363 cells. Collectively, these results indicate that KIAA1363 contributes to the pathogenic properties of cancer cells in vitro and in vivo, possibly through regulating the levels of the bioactive lipid LPA.

We have determined by integrated enzyme and small-molecule profiling that KIAA1363, a protein of previously unknown function, is a 2-acetyl MAGE hydrolase that serves as a key regulator of a lipid signaling network that contributes to cancer pathogenesis. Although we cannot yet conclude which of the specific metabolites regulated by KIAA1363 supports tumor growth in vivo, the rescue of the reduced migratory phenotype of shKIAA1363 cancer cells by LPA is consistent with previous reports showing that this lipid signals through a family of G protein-coupled receptors to promote cancer cell migration and invasion 2829 and 30. LPA is also an established biomarker in ovarian cancer, and the levels of this metabolite are elevated nearly 10-fold in ascites fluid and plasma of patients with ovarian cancer [31]. Our results suggest that additional components in the KIAA1363-ether lipid network, including MAGE, alkyl LPC, and KIAA1363 itself, might also merit consideration as potential diagnostic markers for ovarian cancer. Consistent with this premise, our preliminary analyses have revealed highly elevated levels of KIAA1363 in primary human ovarian tumors compared to normal ovarian tissues (data not shown). The heightened expression of KIAA1363 in several other cancers, including breast 18 and 32, melanoma [18], and pancreatic cancer [33], indicates that alterations in the KIAA1363-ether lipid network may be a conserved feature of tumorigenesis. Considering further that reductions in KIAA1363 activity were found to impair tumor growth of both ovarian and breast cancer cells, it is possible that inhibitors of this enzyme may prove to be of value for the treatment of multiple types of cancer.

 

7.6.6 Peroxisomes – A Nexus for Lipid Metabolism and Cellular Signaling

Lodhi IJ, Semenkovich CF
Cell Metab. 2014 Mar 4; 19(3):380-92
http://dx.doi.org/10.1016%2Fj.cmet.2014.01.002

Peroxisomes are often dismissed as the cellular hoi polloi, relegated to cleaning up reactive oxygen chemical debris discarded by other organelles. However, their functions extend far beyond hydrogen peroxide metabolism. Peroxisomes are intimately associated with lipid droplets and mitochondria, and their ability to carry out fatty acid oxidation and lipid synthesis, especially the production of ether lipids, may be critical for generating cellular signals required for normal physiology. Here we review the biology of peroxisomes and their potential relevance to human disorders including cancer, obesity-related diabetes, and degenerative neurologic disease.

Peroxisomes are multifunctional organelles present in virtually all eukaryotic cells. In addition to being ubiquitous, they are also highly plastic, responding rapidly to cellular or environmental cues by modifying their size, number, morphology, and function (Schrader et al., 2013). Early ultrastructural studies of kidney and liver cells revealed cytoplasmic particles enclosed by a single membrane containing granular matrix and a crystalline core (Rhodin, 1958). These particles were linked with the term “peroxisome” by Christian de Duve, who first identified the organelle in mammalian cells when enzymes such as oxidases and catalases involved in hydrogen peroxide metabolism co-sedimented in equilibrium density gradients (De Duve and Baudhuin, 1966). Based on these studies, it was originally thought that the primary function of these organelles was the metabolism of hydrogen peroxide. Novikoff and colleagues observed a large number of peroxisomes in tissues active in lipid metabolism such as liver, brain, intestinal mucosa, and adipose tissue (Novikoff and Novikoff, 1982;Novikoff et al., 1980). Peroxisomes in different tissues vary greatly in shape and size, ranging from 0.1-0.5 μM in diameter. In adipocytes, peroxisomes tend to be small in size and localized in the vicinity of lipid droplets. Notably, a striking increase in the number of peroxisomes was observed during differentiation of adipogenic cells in culture (Novikoff and Novikoff, 1982). These findings suggest that peroxisomes may be involved in lipid metabolism.

Lazarow and de Duve hypothesized that peroxisomes in animal cells were capable of carrying out fatty acid oxidation. This was confirmed when they showed that purified rat liver peroxisomes contained fatty acid oxidation activity that was robustly increased by treatment of animals with clofibrate (Lazarow and De Duve, 1976). In a series of experiments, Hajra and colleagues discovered that peroxisomes were also capable of lipid synthesis (Hajra and Das, 1996). Over the past three decades, multiple lines of evidence have solidified the concept that peroxisomes play fundamentally important roles in lipid metabolism. In addition to removal of reactive oxygen species, metabolic functions of peroxisomes in mammalian cells include β-oxidation of very long chain fatty acids, α-oxidation of branched chain fatty acids, and synthesis of ether-linked phospholipids as well as bile acids (Figure 1). β-oxidation also occurs in mitochondria, but peroxisomal β-oxidation involves distinctive substrates and complements mitochondrial function; the processes of α-oxidation and ether lipid synthesis are unique to peroxisomes and important for metabolic homeostasis.

Structure and functions of peroxisomes

Structure and functions of peroxisomes

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951609/bin/nihms-555068-f0001.jpg

Figure 1 Structure and functions of peroxisomes

The peroxisome is a single membrane-enclosed organelle that plays an important role in metabolism. The main metabolic functions of peroxisomes in mammalian cells include β-oxidation of very long chain fatty acids, α-oxidation of branched chain fatty acids, synthesis of bile acids and ether-linked phospholipids and removal of reactive oxygen species. Peroxisomes in many, but not all, cell types contain a dense crystalline core of oxidative enzymes.

Here we highlight the established role of peroxisomes in lipid metabolism and their emerging role in cellular signaling relevant to metabolism. We describe the origin of peroxisomes and factors involved in their assembly, division, and function. We address the interaction of peroxisomes with lipid droplets and implications of this interaction for lipid metabolism. We consider fatty acid oxidation and lipid synthesis in peroxisomes and their importance in brown and white adipose tissue (sites relevant to lipid oxidation and synthesis) and disease pathogenesis.

peroxisomal biogenesis and protein import

peroxisomal biogenesis and protein import

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951609/bin/nihms-555068-f0002.jpg

Potential pathways to peroxisomal biogenesis. Peroxisomes are generated autonomously through division of pre-existing organelles (top) or through a de novo process involving budding from the ER followed by import of matrix proteins (bottom). B. Peroxisomal membrane protein import. Peroxisomal membrane proteins (PMPs) are imported post-translationally to the peroxisomal membrane. Pex19 is a soluble chaperone that binds to PMPs and transports them to the peroxisomal membrane, where it docks with a complex containing Pex16 and Pex3. Following insertion of the PMP, Pex19 is recycled back to the cytosol.

Regardless of their origin, peroxisomes require a group of proteins called peroxins for their assembly, division, and inheritance. Over 30 peroxins, encoded by Pex genes, have been identified in yeast (Dimitrov et al., 2013). At least a dozen of these proteins are conserved in mammals, where they regulate various aspects of peroxisomal biogenesis, including factors that control assembly of the peroxisomal membrane, factors that interact with peroxisomal targeting sequences allowing proteins to be shuttled to peroxisomes, and factors that act as docking receptors for peroxisomal proteins.

At least three peroxins (Pex3, Pex16 and Pex19) appear to be critical for assembly of the peroxisomal membrane and import of peroxisomal membrane proteins (PMPs) (Figure 2B). Pex19 is a soluble chaperone and import receptor for newly synthesized PMPs (Jones et al., 2004). Pex3 buds from the ER in a pre-peroxisomal vesicle and functions as a docking receptor for Pex19 (Fang et al., 2004). Pex16 acts as a docking site on the peroxisomal membrane for recruitment of Pex3 (Matsuzaki and Fujiki, 2008). Peroxisomal matrix proteins are translated on free ribosomes in the cytoplasm prior to their import. These proteins have specific peroxisomal targeting sequences (PTS) located either at the carboxyl (PTS1) or amino (PTS2) terminus (Gould et al., 1987Swinkels et al., 1991).

 

7.6.7 A nexus for cellular homeostasis- the interplay between metabolic and signal transduction pathways

Ana P Gomes, John Blenis
Current Opinion in Biotechnology Aug 2015; 34:110–117
http://dx.doi.org/10.1016/j.copbio.2014.12.007

Highlights

  • Signaling networks sense intracellular and extracellular cues to maintain homeostasis.
  • PI3K/AKT and Ras/ERK signaling induces anabolic reprogramming.
  • mTORC1 is a master node of signaling integration that promotes anabolism.
  • AMPK and SIRT1 fine tune signaling networks in response to energetic status.

In multicellular organisms, individual cells have evolved to sense external and internal cues in order to maintain cellular homeostasis and survive under different environmental conditions. Cells efficiently adjust their metabolism to reflect the abundance of nutrients, energy and growth factors. The ability to rewire cellular metabolism between anabolic and catabolic processes is crucial for cells to thrive. Thus, cells have developed, through evolution, metabolic networks that are highly plastic and tightly regulated to meet the requirements necessary to maintain cellular homeostasis. The plasticity of these cellular systems is tightly regulated by complex signaling networks that integrate the intracellular and extracellular information. The coordination of signal transduction and metabolic pathways is essential in maintaining a healthy and rapidly responsive cellular state.

AMPK and SIRT1 fine tune signaling networks in response to energetic status

AMPK and SIRT1 fine tune signaling networks in response to energetic status

 

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AMPK and SIRT1 fine tune signaling networks in response to energetic status

 

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mTORC1 is a master node of signaling integration that promotes anabolism.

 

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Fine-tuning signaling networks

 PI3K/Akt signaling-induced anabolic reprogramming

Growth factors and other ligands activate PI3K signaling upon binding and consequent activation of their cell surface receptors, such as receptor tyrosine kinases (RTKs) and G protein-coupled
receptors (GPCRs). This leads to the phosphorylation of membrane phosphatidylinositiol lipids and the recruitment and activation of several protein kinases, which perpetuate the extracellular
signals to modulate intracellular processes [3,4]. One of the most crucial signal propagators regulated by PI3K signaling is protein kinase B/Akt [3,4]. Indeed, Akt rewires metabolism in response
to environmental cues by three distinct means;
(i) by the direct phosphorylation and regulation of metabolic enzymes,
(ii) by activating/inactivating metabolism altering transcriptional factors, and
(iii) by modulating other kinases that themselves regulate metabolism [5].
Akt regulates glucose metabolism, inducing both glucose uptake and glycolytic flux by increasing the expression of the glucose transporter genes and regulating the activity of glycolytic enzymes,
respectively [6–8]. Moreover, the ability of Akt to induce glycolysis is also mediated by the regulation of Hexokinase (HK). HK performs the first step of glycolysis.

Figure 1 Anabolic rewiring induced by PI3K/Akt, Ras/ERK and mTORC1 signaling.
Extracellular signals activate two major signaling cascades controlled by the activation of PI3K and Ras. PI3K and Ras regulate Akt and ERK, which in turn induce changes in intermediate metabolism
to promote anabolic processes. In addition, they also induce the activation of  mTORC1, thus further supporting the rewiring of cellular metabolism towards anabolic processes. Through various mechanisms
Akt, ERK and mTORC1 stimulate mRNA translation, aerobic glycolysis, glutamine anaplerosis, lipid synthesis, the pentose phosphate and pyrimidine synthesis, thus producing the major components
necessary for cell growth and proliferation.

Figure 2. Regulation of intermediate metabolism by nutrient and energy sensors.
Nutrient and energy-responsive pathways fine-tune the output of signaling cascades, allowing for the correct balance between the availability of nutrients and the cellular capacity to use them effectively.
AMPK and SIRT1 respond to the energy status of the cells through sensing of AMP and NAD+ levels respectively. When energy is scarce, these sensors are activated inducing a rewiring of intermediate
metabolism to catabolic processes in order to produce energy and restore homeostasis. When nutrients (such as glucose and amino acids) and energy are available, AMPK, SIRT1, SIRT3 and SIRT6 are
repressed and mTORC1 is active, thus promoting a shift towards anabolic processes and energy production. These networks of signaling cascades, their interconnection and regulation allow the cells
to maintain energetic balance and allow for the physiological adaptation to the ever-changing environment.

 

7.6.8 Mechanisms-of-intercellular-signaling

7.6.8.1 Activation and signaling of the p38 MAP kinase pathway

Tyler Zarubin1 and Jiahuai Han
Cell Research (2005) 15, 11–18
http://dx.doi.org:/10.1038/sj.cr.7290257

The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.

Cellular behavior in response to extracellular stimuli is mediated through intracellular signaling pathways such as the mitogen-activated protein (MAP) kinase pathways 1. MAP kinases are members of discrete signaling cascades and serve as focal points in response to a variety of extracellular stimuli. Four distinct subgroups within the MAP kinase family have been described:

  • extracellular signal-regulated kinases (ERKs),
  • c-jun N-terminal or stress-activated protein kinases (JNK/SAPK),
  • ERK/big MAP kinase 1 (BMK1), and
  • the p38 group of protein kinases.

The focus of this review will be to highlight the characteristics of

  • the p38 kinases,
  • components of this kinase cascade,
  • activation of this pathway, and
  • the biological consequences of its activation.

p38 (p38) was first isolated as a 38-kDa protein rapidly tyrosine phosphorylated in response to LPS stimulation 23. p38 cDNA was also cloned as a molecule that binds puridinyl imidazole derivatives which are known to inhibit biosynthesis of inflammatory cytokines such as interleukin-1 (IL-1) and tumor-necrosis factor (TNF) in LPS stimulated monocytes 4. To date, four splice variants of the p38 family have been identified: p38, p38 5, p38 (ERK6, SAPK3) 67, and p38(SAPK4) 89. Of these, p38 and p38 are ubiquitously expressed while p38 and p38 are differentially expressed depending on tissue type. All p38 kinases can be categorized by a Thr-Gly-Tyr (TGY) dual phosphorylation motif 10. Sequence comparisons have revealed that each p38 isoform shares 60% identity within the p38 group but only 40–45% to the other three MAP kinase family members.

Mammalian p38s activation has been shown to occur in response to extracellular stimuli such as UV light, heat, osmotic shock, inflammatory cytokines (TNF- & IL-1), and growth factors (CSF-1) 13151617,18192021. This plethora of activators conveys the complexity of the p38 pathway and this matter is further complicated by the observation that activation of p38 is not only dependent on stimulus, but on cell type as well. For example, insulin can stimulate p38 in 3T3-L1 adipocytes 22, but downregulates p38 activity in chick forebrain neuron cells 23. The activation of p38 isoforms can be specifically controlled through different regulators and coactivated by various combinations of upstream regulators 2426.

Like all MAP kinases, p38 kinases are activated by dual kinases termed the MAP kinase kinases (MKKs). However, despite conserved dual phosphorylation sites among p38 isoforms, selective activation by distinct MKKs has been observed. There are two main MAPKKs that are known to activate p38, MKK3 and MKK6. It is proposed that upstream kinases can differentially regulate p38 isoforms as evidenced by the inability of MKK3 to effectively activate p38 while MKK6 is a potent activator despite 80% homology between these two MKKs 27. Also, it has been shown that MKK4, an upstream kinase of JNK, can aid in the activation of p38 and p38 in specific cell types 8. This data suggests then, that activation of p38 isoforms can be specifically controlled through different regulators and coactivated by various combinations of upstream regulators. Furthermore, substrate selectivity may be a reason why each MKK has a distinct function. In addition to the activation by upstream kinases, there is a MAPKK-independent mechanism of p38 MAPK activation involving TAB1 (transforming growth factor–activated protein kinase 1 (TAK1)-binding protein) 28. The activation of p38 in this pathway is achieved by the autophosphorylation of p38 after interaction with TAB1.

The activation of p38 in response to the wide range of extracellular stimuli can be seen in part by the diverse range of MKK kinases (MAP3K) that participate in p38 activation. These include TAK1 33, ASK1/MAPKKK5 34, DLK/MUK/ZPK 3536, and MEKK4 353738. Overexpression of these MAP3Ks leads to activation of both p38 and JNK pathways which is possibly one reason why these two pathways are often co-activated. Also contributing to p38 activation upstream of MAPK kinases are low molecular weight GTP-binding proteins in the Rho family such as Rac1 and Cdc42 4041. Rac1 can bind to MEKK1 or MLK1 while Cdc42 can only bind to MLK1 and both result in activation of p38 via MAP3Ks 3542.

Dephosphorylation, would seem to play a major role in the downregulation of MAP kinase activity. Many dual-specificity phosphatases have been identified that act upon various members of the MAP kinase pathway and are grouped as the MAP kinase phosphatase (MKP) family 45. Several members can efficiently dephosphorylate p38 and p38 4647; however, p38 and p38 are resistant to all known MKP family members.

The first p38 substrate identified was the MAP kinase-activated protein kinase 2 (MAPKAPK2 or MK2) 11552. This substrate, along with its closely related family member MK3 (3pk), were both shown to activate various substrates including small heat shock protein 27 (HSP27) 53, lymphocyte-specific protein 1 (LSP1) 54, cAMP response element-binding protein (CREB) 55, transcription factor ATF1 55, SRF 56, and tyrosine hydroxylase 57. p38 regulated/activated kinase (PRAK) is a p38 and/or p38activated kinase that shares 20-30% sequence identity to MK2 and is thought to regulate heat shock protein 27 (HSP27) 61. Mitogen- and stress-activated protein kinase-1 (MSK1) can be directly activated by p38 and ERK, and may mediate activation of CREB 626364.

Another group of substrates that are activated by p38 comprise transcription factors. Many transcription factors encompassing a broad range of action have been shown to be phosphorylated and subsequently activated by p38. Examples include activating transcription factor 1, 2 & 6 (ATF-1/2/6), SRF accessory protein (Sap1), CHOP (growth arrest and DNA damage inducible gene 153, or GADD153), p53, C/EBP, myocyte enhance factor 2C (MEF2C), MEF2A, MITF1, DDIT3, ELK1, NFAT, and high mobility group-box protein 1 (HBP1) 175566676869707172,73747576. An important cis-element, AP-1 appears to be influenced by p38 through several different mechanisms.  Taken together, all the data suggest that the p38 pathway has a wide variety of functions.

Abundant evidence for p38 involvement in apoptosis exists to date and is based on concomitant activation of p38 and apoptosis induced by a variety of agents such as NGF withdrawal and Fas ligation 959697. Cysteine proteases (caspases) are central to the apoptotic pathway and are expressed as inactive zymogens 98,99. Caspase inhibitors then can block p38 activation through Fas cross-linking, suggesting p38 functions downstream of caspase activation 97100. However, overexpression of dominant active MKK6b can also induce caspase activity and cell death thus implying that p38 may function both upstream and downstream of caspases in apoptosis 101102. It must be mentioned that the role of p38 in apoptosis is cell type and stimulus dependent. While p38 signaling has been shown to promote cell death in some cell lines, in different cell lines p38 has been shown to enhance survival, cell growth, and differentiation.

p38 now seems to have a role in tumorigenesis and sensescence. There have been reports that activation of MKK6 and MKK3 led to a senescent phenotype dependent upon p38 MAPK activity. Also, p38 MAPK activity was shown responsible for senescence in response to telomere shortening, H2O2 exposure, and chronic RAS oncogene signaling 117118119. A common feature of tumor cells is a loss of senescence and p38 may be linked to tumorigenesis in certain cells. It has been reported that p38 activation may be reduced in tumors and that loss of components of the p38 pathway such as MKK3 and MKK6 resulted in increased proliferation and likelihood of tumorigenic conversion regardless of the cell line or the tumor induction agent used in these studies 29.

Although all research done on the p38 pathway cannot be reviewed here, certain conclusions can still be made regarding the operation of p38 as a signal transduction mediator. The p38 family (,,,) is activated by both stress and mitogenic stimuli in a cell dependent manner and certain isoforms can either directly or indirectly target proteins to control pre/post transcription. p38 MAPKs also have the ability to activate other kinases and consequently regulate numerous cellular responses. Because p38 signaling has been implicated in cellular responses including inflammation, cell cycle, cell death, development, cell differentiation, senescence, and tumorigenesis, emphasis must be placed on p38 function with respect to specific cell types.

Regulation of the p38 pathway is not an isolated cascade and many different upstream signals can lead to p38 activation. These signals may be p38 specific (MKK3/6), general MAPKKs (MKK4), or MAPKK independent signals (TAB1). Downstream signaling pathways of p38 are quite divergent and each component may interact with other cellular components, both upstream and downstream, to coordinate cellular processes such as feedback mechanisms. Furthermore, in vivo p38 is not an isolated event and exists in the presence of other MAP kinases and a plethora of other signaling pathways. The subcellular location of p38 activation may also play a critical role determining the resulting effect and may add yet another order of complexity to the investigation of p38 function.

 

7.6.8.2 Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases

Gary L. Johnson and Razvan Lapadat
Science 6 Dec 2002; 298: 1911-1912.

Multicellular organisms have three well-characterized subfamilies of mitogen activated protein kinases (MAPKs) that control a vast array of physiological processes. These enzymes are regulated by a characteristic phosphorelay system in which a series of three protein kinases phosphorylate and activate one another. The extracellular signal–regulated kinases (ERKs) function in the control of cell division, and inhibitors of these enzymes are being explored as anticancer agents. The c-Jun amino-terminal kinases ( JNKs) are critical regulators of transcription, and JNK inhibitors may be effective in control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses.

Protein kinases are enzymes that covalently attach phosphate to the side chain of either serine, threonine, or tyrosine of specific proteins inside cells. Such phosphorylation of proteins can control their enzymatic activity, their interaction with other proteins and molecules, their location in the cell, and their propensity for degradation by proteases. Mitogen-activated protein kinases (MAPKs) compose a family of protein kinases whose function and regulation have been conserved during evolution from unicellular organisms such as brewers’ yeast to complex organisms including humans (1). MAPKs phosphorylate specific serines and threonines of target protein substrates and regulate cellular activities ranging from gene expression, mitosis, movement, metabolism, and programmed death. Because of the many important cellular functions controlled by MAPKs, they have been studied extensively to define their roles in physiology and human disease. MAPK-catalyzed phosphorylation of substrate proteins functions as a switch to turn on or off the activity of the substrate protein.

MAPKs are part of a phosphorelay system composed of three sequentially activated kinases, and, like their substrates, MAPKs are regulated by phosphorylation (Fig. 1) (2). MKK-catalyzed phosphorylation activates the MAPK and increases its activity in catalyzing the phosphorylation of its own substrates. MAPK phosphatases reverse the phosphorylation and return the MAPK to an inactive state. MKKs are highly selective in phosphorylating specific MAPKs. MAPK kinase kinases (MKKKs) are the third component of the phosphorelay system. MKKKs phosphorylate and activate specific MKKs. MKKKs have distinct motifs in their sequences that selectively confer their activation in response to different stimuli.

Fig. 1. MAPK phosphorelay systems.

The modules shown are representative of pathway connections for the respective MAPK phosphorelay systems.There are multiple component MKKKs, MKKs, and MAPKs for each system.For example, there are three Raf proteins (c-Raf1, B-Raf, A-Raf), two MKKs (MKK1 and MKK2), and two ERKs (ERK1 and ERK2) that can compose MAPK phosphorelay systems responsive to growth factors.The ERK, JNK, and p39 pathways in the STKE Connections Map demonstrate the potential complexity of these systems.

ERKs 1 and 2 are both components of a three-kinase phosphorelay module that includes the MKKK c-Raf1, B-Raf, or A-Raf, which can be activated by the proto-oncogene Ras. Mutations that convert Ras to an activated oncogene are common oncogenic mutations in many human tumors. Oncogenic Ras persistently activates the ERK1 and ERK2 pathways, which contributes to the increased proliferative rate of tumor cells. For this reason, inhibitors of the ERK pathways are entering clinical trials as potential anticancer agents.

Regulation of the JNK pathway is extremely complex and is influenced by many MKKKs. As depicted in the STKE JNK Pathway Connections Map, there are 13 MKKKs that regulate the JNKs. This diversity of MKKKs allows a wide range of stimuli to activate this MAPK pathway. JNKs are important in controlling programmed cell death or apoptosis (9). The inhibition of JNKs enhances chemotherapy-induced inhibition of tumor cell growth, suggesting that JNKs may provide a molecular target for the treatment of cancer. The pharmaceutical industry is bringing JNK inhibitors into clinical trials.

Recently, a major paradigm shift for MAPK regulation was developed for p38. The p38 enzyme is activated by the protein TAB1 (12), but TAB1 is not a MKK. Rather, TAB1 appears to be an adaptor or scaffolding protein and has no known catalytic activity. This is the first demonstration that another mechanism exists for the regulation of MAPKs in addition to the MKKK-MKKMAPK regulatory module.

The importance of MAPKs in controlling cellular responses to the environment and in regulating gene expression, cell growth, and apoptosis has made them a priority for research related to many human diseases. The ERK, JNK, and p38 pathways are all molecular targets for drug development, and inhibitors of MAPKs will undoubtedly be one of the next group of drugs developed for the treatment of human disease (13).

7.6.9 Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

B Bian, S Mongrain, S Cagnol, Marie-Josée Langlois, J Boulanger, et al.
Molec Carcinogen 25 Mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22312

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non-permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27Kip1 were observed in cathepsin B-deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy

Colorectal cancer (CRC),a major malignancy worldwide and the second leading cause of cancer death in North America, develops through multiple steps. The ability of cancers to invade and metastasize depends on the action of proteases actively taking center stage in extracellular proteolysis [2]. Of all the proteases, the cysteine protease cathepsin B is of significant importance [3]. Cathepsin B primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during malignant transformation cathepsin B can be upregulated [3, 4]. Cathepsin B in tumors can either be secreted, bound to the cell membrane or released by shedding vesicles [4]. Expression and redistribution of active cathepsin B to the basal plasma membrane occurs in late colon adenomas [5, 6] coincident with the activation of KRAS [1]. In line with these results, Cavallo-Medved et al. [7] have demonstrated that trafficking of cathepsin B to caveolae and its secretion are regulated by active KRAS in CRC cells in culture. Accordingly, secretion of cathepsin B, increased in the extracellular environment of CRC [8, 9], is suspected to play an essential role in disrupting extracellular matrix barriers, facilitating invasion and metastasis [10-12]. These data are consistent with the link between cathepsin B protein expression in colorectal carcinomas and shortened patient survival [6].

In a recent prospective cohort study of 558 men and women with colonic tumors [13] 82% of patients had tumors that expressed cathepsin B, irrespective of stage, while the remaining 18% had tumors that did not express cathepsin B. Other studies have suggested that cathepsin B expression or activity may actually peak during early stage cancer and subsequently decline with advanced disease [14, 15]. This points to a possible role of cathepsin B in both early and late alterations leading to colonic cancer.

This study used two strategies to specifically counteract the action of cathepsin B. The first involved the use of RNA interference (RNAi) to inhibit the expression of cathepsin B protein into CRC cells while the second approach employed the highly selective cathepsin inhibitor Ca074 to block extracellular cathepsin B activity. Results suggest that extracellular cathepsin B is involved in cell invasion whereas intracellular cathepsin B controls malignant properties of CRC cells. Further, biochemical analysis suggests that intracellular cathepsin B regulates tumorigenesis by degrading the p27Kip1 cell cycle inhibitor.

mRNA and Activated Levels of Cathepsin B Are Increased in Adenomas and in Colorectal Tumors of All Stages

Cathepsin B expression was analyzed at both the mRNA and protein levels in a series of human paired specimens at various tumor stages. As shown in Figure 1A, increased transcript levels of cathepsin B were observed in colorectal tumors, regardless of tumor stage, including in adenomas. Of note, increased cathepsin B expression was more prominent in tumors exhibiting APC mutations. By contrast, there did not appear to be a significant difference relative to KRAS mutations (Figure 1B). To establish whether these increased mRNA levels could be correlated with increased cathepsin B protein levels and more importantly with increased activity, expression of the active processed forms of the protease (25 and 30 kDa) was analyzed by Western blot. Both pro-cathepsin B and active cathepsin B were also increased in colorectal tumors compared to normal tissues (Figure 1C and D). These data hence suggest that increased transcription contributes to a greater expression of active cathepsin B in CRC.

Extracellular Cathepsin B Contributes to Invasiveness of Human CRC Cells but is Dispensable for Their Growth in Soft Agar

Cathepsin B protein levels were next examined in lysates obtained from various human CRC cell lines. As shown in Figure 2A, the proactive and catalytically active processed forms of cathepsin B were detected at various levels in CRC cell lines. Selected cathepsin B presence was also confirmed in conditioned culture medium of CRC cells, again at various levels (Figure 2A, lower panel). However, while the pro-form of cathepsin B was readily observed in conditioned culture medium of all CRC cells, the catalytically-active processed forms of cathepsin B were not detected in Western blot analyses. Additionally, using a fluorescence-based enzymatic assay, no cathepsin B enzyme activity was detected in conditioned medium. Since the pro-protease form might be activated under acidic pH conditions (peri- or extracellular) and by extracellular components of the extracellular matrix, the impact of extracellular inhibition of cathepsin B activation on CRC cell invasion was verified using Biocoat Matrigel chambers. HT-29, DLD1, and SW480 CRC cell lines secreting different levels of pro-cathepsin B (Figure 2A) were tested. Experiments were performed using the highly selective and non-permeant inhibitor Ca074 to reduce extracellular cathepsin B activity. At 10 μM, Ca074 produced a >99% inhibition of recombinant cathepsin B levels while barely reducing intracellular cathepsin B, that is, 5–8%, even upon 12 h exposure to the inhibitor (data not shown). Of note, treatment with 10 μM Ca074 significantly inhibited Matrigel invasion by approximately 45–60% in HT29, DLD1, and SW480 CRC cell lines (Figure 2B). By contrast, treatment with Ca074 had no significant effect on their capacity to form colonies in soft agarose (Figure 2C).

Cathepsin B Silencing in Human CRC Cells Inhibits Tumorigenicity and Metastasis in Immunodeficient Mice

Suppression of cathepsin B expression was found to significantly attenuate the metastatic potential of CRC cells in vivo in experimental metastasis assays. Indeed, immunodeficient mice injected with control CRC cells into the tail vein showed extensive lung metastasis within 28 d, whereas cells expressing shRNA against cathepsin B exhibited reduced lung colonization (Figure 4A). Cathepsin B silencing also altered the capacity of CRC cells to form tumors in mice as assessed by subcutaneous xenograft assays. HT29 cells induced palpable tumors with a short latency period of 9 d after their injection while downregulation of cathepsin B expression in these cells severely impaired their capacity to grow as tumors (Figure 4B).

Cathepsin B Silencing in Human CRC Cells Inhibits Growth in Soft Agar and Invasion Capacity

Recombinant lentiviruses encoding anti-cathepsin B short hairpin RNA (shRNA) were developed in order to stably suppress cathepsin B expression in CRC cells. As shown in Figure 3A, intracellular cathepsin B mRNA and protein levels were decreased in HT29 and DLD1 cells in comparison to a control shRNA which had no effect. Reduction of cathepsin B expression modestly slowed the proliferation rate of HT29 and DLD1 populations in 2D cell culture (Figure 3B). Conversely, cathepsin B silencing significantly reduced the ability of HT29 and DLD1 cells to form colonies in soft agarose (Figure 3C). This indicates that intracellular cathepsin B controls anchorage-independent growth of CRC cells given the absence of Ca074 effect (Figure 2C). Moreover, cathepsin B silencing also reduced the number of invading HT29 and DLD1 cells to a similar extent as Ca074 treatment (Figure 3D vs. Figure 2B).

Cathepsin B Silencing in Human CRC Cells Inhibits Tumorigenicity and Metastasis in Immunodeficient Mice

Suppression of cathepsin B expression was found to significantly attenuate the metastatic potential of CRC cells in vivo in experimental metastasis assays. Indeed, immunodeficient mice injected with control CRC cells into the tail vein showed extensive lung metastasis within 28 d, whereas cells expressing shRNA against cathepsin B exhibited reduced lung colonization (Figure 4A). Cathepsin B silencing also altered the capacity of CRC cells to form tumors in mice as assessed by subcutaneous xenograft assays. HT29 cells induced palpable tumors with a short latency period of 9 d after their injection while downregulation of cathepsin B expression in these cells severely impaired their capacity to grow as tumors (Figure 4B).

Cathepsin B Cleaves the Cell Cycle Inhibitor p27Kip1

In order to verify whether p27Kip1 is in fact a substrate for cathepsin B, both proteins were first overexpressed in 293 T cells and cells subsequently lysed 2 d later for Western blot analysis of their respective expression. As shown in Figure 5A, forced expression of cathepsin B in 293 T cells dose-dependently reduced p27Kip1 protein levels. Next, to determine whether p27Kip1 could be degraded by cathepsin B in vitro, lysates from 293 T cells overexpressing HA-tagged p27Kip1 were incubated with purified cathepsin B and analyzed by Western blot. Figure 5B and C shows that cathepsin B degraded p27Kip1 in a time-dependent manner as visualized by the accumulation of three lower molecular mass species (26, 20, and 12 kDa) in addition to the full-length p27Kip1 protein (see arrows versus arrowhead).

Cathepsin B is capable of endopeptidase, peptidyl-dipeptidase, and carboxydipeptidase activities [18-20]. Cathepsin B also possesses a basic amino acid in the catalytic subsite in position S2 enabling the protease to preferentially split its substrates after Arg–Arg or Lys–Arg or Arg–Lys sequences. At least five of these sequences can be found within the human p27Kip1 sequence (Figure 5D). Therefore, the first amino acid of these doublets was mutated into alanine to test whether it would affect the degradation by cathepsin B. Mutation of arginine 58 (Figure 5E) and lysine 189 (Figure 5F) did not alter the cleavage profile of p27Kip1 by cathepsin B. Mutation of lysine 165 and arginine 194 also had no altering effect (not shown). On the other hand, mutation of arginine 152 into alanine markedly reduced the detection of the 20-kDa fragment (Figure 5E).

The protein stability of wild-type p27Kip1 was then compared to that of the p27Kip1 R152A/Δ189–198 mutant, which is more resistant to cathepsin B cleavage. 293T cells were transiently transfected with either wild-type p27Kip1 or p27Kip1 mutant and subsequently treated with cycloheximide to inhibit protein neosynthesis. Thereafter, cells were lysed at different time intervals in order to analyze protein expression levels of p27Kip1 forms. As shown in Figure 6A, following cycloheximide treatment, protein levels of the p27Kip1 mutant decreased much more slowly than that of wild-type protein. Specifically, 10 h after cycloheximide addition, expression of p27Kip1 protein was clearly decreased while expression of the p27Kip1 mutant remained at control (time 0) levels. Of note, forced expression of cathepsin B in 293 T cells dose-dependently reduced the wild-type form of p27Kip1 protein levels while expression of p27Kip1 R152A/Δ189–198 mutant was only very slightly affected (Figure 6B).

Colocalization of Endogenous p27Kip1 With Cathepsin B Into Lysosomes

As shown in Figure 7A, the anti-cathepsin B antibody confirmed the colocalization of cathepsin B (in green) with the lysosomal acidotropic probe LysoTracker (in red). As expected, most of p27Kip1 staining (in green) was observed in the cell nucleus (Figure 7B). However, certain areas of colocalization were observed between endogenous p27Kip1 (in green) and cathepsin B (in red) (Figure 7B, asterisks). Moreover, Western blot analyses revealed the presence of p27Kip1 protein in lysosome-enriched fractions obtained from differential centrifugation of Caco-2/15 and SW480 cell lysates (Figure 7C and D). These lysosomal fractions were enriched in lysosome-associated membrane protein 1 (LAMP1) and exhibited very low or undetectable levels of the nuclear lamin B protein.

The most extensive literature to date regarding cathepsin B highlights a key role of this protease in the invasiveness and metastasis of various carcinoma cells [3, 8, 10-12]. The present findings demonstrate that cathepsin B has not only a role in facilitating CRC invasion and metastasis, but also in mediating early premalignant processes. Results herein show that cathepsin B promotes anchorage-independent CRC cell growth, which translates in vivo to enhanced tumor growth. In addition, cathepsin B was identified as a new protease capable of proteolytic cleavage of the cell cycle inhibitor p27Kip1. This is especially relevant since the loss of p27Kip1 expression has been strongly associated with aggressive tumor behavior and poor clinical outcome in CRC [22, 23].

These data are reminiscent of the immunohistochemistry data reported by Chan et al. [13] showing that cathepsin B protein was expressed in the vast majority of colon cancers analyzed (558 tumors), which was also independent of tumor stage. The present data also revealed that increased transcription of cathepsin B was associated with the presence of mutations in APC but not in KRAS, thus emphasizing the fact that cathepsin B gene expression is already deregulated in early stages of colorectal carcinoma. Indeed, most CRCs acquire loss-of-function mutations in both copies of the APC gene, resulting in inefficient breakdown of intracellular β-catenin and enhanced nuclear signaling [27]. Given the importance of the Wnt/APC/β-catenin pathway in human tumorigenesis initiation, the present data showing an association between cathepsin B expression and APC mutations are particularly noteworthy.

 

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Refined Warburg hypothesis -2.1.2

Writer and Curator: Larry H. Bernstein, MD, FCAP

Refined Warburg Hypothesis -2.1.2

The Warburg discoveries from 1922 on, and the influence on metabolic studies for the next 50 years was immense, and then the revelations of the genetic code took precedence.  Throughout this period, however, the brilliant work of Briton Chance, a giant of biochemistry at the University of Pennsylvania, opened new avenues of exploration that led to a recent resurgence in this vital need for answers in cancer research. The next two series of presentations will open up this resurgence of fundamental metabolic research in cancer and even neurodegenerative diseases.

2.1.2.1 Cancer Cell Metabolism. Warburg and Beyond

Hsu PP, Sabatini DM
Cell, Sep 5, 2008; 134:703-707
http://dx.doi.org:/10.016/j.cell.2008.08.021

Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.

It is hard to begin a discussion of cancer cell metabolism without first mentioning Otto Warburg. A pioneer in the study of respiration, Warburg made a striking discovery in the 1920s. He found that, even in the presence of ample oxygen, cancer cells prefer to metabolize glucose by glycolysis, a seeming paradox as glycolysis, when compared to oxidative phosphorylation, is a less efficient pathway for producing ATP (Warburg, 1956). The Warburg effect has since been demonstrated in different types of tumors and the concomitant increase in glucose uptake has been exploited clinically for the detection of tumors by fluorodeoxyglucose positron emission tomography (FDG-PET). Although aerobic glycolysis has now been generally accepted as a metabolic hallmark of cancer, its causal relationship with cancer progression is still unclear. In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

Figure 1. The Altered Metabolism of Cancer Cells

Drivers (A and B). The metabolic derangements in cancer cells may arise either from the selection of cells that have adapted to the tumor microenvironment or from aberrant signaling due to oncogene activation. The tumor microenvironment is spatially and temporally heterogeneous, containing regions of low oxygen and low pH (purple). Moreover, many canonical cancer-associated signaling pathways induce metabolic reprogramming. Target genes activated by hypoxia inducible factor (HIF) decrease the dependence of the cell on oxygen, whereas Ras, Myc, and Akt can also upregulate glucose consumption and glycolysis. Loss of p53 may also recapitulate the features of the Warburg effect, that is, the uncoupling of glycolysis from oxygen levels. Advantages (C–E). The altered metabolism of cancer cells is likely to imbue them with several proliferative and survival advantages, such as enabling cancer cells to execute the biosynthesis of macromolecules (C), to avoid apoptosis (D), and to engage in local metabolite-based paracrine and autocrine signaling (E). Potential Liabilities (F and G). This altered metabolism, however, may also confer several vulnerabilities on cancer cells. For example, an upregulated metabolism may result in the build up of toxic metabolites, including lactate and noncanonical nucleotides, which must be disposed of (F). Moreover, cancer cells may also exhibit a high energetic demand, for which they must either increase flux through normal ATP-generating processes, or else rely on an increased diversity of fuel sources (G).

The Tumor Microenvironment Selects for Altered Metabolism

One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment (Wiesener et al., 2001; Zhong et al., 1999). Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF.

Recent work has demonstrated that the key components of the Warburg effect—increased glucose consumption, decreased oxidative phosphorylation, and accompanying lactate production—are also distinguishing features of oncogene activation. The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Samanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes. Loss of the tumor suppressor protein p53 prevents expression of the gene encoding SCO2 (the synthesis of cytochrome c oxidase protein), which interferes with the function of the mitochondrial respiratory chain (Matoba et al., 2006). A second p53 effector, TIGAR (TP53-induced glycolysis and apoptosis regulator), inhibits glycolysis by decreasing levels of fructose-2,6-bisphosphate, a potent stimulator of glycolysis and inhibitor of gluconeogenesis (Bensaad et al., 2006). Other work also suggests that p53-mediated regulation of glucose metabolism may be dependent on the transcription factor NF-κB (Kawauchi et al., 2008).
It has been shown that inhibition of lactate dehydrogenase A (LDH-A) prevents the Warburg effect and forces cancer cells to revert to oxidative phosphorylation in order to reoxidize NADH and produce ATP (Fantin et al., 2006; Shim et al., 1997). While the cells are respiratory competent, they exhibit attenuated tumor growth, suggesting that aerobic glycolysis might be essential for cancer progression. In a primary fibroblast cell culture model of stepwise malignant transformation through overexpression of telomerase, large and small T antigen, and the H-Ras oncogene, increasing tumorigenicity correlates with sensitivity to glycolytic inhibition. This finding suggests that the Warburg effect might be inherent to the molecular events of transformation (Ramanathan et al., 2005). However, the introduction of similar defined factors into human mesenchymal stem cells (MSCs) revealed that transformation can be associated with increased dependence on oxidative phosphorylation (Funes et al., 2007). Interestingly, when introduced in vivo these transformed MSCs do upregulate glycolytic genes, an effect that is reversed when the cells are explanted and cultured under normoxic conditions. These contrasting models suggest that the Warburg effect may be context dependent, in some cases driven by genetic changes and in others by the demands of the microenvironment. Regardless of whether the tumor microenvironment or oncogene activation plays a more important role in driving the development of a distinct cancer metabolism, it is likely that the resulting alterations confer adaptive, proliferative, and survival advantages on the cancer cell.

Altered Metabolism Provides Substrates for Biosynthetic Pathways

Although studies in cancer metabolism have largely been energy-centric, rapidly dividing cells have diverse requirements. Proliferating cells require not only ATP but also nucleotides, fatty acids, membrane lipids, and proteins, and a reprogrammed metabolism may serve to support synthesis of macromolecules. Recent studies have shown that several steps in lipid synthesis are required for and may even actively promote tumorigenesis. Inhibition of ATP citrate lyase, the distal enzyme that converts mitochondrial-derived citrate into cytosolic acetyl coenzyme A, the precursor for many lipid species, prevents cancer cell proliferation and tumor growth (Hatzivassiliou et al., 2005). Fatty acid synthase, expressed at low levels in normal tissues, is upregulated in cancer and may also be required for tumorigenesis (reviewed in Menendez and Lupu, 2007). Furthermore, cancer cells may also enhance their biosynthetic capabilities by expressing a tumor-specific form of pyruvate kinase (PK), M2-PK. Pyruvate kinase catalyzes the third irreversible reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, the M2-PK of cancer cells is thought to be less active in the conversion of PEP to pyruvate and thus less efficient at ATP production (reviewed in Mazurek et al., 2005). A major advantage to the cancer cell, however, is that the glycolytic intermediates upstream of PEP might be shunted into synthetic processes.

Biosynthesis, in addition to causing an inherent increase in ATP demand in order to execute synthetic reactions, should also cause a decrease in ATP supply as various glycolytic and Krebs cycle intermediates are diverted. Lipid synthesis, for example, requires the cooperation of glycolysis, the Krebs cycle, and the pentose phosphate shunt. As pyruvate must enter the mitochondria in this case, it avoids conversion to lactate and therefore cannot contribute to glycolysis-derived ATP. Moreover, whereas increased biosynthesis may explain the glucose hunger of cancer cells, it cannot explain the increase in lactic acid production originally described by Warburg, suggesting that lactate must also result from the metabolism of non-glucose substrates. Recently, it has been demonstrated that glutamine may be metabolized by the citric acid cycle in cancer cells and converted into lactate, producing NADPH for lipid biosynthesis and oxaloacetate for replenishment of Krebs cycle intermediates (DeBerardinis et al., 2007).

Metabolic Pathways Regulate Apoptosis

In addition to involvement in proliferation, altered metabolism may promote another cancer-essential function: the avoidance of apoptosis. Loss of the p53 target TIGAR sensitizes cancer cells to apoptosis, most likely by causing an increase in reactive oxygen species (Bensaad et al., 2006). On the other hand, overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents caspase-independent cell death, presumably by stimulating glycolysis, increasing cellular ATP levels, and promoting autophagy (Colell et al., 2007). Whether or not GAPDH plays a physiological role in the regulation of cell death remains to be determined. Intriguingly, Bonnet et al. (2007) have reported that treating cancer cells with dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase kinase, has striking effects on their survival and on xenograft tumor growth.

DCA, a currently approved treatment for congenital lactic acidosis, activates oxidative phosphorylation and promotes apoptosis by two mechanisms. First, increased flux through the electron transport chain causes depolarization of the mitochondrial membrane potential (which the authors found to be hyperpolarized specifically in cancer cells) and release of the apoptotic effector cytochrome c. Second, an increase in reactive oxygen species generated by oxidative phosphorylation upregulates the voltage-gated K+ channel, leading to potassium ion efflux and caspase activation. Their work suggests that cancer cells may shift their metabolism to glycolysis in order to prevent cell death and that forcing cancer cells to respire aerobically can counteract this adaptation.

Cancer Cells May Signal Locally in the Tumor Microenvironment

Cancer cells may rewire metabolic pathways to exploit the tumor microenvironment and to support cancer-specific signaling. Without access to the central circulation, it is possible that metabolites can be concentrated locally and reach suprasystemic levels, allowing cancer cells to engage in metabolite-mediated autocrine and paracrine signaling that does not occur in normal tissues. So called androgen-independent prostate cancers may only be independent from exogenous, adrenal-synthesized androgens. Androgen-independent prostate cancer cells still express the androgen receptor and may be capable of autonomously synthesizing their own androgens (Stanbrough et al., 2006).

Metabolism as an Upstream Modulator of Signaling Pathways

Not only is metabolism downstream of oncogenic pathways, but an altered upstream metabolism may affect the activity of signaling pathways that normally sense the state of the cell. Individuals with inherited mutations in succinate dehydrogenase and fumarate hydratase develop highly angiogenic tumors, not unlike those exhibiting loss of the VHL tumor suppressor protein that acts upstream of HIF (reviewed in Kaelin and Ratcliffe, 2008). The mechanism of tumorigenesis in these cancer syndromes is still contentious. However, it has been proposed that loss of succinate dehydrogenase and fumarate hydratase causes an accumulation of succinate or fumarate, respectively, leading to inhibition of the prolyl hydroxylases that mark HIF for VHL-mediated degradation (Isaacs et al., 2005; Pollard et al., 2005; Selak et al., 2005). In this rare case, succinate dehydrogenase and fumarate hydratase are acting as bona fide tumor suppressors.

There are many complex questions to be answered: Is it possible that cancer cells exhibit “metabolite addiction”? Are there unique cancer-specific metabolic pathways, or combinations of pathways, utilized by the cancer cell but not by normal cells? Are different stages of metabolic adaptations required for the cancer cell to progress from the primary tumor stage to invasion to metastasis? How malleable is cancer metabolism?

2.1.2.2 Cancer metabolism. The Warburg effect today

Ferreira LMR
Exp Molec Pathol 2010; 89:372-383.
http://dx.doi.org/10.1016/j.yexmp.2010.08.006

One of the first studies on the energy metabolism of a tumor was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.

Research highlights

► Warburg first established a tumor metabolic pattern in the 1920s. ► Tumors’ increased glucose uptake has been studied since then. ► Cancer bioenergetics’ study provides insights in all its hallmarks. ► New cancer diagnostic and therapeutic techniques focus on cancer metabolism.

Introduction
Contestation to Warburg’s ideas
Glucose’s uptake and intracellular fates
Lactate production and induced acidosis
Hypoxia
Impairment of mitochondrial function
Tumour microenvironment
Proliferating versus cancer cells
More on cancer bioenergetics – integration of metabolism
Perspectives

2.1.2.3 New aspects of the Warburg effect in cancer cell biology

Bensinger SJ, Cristofk HR
Sem Cell Dev Biol 2012; 23:352-361
http://dx.doi.org:/10.1016/j.semcdb.2012.02.003

Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis–also known as the Warburg effect–characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.

Highlights

► This review discusses regulatory mechanisms that contribute to the Warburg effect in cancer. ► We list cancers for which FDG-PET has established applications as well as those cancers for which FDG-PET has not been established. ► PKM2 is highlighted as an important integrator of diverse cellular stimuli to modulate metabolic flux and cancer cell proliferation. ► We discuss how cancer metabolism can directly influence gene expression programs. ► Contribution of aerobic glycolysis to the cancer microenvironment and chemotherapeutic resistance/susceptibility is also discussed.

Regulation of the Warburg effect

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Fig. 1. PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Metabolism can directly influence gene expression programs

Metabolism can directly influence gene expression programs

Fig. 2. Metabolism can directly influence gene expression programs. A schematic representation of how metabolism can intrinsically influence epigenetics resulting in durable and heritable gene expression programs in progeny.

2.1.2.4 Choosing between glycolysis and oxidative phosphorylation. A tumor’s dilemma

Jose C, Ballance N, Rossignal R
Biochim Biophys Acta 201; 1807(6): 552-561.
http://dx.doi.org/10.1016/j.bbabio.2010.10.012

A considerable amount of knowledge has been produced during the last five years on the bioenergetics of cancer cells, leading to a better understanding of the regulation of energy metabolism during oncogenesis, or in adverse conditions of energy substrate intermittent deprivation. The general enhancement of the glycolytic machinery in various cancer cell lines is well described and recent analyses give a better view of the changes in mitochondrial oxidative phosphorylation during oncogenesis. While some studies demonstrate a reduction of oxidative phosphorylation (OXPHOS) capacity in different types of cancer cells, other investigations revealed contradictory modifications with the upregulation of OXPHOS components and a larger dependency of cancer cells on oxidative energy substrates for anabolism and energy production. This apparent conflictual picture is explained by differences in tumor size, hypoxia, and the sequence of oncogenes activated. The role of p53, C-MYC, Oct and RAS on the control of mitochondrial respiration and glutamine utilization has been explained recently on artificial models of tumorigenesis. Likewise, the generation of induced pluripotent stem cells from oncogene activation also showed the role of C-MYC and Oct in the regulation of mitochondrial biogenesis and ROS generation. In this review article we put emphasis on the description of various bioenergetic types of tumors, from exclusively glycolytic to mainly OXPHOS, and the modulation of both the metabolic apparatus and the modalities of energy substrate utilization according to tumor stage, serial oncogene activation and associated or not fluctuating microenvironmental substrate conditions. We conclude on the importance of a dynamic view of tumor bioenergetics.

Research Highlights

►The bioenergetics of cancer cells differs from normals. ►Warburg hypothesis is not verified in tumors using mitochondria to synthesize ATP. ►Different oncogenes can either switch on or switch off OXPHOS. ►Bioenergetic profiling is a prerequisite to metabolic therapy. ►Aerobic glycolysis and OXPHOS cooperate during cancer progression.

  1. Cancer cell variable bioenergetics

Cancer cells exhibit profound genetic, bioenergetic and histological differences as compared to their non-transformed counterpart. All these modifications are associated with unlimited cell growth, inhibition of apoptosis and intense anabolism. Transformation from a normal cell to a malignant cancer cell is a multi-step pathogenic process which includes a permanent interaction between cancer gene activation (oncogenes and/or tumor-suppressor genes), metabolic reprogramming and tumor-induced changes in microenvironment. As for the individual genetic mapping of human tumors, their metabolic characterization (metabolic–bioenergetic profiling) has evidenced a cancer cell-type bioenergetic signature which depends on the history of the tumor, as composed by the sequence of oncogenes activated and the confrontation to intermittent changes in oxygen, glucose and amino-acid delivery.

In the last decade, bioenergetic studies have highlighted the variability among cancer types and even inside a cancer type as regards to the mechanisms and the substrates preferentially used for deriving the vital energy. The more popular metabolic remodeling described in tumor cells is an increase in glucose uptake, the enhancement of glycolytic capacity and a high lactate production, along with the absence of respiration despite the presence of high oxygen concentration (Warburg effect) [1]. To explain this abnormal bioenergetic phenotype pioneering hypotheses proposed the impairment of mitochondrial function in rapidly growing cancer cells [2].

Although the increased consumption of glucose by tumor cells was confirmed in vivo by positron emission tomography (PET) using the glucose analog 2-(18F)-fluoro-2-deoxy-d-glucose (FDG), the actual utilization of glycolysis and oxidative phosphorylation (OXPHOS) cannot be evaluated with this technique. Nowadays, Warburg’s “aerobic-glycolysis” hypothesis has been challenged by a growing number of studies showing that mitochondria in tumor cells are not inactive per se but operate at low capacity [3] or, in striking contrast, supply most of the ATP to the cancer cells [4]. Intense glycolysis is effectively not observed in all tumor types. Indeed not all cancer cells grow fast and intense anabolism is not mandatory for all cancer cells. Rapidly growing tumor cells rely more on glycolysis than slowly growing tumor cells. This is why a treatment with bromopyruvate, for example is very efficient only on rapidly growing cells and barely useful to decrease the growth rate of tumor cells when their normal proliferation is slow. Already in 1979, Reitzer and colleagues published an article entitled “Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells”, which demonstrated that oxidative phosphorylation was used preferentially to produce ATP in cervical carcinoma cells [5]. Griguer et al. also identified several glioma cell lines that were highly dependent on mitochondrial OXPHOS pathway to produce ATP [6]. Furthermore, a subclass of glioma cells which utilize glycolysis preferentially (i.e., glycolytic gliomas) can also switch from aerobic glycolysis to OXPHOS under limiting glucose conditions  [7] and [8], as observed in cervical cancer cells, breast carcinoma cells, hepatoma cells and pancreatic cancer cells [9][10] and [11]. This flexibility shows the interplay between glycolysis and OXPHOS to adapt the mechanisms of energy production to microenvironmental changes as well as differences in tumor energy needs or biosynthetic activity. Herst and Berridge also demonstrated that a variety of human and mouse leukemic and tumor cell lines (HL60, HeLa, 143B, and U937) utilize mitochondrial respiration to support their growth [12]. Recently, the measurement of OXPHOS contribution to the cellular ATP supply revealed that mitochondria generate 79% of the cellular ATP in HeLa cells, and that upon hypoxia this contribution is reduced to 30% [4]. Again, metabolic flexibility is used to survive under hypoxia. All these studies demonstrate that mitochondria are efficient to synthesize ATP in a large variety of cancer cells, as reviewed by Moreno-Sanchez [13]. Despite the observed reduction of the mitochondrial content in tumors [3][14][15][16][17][18] and [19], cancer cells maintain a significant level of OXPHOS capacity to rapidly switch from glycolysis to OXPHOS during carcinogenesis. This switch is also observed at the level of glutamine oxidation which can occur through two modes, “OXPHOS-linked” or “anoxic”, allowing to derive energy from glutamine or serine regardless of hypoxia or respiratory chain reduced activity [20].
While glutamine, glycine, alanine, glutamate, and proline are typically oxidized in normal and tumor mitochondria, alternative substrate oxidations may also contribute to ATP supply by OXPHOS. Those include for instance the oxidation of fatty-acids, ketone bodies, short-chain carboxylic acids, propionate, acetate and butyrate (as recently reviewed in [21]).

  1. Varying degree of mitochondrial utilization during tumorigenesis

In vivo metabolomic analyses suggest the existence of a continuum of bioenergetic remodeling in rat tumors according to tumor size and its rate of growth [22]. Peter Vaupel’s group showed that small tumors were characterized by a low conversion of glucose to lactate whereas the conversion of glutamine to lactate was high. In medium sized tumors the flow of glucose to lactate as well as oxygen utilization was increased whereas glutamine and serine consumption were reduced. At this stage tumor cells started with glutamate and alanine production. Large tumors were characterized by a low oxygen and glucose supply but a high glucose and oxygen utilization rate. The conversion of glucose to glycine, alanine, glutamate, glutamine, and proline reached high values and the amino acids were released [22]. Certainly, in the inner layers constituting solid tumors, substrate and oxygen limitation is frequently observed. Experimental studies tried to reproduce these conditions in vitro and revealed that nutrients and oxygen limitation does not affect OXPHOS and cellular ATP levels in human cervix tumor [23]. Furthermore, the growth of HeLa cells, HepG2 cells and HTB126 (breast cancer) in aglycemia and/or hypoxia even triggered a compensatory increase in OXPHOS capacity, as discussed above. Yet, the impact of hypoxia might be variable depending on cell type and both the extent and the duration of oxygen limitation.
In two models of sequential oncogenesis, the successive activation of specific oncogenes in non-cancer cells evidenced the need for active OXPHOS to pursue tumorigenesis. Funes et al. showed that the transformation of human mesenchymal stem cells increases their dependency on OXPHOS for energy production [24], while Ferbeyre et al. showed that cells expressing oncogenic RAS display an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest [25]. Such observations suggest that waves of gene regulation could suppress and then restore OXPHOS in cancer cells during tumorigenesis [20]. Therefore, the definition of cancer by Hanahan and Weinberg [26] restricted to six hallmarks (1—self-sufficiency in growth signals, 2—insensitivity to growth-inhibitory (antigrowth) signals, 3—evasion of programmed cell death (apoptosis), 4—limitless replicative potential, 5—sustained angiogenesis, and 6—tissue invasion and metastases) should also include metabolic reprogramming, as the seventh hallmark of cancer. This amendment was already proposed by Tennant et al. in 2009 [27]. In 2006, the review Science published a debate on the controversial views of Warburg theory [28], in support of a more realistic description of cancer cell’s variable bioenergetic profile. The pros think that high glycolysis is an obligatory feature of human tumors, while the cons propose that high glycolysis is not exclusive and that tumors can use OXPHOS to derive energy. A unifying theory closer to reality might consider that OXPHOS and glycolysis cooperate to sustain energy needs along tumorigenesis [20]. The concept of oxidative tumors, against Warburg’s proposal, was introduced by Guppy and colleagues, based on the observation that breast cancer cells can generate 80% of their ATP by the mitochondrion [29]. The comparison of different cancer cell lines and excised tumors revealed a variety of cancer cell’s bioenergetic signatures which raised the question of the mechanisms underlying tumor cell metabolic reprogramming, and the relative contribution of oncogenesis and microenvironment in this process. It is now widely accepted that rapidly growing cancer cells within solid tumors suffer from a lack of oxygen and nutrients as tumor grows. In such situation of compromised energy substrate delivery, cancer cell’s metabolic reprogramming is further used to sustain anabolism (Fig. 1), through the deviation of glycolysis, Krebs cycle truncation and OXPHOS redirection toward lipid and protein synthesis, as needed to support uncontrolled tumor growth and survival [30] and [31]. Again, these features are not exclusive to all tumors, as Krebs cycle truncation was only observed in some cancer cells, while other studies indicated that tumor cells can maintain a complete Krebs cycle [13] in parallel with an active citrate efflux. Likewise, generalizations should be avoided to prevent over-interpretations.
Fig. 1. Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

The oncogene C-MYC participate to these changes via the stimulation of glutamine utilization through the coordinate expression of genes necessary for cells to engage in glutamine catabolism [30]. According to Newsholme EA and Board M [32] both glycolysis and glutaminolysis not only serve for ATP production, but also provide precious metabolic intermediates such as glucose-6-phosphate, ammonia and aspartate required for the synthesis of purine and pyrimidine nucleotides (Fig. 1). In this manner, the observed apparent excess in the rates of glycolysis and glutaminolysis as compared to the requirement for energy production could be explained by the need for biosynthetic processes. Yet, one should not reduce the shift from glycolysis to OXPHOS utilization to the sole activation of glutaminolysis, as several other energy substrates can be used by tumor mitochondria to generate ATP [21]. The contribution of these different fuels to ATP synthesis remains poorly investigated in human tumors.

  1. The metabolism of pre-cancer cells and its ongoing modulation by carcinogenesis

At the beginning of cancer, there might have been a cancer stem cell hit by an oncogenic event, such as alterations in mitogen signaling to extracellular growth factor receptors (EGFR), oncogenic activation of these receptors, or oncogenic alterations of downstream targets in the pathways that leads to cell proliferation (RAS–Raf–ERK and PI3K–AKT, both leading to m-TOR activation stimulating cell growth). Alterations of checkpoint genes controlling the cell cycle progression like Rb also participate in cell proliferation (Fig. 2) and this re-entry in the cell cycle implies three major needs to fill in: 1) supplying enough energy to grow and 2) synthesize building blocks de novo and 3) keep vital oxygen and nutrients available. However, the bioenergetic status of the pre-cancer cell could determine in part the evolution of carcinogenesis, as shown on mouse embryonic stem cells. In this study, Schieke et al. showed that mitochondrial energy metabolism modulates both the differentiation and tumor formation capacity of mouse embryonic stem cells [37]. The idea that cancer derives from a single cell, known as the cancer stem cell hypothesis, was introduced by observations performed on leukemia which appeared to be organized as origination from a primitive hematopoietic cell [38]. Nowadays cancer stem cells were discovered for all types of tumors [39][40][41] and [42], but little is known of their bioenergetic properties and their metabolic adaptation to the microenvironment. This question is crucial as regards the understanding of what determines the wide variety of cancer cell’s metabolic profile.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Fig. 2. Impact of different oncogenes on tumor progression and energy metabolism remodeling.

The analysis of the metabolic changes that occur during the transformation of adult mesenchymal stem cells revealed that these cells did not switch to aerobic glycolysis, but their dependency on OXPHOS was even increased [24]. Hence, mitochondrial energy metabolism could be critical for tumorigenesis, in contrast with Warburg’s hypothesis. As discussed above, the oncogene C-MYC also stimulates OXPHOS [30]. Furthermore, it was recently demonstrated that cells chronically treated with oligomycin repress OXPHOS and produce larger tumors with higher malignancy [19]. Likewise, alteration of OXPHOS by mutations in mtDNA increases tumorigenicity in different types of cancer cells [43][44] and [45].

Recently, it was proposed that mitochondrial energy metabolism is required to generate reactive oxygen species used for the carcinogenetic process induced by the K-RAS mutation [46]. This could explain the large number of mitochondrial DNA mutations found in several tumors. The analysis of mitochondria in human embryonic cells which derive energy exclusively from anaerobic glycolysis have demonstrated an immature mitochondrial network characterized by few organelles with poorly developed cristae and peri-nuclear distribution [47] and [48]. The generation of human induced pluripotent stem cell by the introduction of different oncogenes as C-MYC and Oct4 reproduced this reduction of mitochondrial OXPHOS capacity[49] and [50]. This indicates again the impact of oncogenes on the control of OXPHOS and might explain the existence of pre-cancer stem cells with different bioenergetic backgrounds, as modeled by variable sequences of oncogene activation. Accordingly, the inhibition of mitochondrial respiratory chain has been recently found associated with enhancement of hESC pluripotency [51].

Based on the experimental evidence discussed above, one can argue that 1) glycolysis is indeed a feature of several tumors and associates with faster growth in high glucose environment, but 2) active OXPHOS is also an important feature of (other) tumors taken at a particular stage of carcinogenesis which might be more advantageous than a “glycolysis-only” type of metabolism in conditions of intermittent shortage in glucose delivery. The metabolic apparatus of cancer cells is not fixed during carcinogenesis and might depend both on the nature of the oncogenes activated and the microenvironment. It was indeed shown that cancer cells with predominant glycolytic metabolism present a higher malignancy when submitted to carcinogenetic induction and analysed under fixed experimental conditions of high glucose [19]. Yet, if one grows these cells in a glucose-deprived medium they shift their metabolism toward predominant OXPHOS, as shown in HeLa cells and other cell types [9]. Therefore, one might conclude that glycolytic cells have a higher propensity to generate aggressive tumors when glucose availability is high. However, these cells can become OXPHOS during tumor progression [24] and [52]. All these observations indicate again the importance of maintaining an active OXPHOS metabolism to permit evolution of both embryogenesis and carcinogenesis, which emphasizes the importance of targeting mitochondria to alter this malignant process.

  1. Oncogenes and the modulation of energy metabolism

Several oncogenes and associated proteins such as HIF-1α, RAS, C-MYC, SRC, and p53 can influence energy substrate utilization by affecting cellular targets, leading to metabolic changes that favor cancer cell survival, independently of the control of cell proliferation. These oncogenes stimulate the enhancement of aerobic glycolysis, and an increasing number of studies demonstrate that at least some of them can also target directly the OXPHOS machinery, as discussed in this article (Fig. 2). For instance, C-MYC can concurrently drive aerobic glycolysis and/or OXPHOS according to the tumor cell microenvironment, via the expression of glycolytic genes or the activation of mitochondrial oxidation of glutamine [53]. The oncogene RAS has been shown to increase OXPHOS activity in early transformed cells [24][52] and [54] and p53 modulates OXPHOS capacity via the regulation of cytochrome c oxidase assembly [55]. Hence, carcinogenic p53 deficiency results in a decreased level of COX2 and triggers a shift toward anaerobic metabolism. In this case, lactate synthesis is increased, but cellular ATP levels remain stable [56]. The p53-inducible isoform of phosphofructokinase, termed TP53-induced glycolysis and apoptotic regulator, TIGAR, a predominant phosphatase activity isoform of PFK-2, has also been identified as an important regulator of energy metabolism in tumors [57].

  1. Tumor specific isoforms (or mutated forms) of energy genes

Tumors are generally characterized by a modification of the glycolytic system where the level of some glycolytic enzymes is increased, some fetal-like isozymes with different kinetic and regulatory properties are produced, and the reverse and back-reactions of the glycolysis are strongly reduced [60]. The GAPDH marker of the glycolytic pathway is also increased in breast, gastric, lung, kidney and colon tumors [18], and the expression of glucose transporter GLUT1 is elevated in most cancer cells. The group of Cuezva J.M. developed the concept of cancer bioenergetic signature and of bioenergetic index to describe the metabolic profile of cancer cells and tumors [18], [61], [64], [65]. This signature describes the changes in the expression level of proteins involved in glycolysis and OXPHOS, while the BEC index gives a ratio of OXPHOS protein content to glycolytic protein content, in good correlation with cancer prognostic[61]. Recently, this group showed that the beta-subunit of the mitochondrial F1F0-ATP synthase is downregulated in a large number of tumors, thus contributing to the Warburg effect [64] and [65]. It was also shown that IF1 expression levels were increased in hepatocellular carcinomas, possibly to prevent the hydrolysis of glytolytic ATP [66]. Numerous changes occur at the level of OXPHOS and mitochondrial biogenesis in human tumors, as we reviewed previously [67]. Yet the actual impact of these changes in OXPHOS protein expression level or catalytic activities remains to be evaluated on the overall fluxes of respiration and ATP synthesis. Indeed, the metabolic control analysis and its extension indicate that it is often required to inhibit activity beyond a threshold of 70–85% to affect the metabolic fluxes [68] and [69]. Another important feature of cancer cells is the higher level of hexokinase II bound to mitochondrial membrane (50% in tumor cells). A study performed on human gliomas (brain) estimated the mitochondrial bound HK fraction (mHK) at 69% of total, as compared to 9% for normal brain [70]. This is consistent with the 5-fold amplification of the type II HK gene observed by Rempel et al. in the rapidly growing rat AS-30D hepatoma cell line, relative to normal hepatocytes [71]. HKII subcellular fractionation in cancer cells was described in several studies [72][73] and [74]. The group led by Pete Pedersen explained that mHK contributes to (i) the high glycolytic capacity by utilizing mitochondrially regenerated ATP rather than cytosolic ATP (nucleotide channelling) and (ii) the lowering of OXPHOS capacity by limiting Pi and ADP delivery to the organelle [75] and [76].

All these observations are consistent with the increased rate of FDG uptake observed by PET in living tumors which could result from both an increase in glucose transport, and/or an increase in hexokinase activity. However, FDG is not a complete substrate for glycolysis (it is only transformed into FDG-6P by hexokinase before to be eliminated) and cannot be used to evidence a general increase in the glycolytic flux. Moreover, FDG-PET scan also gives false positive and false negative results, indicating that some tumors do not depend on, or do not have, an increased glycolytic capacity. The fast glycolytic system described above is further accommodated in cancer cells by an increase in the lactate dehydrogenase isoform A (LDH-A) expression level. This isoform presents a higher Vmax useful to prevent the inhibition of high glycolysis by its end product (pyruvate) accumulation. Recently, Fantin et al. showed that inhibition of LDH-A in tumors diminishes tumorigenicity and was associated with the stimulation of mitochondrial respiration [79]. The preferential expression of the glycolytic pyruvate kinase isoenzyme M2 (PKM2) in tumor cells, determines whether glucose is converted to lactate for regeneration of energy (active tetrameric form, Warburg effect) or used for the synthesis of cell building blocks (nearly inactive dimeric form) [80]. In the last five years, mutations in proteins of the respiratory system (SDH, FH) and of the TCA cycle (IDH1,2) leading to the accumulation of metabolite and the subsequent activation of HIF-1α were reported in a variety of human tumors [81], [82] and [83].

  1. Tumor microenvironment modulates cancer cell’s bioenergetics

It was extensively described how hypoxia activates HIF-1α which stimulates in turn the expression of several glycolytic enzymes such as HK2, PFK, PGM, enolase, PK, LDH-A, MCT4 and glucose transporters Glut 1 and Glut 3. It was also shown that HIF-1α can reduce OXPHOS capacity by inhibiting mitochondrial biogenesis [14] and [15], PDH activity [87] and respiratory chain activity [88]. The low efficiency and uneven distribution of the vascular system surrounding solid tumors can lead to abrupt changes in oxygen (intermittent hypoxia) but also energy substrate delivery. .. The removal of glucose, or the inhibition of glycolysis by iodoacetate led to a switch toward glutamine utilization without delay followed by a rapid decrease in acid release. This illustrates once again how tumors and human cancer cell lines can utilize alternative energy pathway such as glutaminolysis to deal with glucose limitation, provided the presence of oxygen. It was also observed that in situations of glucose limitation, tumor derived-cells can adapt to survive by using exclusively an oxidative energy substrate [9] and [10]. This is typically associated with an enhancement of the OXPHOS system. … In summary, cancer cells can survive by using exclusively OXPHOS for ATP production, by altering significantly mitochondrial composition and form to facilitate optimal use of the available substrate (Fig. 3). Yet, glucose is needed to feed the pentose phosphate pathway and generate ribose essential for nucleotide biosynthesis. This raises the question of how cancer cells can survive in the growth medium which do not contain glucose (so-called “galactose medium” with dialysed serum [9]). In the OXPHOS mode, pyruvate, glutamate and aspartate can be derived from glutamine, as glutaminolysis can replenish Krebs cycle metabolic pool and support the synthesis of alanine and NADPH [31]. Glutamine is a major source for oxaloacetate (OAA) essential for citrate synthesis. Moreover, the conversion of glutamine to pyruvate is associated with the reduction of NADP+ to NADPH by malic enzyme. Such NADPH is a required electron donor for reductive steps in lipid synthesis, nucleotide metabolism and GSH reduction. In glioblastoma cells the malic enzyme flux was estimated to be high enough to supply all of the reductive power needed for lipid synthesis [31].

Fig. 3. Interplay between energy metabolism, oncogenes and tumor microenvironment during tumorigenesis (the “metabolic wave model”).

Interplay between energy metabolism, oncogenes and tumor microenvironment

Interplay between energy metabolism, oncogenes and tumor microenvironment

While the mechanisms leading to the enhancement of glycolytic capacity in tumors are well documented, less is known about the parallel OXPHOS changes. Both phenomena could result from a selection of pre-malignant cells forced to survive under hypoxia and limited glucose delivery, followed by an adaptation to intermittent hypoxia, pseudo-hypoxia, substrate limitation and acidic environment. This hypothesis was first proposed by Gatenby and Gillies to explain the high glycolytic phenotype of tumors [91], [92] and [93], but several lines of evidence suggest that it could also be used to explain the mitochondrial modifications observed in cancer cells.

  1. Aerobic glycolysis and mitochondria cooperate during cancer progression

Metabolic flexibility considers the possibility for a given cell to alternate between glycolysis and OXPHOS in response to physiological needs. Louis Pasteur found that in most mammalian cells the rate of glycolysis decreases significantly in the presence of oxygen (Pasteur effect). Moreover, energy metabolism of normal cell can vary widely according to the tissue of origin, as we showed with the comparison of five rat tissues[94]. During stem cell differentiation, cell proliferation induces a switch from OXPHOS to aerobic glycolysis which might generate ATP more rapidly, as demonstrated in HepG2 cells [95] or in non-cancer cells[96] and [97]. Thus, normal cellular energy metabolism can adapt widely according to the activity of the cell and its surrounding microenvironment (energy substrate availability and diversity). Support for this view came from numerous studies showing that in vitro growth conditions can alter energy metabolism contributing to a dependency on glycolysis for ATP production [98].

Yet, Zu and Guppy analysed numerous studies and showed that aerobic glycolysis is not inherent to cancer but more a consequence of hypoxia[99].

Table 1. Impact of different oncogenes on energy metabolism

Impact of different oncogenes on energy metabolism.

Impact of different oncogenes on energy metabolism.

2.1.2.5 Mitohormesis

Yun J, Finkel T
Cell Metab May 2014; 19(5):757–766
http://dx.doi.org/10.1016/j.cmet.2014.01.011

For many years, mitochondria were viewed as semiautonomous organelles, required only for cellular energetics. This view has been largely supplanted by the concept that mitochondria are fully integrated into the cell and that mitochondrial stresses rapidly activate cytosolic signaling pathways that ultimately alter nuclear gene expression. Remarkably, this coordinated response to mild mitochondrial stress appears to leave the cell less susceptible to subsequent perturbations. This response, termed mitohormesis, is being rapidly dissected in many model organisms. A fuller understanding of mitohormesis promises to provide insight into our susceptibility for disease and potentially provide a unifying hypothesis for why we age.

Figure 1. The Basis of Mitohormesis. Any of a number of endogenous or exogenous stresses can perturb mitochondrial function. These perturbations are relayed to the cytosol through, at present, poorly understood mechanisms that may involve mitochondrial ROS as well as other mediators. These cytoplasmic signaling pathways and subsequent nuclear transcriptional changes induce various long-lasting cytoprotective pathways. This augmented stress resistance allows for protection from a wide array of subsequent stresses.

Figure 2. Potential Parallels between the Mitochondrial Unfolded Protein Response and Quorum Sensing in Gram-Positive Bacteria. In the C. elegans UPRmt response, mitochondrial proteins (indicated by blue swirls) are degraded by matrix proteases, and the oligopeptides that are generated are then exported through the ABC transporter family member HAF-1. Once in the cytosol, these peptides can influence the subcellular localization of the transcription factor ATFS-1. Nuclear ATFS-1 is capable of orchestrating a broad transcriptional response to mitochondrial stress. As such, this pathway establishes a method for mitochondrial and nuclear genomes to communicate. In some gram-positive bacteria, intracellularly generated peptides can be similarly exported through an ABC transporter protein. These peptides can be detected in the environment by a membrane-bound histidine kinases (HK) sensor. The activation of the HK sensor leads to phosphorylation of a response regulator (RR) protein that, in turn, can alter gene expression. This program allows communication between dispersed gram-positive bacteria and thus coordinated behavior of widely dispersed bacterial genomes.

Figure 3. The Complexity of Mitochondrial Stresses and Responses. A wide array of extrinsic and intrinsic mitochondrial perturbations can elicit cellular responses. As detailed in the text, genetic or pharmacological disruption of electron transport, incorrect folding of mitochondrial proteins, stalled mitochondrial ribosomes, alterations in signaling pathways, or exposure to toxins all appear to elicit specific cytoprotective programs within the cell. These adaptive responses include increased mitochondrial number (biogenesis), alterations in metabolism, increased antioxidant defenses, and augmented protein chaperone expression. The cumulative effect of these adaptive mechanisms might be an extension of lifespan and a decreased incidence of age-related pathologies.

2.1.2.6 Mitochondrial function and energy metabolism in cancer cells. Past overview and future perspectives

Mayevsky A
Mitochondrion. 2009 Jun; 9(3):165-79
http://dx.doi.org:/10.1016/j.mito.2009.01.009

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.

 Energy metabolism in mammalian cells

Fig. 1. Schematic representation of cellular energy metabolism and its relationship to microcirculatory blood flow and hemoglobin oxygenation.

Fig. 2. Schematic representation of the central role of the mitochondrion in the various processes involved in the pathology of cancer cells and tumors. Six issues marked as 1–6 are discussed in details in the text.

In vivo monitoring of tissue energy metabolism in mammalian cells

Fig. 3. Schematic presentation of the six parameters that could be monitored for the evaluation of tissue energy metabolism (see text for details).

Optical spectroscopy of tissue energy metabolism in vivo

Multiparametric monitoring system

Fig. 4. (A) Schematic representation of the Time Sharing Fluorometer Reflectometer (TSFR) combined with the laser Doppler flowmeter (D) for blood flow monitoring. The time sharing system includes a wheel that rotates at a speed of3000 rpm wit height filters: four for the measurements of mitochondrial NADH(366 nm and 450 nm)and four for oxy-hemoglobin measurements (585 nm and 577 nm) as seen in (C). The source of light is a mercury lamp. The probe includes optical fibers for NADH excitation (Ex) and emission (Em), laser Doppler excitation (LD in), laser Doppler emission (LD out) as seen in part E The absorption spectrum of Oxy- and Deoxy- Hemoglobin indicating the two wave length used (C).

Fig. 7. Comparison between mitochondrial metabolic states in vitro and the typical tissue metabolic states in vivo evaluated by NADH redox state, tissue blood flow and hemoglobin oxygenation as could be measured by the suggested monitoring system.

(very important)

2.1.2.7 Metabolic Reprogramming. Cancer Hallmark Even Warburg Did Not Anticipate

Ward PS, Thompson CB.
Cancer Cell 2012; 21(3):297-308
http://dx.doi.org/10.1016/j.ccr.2012.02.014

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer.

The propensity for proliferating cells to secrete a significant fraction of glucose carbon through fermentation was first elucidated in yeast. Otto Warburg extended these observations to mammalian cells, finding that proliferating ascites tumor cells converted the majority of their glucose carbon to lactate, even in oxygen-rich conditions. Warburg hypothesized that this altered metabolism was specific to cancer cells, and that it arose from mitochondrial defects that inhibited their ability to effectively oxidize glucose carbon to CO2. An extension of this hypothesis was that dysfunctional mitochondria caused cancer (Koppenol et al., 2011). Warburg’s seminal finding has been observed in a wide variety of cancers. These observations have been exploited clinically using 18F-deoxyglucose positron emission tomography (FDG-PET). However, in contrast to Warburg’s original hypothesis, damaged mitochondria are not at the root of the aerobic glycolysis exhibited by most tumor cells. Most tumor mitochondria are not defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells mitochondrial metabolism is reprogrammed to meet the challenges of macromolecular synthesis. This possibility was never considered by Warburg and his contemporaries.

Advances in cancer metabolism research over the last decade have enhanced our understanding of how aerobic glycolysis and other metabolic alterations observed in cancer cells support the anabolic requirements associated with cell growth and proliferation. It has become clear that anabolic metabolism is under complex regulatory control directed by growth factor signal transduction in non-transformed cells. Yet despite these advances, the repeated refrain from traditional biochemists is that altered metabolism is merely an indirect phenomenon in cancer, a secondary effect that pales in importance to the activation of primary proliferation and survival signals (Hanahan and Weinberg, 2011). Most proto-oncogenes and tumor suppressor genes encode components of signal transduction pathways. Their roles in carcinogenesis have traditionally been attributed to their ability to regulate the cell cycle and sustain proliferative signaling while also helping cells evade growth suppression and/or cell death (Hanahan and Weinberg, 2011). But evidence for an alternative concept, that the primary functions of activated oncogenes and inactivated tumor suppressors are to reprogram cellular metabolism, has continued to build over the past several years. Evidence is also developing for the proposal that proto-oncogenes and tumor suppressors primarily evolved to regulate metabolism.

We begin this review by discussing how proliferative cell metabolism differs from quiescent cell metabolism on the basis of active metabolic reprogramming by oncogenes and tumor suppressors. Much of this reprogramming depends on utilizing mitochondria as functional biosynthetic organelles. We then further develop the idea that altered metabolism is a primary feature selected for during tumorigenesis. Recent advances have demonstrated that altered metabolism in cancer extends beyond adaptations to meet the increased anabolic requirements of a growing and dividing cell. Changes in cancer cell metabolism can also influence cellular differentiation status, and in some cases these changes arise from oncogenic alterations in metabolic enzymes themselves.

Metabolism in quiescent vs. proliferating cells nihms-360138-f0001

Metabolism in quiescent vs. proliferating cells: both use mitochondria.
(A) In the absence of instructional growth factor signaling, cells in multicellular organisms lack the ability to take up sufficient nutrients to maintain themselves. Neglected cells will undergo autophagy and catabolize amino acids and lipids through the TCA cycle, assuming sufficient oxygen is available. This oxidative metabolism maximizes ATP production. (B) Cells that receive instructional growth factor signaling are directed to increase their uptake of nutrients, most notably glucose and glutamine. The increased nutrient uptake can then support the anabolic requirements of cell growth: mainly lipid, protein, and nucleotide synthesis (biomass). Excess carbon is secreted as lactate. Proliferating cells may also use strategies to decrease their ATP production while increasing their ATP consumption. These strategies maintain the ADP:ATP ratio necessary to maintain glycolytic flux. Green arrows represent metabolic pathways, while black arrows represent signaling.

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling.
(A) The traditional demand-based model of how metabolism is altered in proliferating cells. In response to growth factor signaling, increased transcription and translation consume free energy and decrease the ADP:ATP ratio. This leads to enhanced flux of glucose carbon through glycolysis and the TCA cycle for the purpose of producing more ATP. (B) Supply-based model of how metabolism changes in proliferating cells. Growth factor signaling directly reprograms nutrient uptake and metabolism. Increased nutrient flux through glycolysis and the mitochondria in response to growth factor signaling is used for biomass production. Metabolism also impacts transcription and translation through mechanisms independent of ATP availability.

Alterations in classic oncogenes directly reprogram cell metabolism to increase nutrient uptake and biosynthesis. PI3K/Akt signaling downstream of receptor tyrosine kinase (RTK) activation increases glucose uptake through the transporter GLUT1, and increases flux through glycolysis. Branches of glycolytic metabolism contribute to nucleotide and amino acid synthesis. Akt also activates ATP-citrate lyase (ACL), promoting the conversion of mitochondria-derived citrate to acetyl-CoA for lipid synthesis. Mitochondrial citrate can be synthesized when glucose-derived acetyl-CoA, generated by pyruvate dehydrogenase (PDH), condenses with glutamine-derived oxaloacetate (OAA) via the activity of citrate synthase (CS). mTORC1 promotes protein synthesis and mitochondrial metabolism. Myc increases glutamine uptake and the conversion of glutamine into a mitochondrial carbon source by promoting the expression of the enzyme glutaminase (GLS). Myc also promotes mitochondrial biogenesis. In addition, Myc promotes nucleotide and amino acid synthesis, both through direct transcriptional regulation and through increasing the synthesis of mitochondrial metabolite precursors.

Pyruvate kinase M2 (PKM2) expression in proliferating cells is regulated by signaling and mitochondrial metabolism to facilitate macromolecular synthesis. PKM2 is a less active isoform of the terminal glycolytic enzyme pyruvate kinase. It is also uniquely inhibited downstream of tyrosine kinase signaling. The decreased enzymatic activity of PKM2 in the cytoplasm promotes the accumulation of upstream glycolytic intermediates and their shunting into anabolic pathways. These pathways include the serine synthetic pathway that contributes to nucleotide and amino acid production. When mitochondrial metabolism is excessive, reactive oxygen species (ROS) from the mitochondria can feedback to inhibit PKM2 activity. Acetylation of PKM2, dependent on acetyl-CoA availability, may also promote PKM2 degradation and further contribute to increased flux through anabolic synthesis pathways branching off glycolysis.

IDH1 and IDH2 mutants convert glutamine carbon to the oncometabolite 2-hydroxyglutarate to dysregulate epigenetics and cell differentiation. (A) α-ketoglutarate, produced in part by wild-type isocitrate dehydrogenase (IDH), can enter the nucleus and be used as a substrate for dioxygenase enzymes that modify epigenetic marks. These enzymes include the TET2 DNA hydroxylase enzyme which converts 5-methylcytosine to 5-hydroxymethylcytosine, typically at CpG dinucleotides. 5-hydroxymethylcytosine may be an intermediate in either active or passive DNA demethylation. α-ketoglutarate is also a substrate for JmjC domain histone demethylase enzymes that demethylate lysine residues on histone tails. (B) The common feature of cancer-associated mutations in cytosolic IDH1 and mitochondrial IDH2 is the acquisition of a neomorphic enzymatic activity. This activity converts glutamine-derived α-ketoglutarate to the oncometabolite 2HG. 2HG can competitively inhibit α-ketoglutarate-dependent enzymes like TET2 and the JmjC histone demethylases, thereby impairing normal epigenetic regulation. This results in altered histone methylation marks, in some cases DNA hypermethylation at CpG islands, and dysregulated cellular differentiation.

Hypoxia and HIF-1 activation promote an alternative pathway for citrate synthesis through reductive metabolism of glutamine. (A) In proliferating cells under normoxic conditions, citrate is synthesized from both glucose and glutamine. Glucose carbon provides acetyl-CoA through the activity of PDH. Glutamine carbon provides oxaloacetate through oxidative mitochondrial metabolism dependent on NAD+. Glucose-derived acetyl-CoA and glutamine-derived oxaloacetate condense to form citrate via the activity of citrate synthase (CS). Citrate can be exported to the cytosol for lipid synthesis. (B) In cells proliferating in hypoxia and/or with HIF-1 activation, glucose is diverted away from mitochondrial acetyl-CoA and citrate production. Citrate can be maintained through an alternative pathway of reductive carboxylation, which we propose to rely on reverse flux of glutamine-derived α-ketoglutarate through IDH2. This reverse flux in the mitochondria would promote electron export from the mitochondria when the activity of the electron transport chain is inhibited because of the lack of oxygen as an electron acceptor. Mitochondrial reverse flux can be accomplished by NADH conversion to NADPH by mitochondrial transhydrogenase and the resulting NADPH use in α-ketoglutarate carboxylation. When citrate/isocitrate is exported to the cytosol, some may be metabolized in the oxidative direction by IDH1 and contribute to a shuttle that produces cytosolic NADPH.

A major paradox remaining with PKM2 is that cells expressing PKM2 produce more glucose-derived pyruvate than PKM1-expressing cells, despite having a form of the pyruvate kinase enzyme that is less active and more sensitive to inhibition. One way to get around the PKM2 bottleneck and maintain/enhance pyruvate production may be through an proposed alternative glycolytic pathway, involving an enzymatic activity not yet purified, that dephosphorylates PEP to pyruvate without the generation of ATP (Vander Heiden et al., 2010). Another answer to this paradox may emanate from the serine synthetic pathway. The decreased enzymatic activity of PKM2 can promote the accumulation of the 3-phosphoglycerate glycolytic intermediate that serves as the entry point for the serine synthetic pathway branch off glycolysis. The little studied enzyme serine dehydratase can then directly convert serine to pyruvate. A third explanation may lie in the oscillatory activity of PKM2 from the inactive dimer to active tetramer form. Regulatory inputs into PKM2 like tyrosine phosphorylation and ROS destabilize the tetrameric form of PKM2 (Anastasiou et al., 2011; Christofk et al., 2008b; Hitosugi et al., 2009), but other inputs present in glycolytic cancer cells like fructose-1,6-bisphosphate and serine can continually allosterically activate and/or promote reformation of the PKM2 tetramer (Ashizawa et al., 1991; Eigenbrodt et al., 1983). Thus, PKM2 may be continually switching from inactive to active forms in cells, resulting in an apparent upregulation of flux through anabolic glycolytic branching pathways while also maintaining reasonable net flux of glucose carbon through PEP to pyruvate. With such an oscillatory system, small changes in the levels of any of the above-mentioned PKM2 regulatory inputs can cause exquisite, rapid, adjustments to glycolytic flux. This would be predicted to be advantageous for proliferating cells in the setting of variable extracellular nutrient availability. The capability for oscillatory regulation of PKM2 could also provide an explanation for why tumor cells do not select for altered glycolytic metabolism upstream of PKM2 through deletions and/or loss of function mutations of other glycolytic enzymes.

IDH1 mutations at R132 are not simply loss-of-function for isocitrate and α-ketoglutarate interconversion, but also acquire a novel reductive activity to convert α-ketoglutarate to 2-hydroxyglutarate (2HG), a rare metabolite found at only trace amounts in mammalian cells under normal conditions (Dang et al., 2009). However, it still remained unclear if 2HG was truly a pathogenic “oncometabolite” resulting from IDH1 mutation, or if it was just the byproduct of a loss of function mutation. Whether 2HG production or the loss of IDH1 normal function played a more important role in tumorigenesis remained uncertain.

A potential answer to whether 2HG production was relevant to tumorigenesis arrived with the study of mutations in IDH2, the mitochondrial homolog of IDH1. Up to this point a small fraction of gliomas lacking IDH1 mutations were known to harbor mutations at IDH2 R172, the analogous residue to IDH1 R132 (Yan et al., 2009). However, given the rarity of these IDH2 mutations, they had not been characterized for 2HG production. The discovery of IDH2 R172 mutations in AML as well as glioma samples prompted the study of whether these mutations also conferred the reductive enzymatic activity to produce 2HG. Enzymatic assays and measurement of 2HG levels in primary AML samples confirmed that these IDH2 R172 mutations result in 2HG elevation (Gross et al., 2010; Ward et al., 2010).

It was then investigated if the measurement of 2HG levels in primary tumor samples with unknown IDH mutation status could serve as a metabolite screening test for both cytosolic IDH1 and mitochondrial IDH2 mutations. AML samples with low to undetectable 2HG were subsequently sequenced and determined to be IDH1 and IDH2 wild-type, and several samples with elevated 2HG were found to have neomorphic mutations at either IDH1 R132 or IDH2 R172 (Gross et al., 2010). However, some 2HG-elevated AML samples lacked IDH1 R132 or IDH2 R172 mutations. When more comprehensive sequencing of IDH1 and IDH2 was performed, it was found that the common feature of this remaining subset of 2HG-elevated AMLs was another mutation in IDH2, occurring at R140 (Ward et al., 2010). This discovery provided additional evidence that 2HG production was the primary feature being selected for in tumors.

In addition to intensifying efforts to find the cellular targets of 2HG, the discovery of the 2HG-producing IDH1 and IDH2 mutations suggested that 2HG measurement might have clinical utility in diagnosis and disease monitoring. While much work is still needed in this area, serum 2HG levels have successfully correlated with IDH1 R132 mutations in AML, and recent data have suggested that 1H magnetic resonance spectroscopy can be applied for 2HG detection in vivo for glioma (Andronesi et al., 2012; Choi et al., 2012; Gross et al., 2010; Pope et al., 2012). These methods may have advantages over relying on invasive solid tumor biopsies or isolating leukemic blast cells to obtain material for sequencing of IDH1 and IDH2. Screening tumors and body fluids by 2HG status also has potentially increased applicability given the recent report that additional IDH mutations can produce 2HG (Ward et al., 2011). These additional alleles may account for the recently described subset of 2HG-elevated chondrosarcoma samples that lacked the most common IDH1 or IDH2 mutations but were not examined for other IDH alterations (Amary et al., 2011). Metabolite screening approaches can also distinguish neomorphic IDH mutations from SNPs and sequencing artifacts with no effect on IDH enzyme activity, as well as from an apparently rare subset of loss-of-function, non 2HG-producing IDH mutations that may play a secondary tumorigenic role in altering cellular redox (Ward et al., 2011).

Will we find other novel oncometabolites like 2HG? We should consider basing the search for new oncometabolites on those metabolites already known to cause disease in pediatric inborn errors of metabolism (IEMs). 2HG exemplifies how advances in research on IEMs can inform research on cancer metabolism, and vice versa. Methods developed by those studying 2HG aciduria were used to demonstrate that R(-)-2HG (also known as D-2HG) is the exclusive 2HG stereoisomer produced by IDH1 and IDH2 mutants (Dang et al., 2009; Ward et al., 2010). Likewise, following the discovery of 2HG-producing IDH2 R140 mutations in leukemia, researchers looked for and successfully found germline IDH2 R140 mutations in D-2HG aciduria. IDH2 R140 mutations now account for nearly half of all cases of this devastating disease (Kranendijk et al., 2010). While interest has surrounded 2HG due to its apparent novelty as a metabolite not found in normal non-diseased cells, there are situations where 2HG appears in the absence of metabolic enzyme mutations. For example, in human cells proliferating in hypoxia, α-ketoglutarate can accumulate and be metabolized through an enhanced reductive activity of wild-type IDH2 in the mitochondria, leading to 2HG accumulation in the absence of IDH mutation (Wise et al., 2011). The ability of 2HG to alter epigenetics may reflect its evolutionary ancient status as a signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency.

With this broadened view of what constitutes an oncometabolite, one could argue that the discoveries of two other oncometabolites, succinate and fumarate, preceded that of 2HG. Loss of function mutations in the TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) have been known for several years to occur in pheochromocytoma, paraganglioma, leiomoyoma, and renal carcinoma. It was initially hypothesized that these mutations contribute to cancer through mitochondrial damage producing elevated ROS (Eng et al., 2003). However, potential tumorigenic effects were soon linked to the elevated levels of succinate and fumarate arising from loss of SDH and FH function, respectively. Succinate was initially found to impair PHD2, the α-ketoglutarate-dependent enzyme regulating HIF stability, through product inhibition (Selak et al., 2005). Subsequent work confirmed that fumarate could inhibit PHD2 (Isaacs et al., 2005), and that succinate could also inhibit the related enzyme PHD3 (Lee et al., 2005). These observations linked the elevated HIF levels observed in SDH and FH deficient tumors to the activity of the succinate and fumarate metabolites. Recent work has suggested that fumarate may have other important roles that predominate in FH deficiency. For example, fumarate can modify cysteine residues to inhibit a negative regulator of the Nrf2 transcription factor. This post-translational modification leads to the upregulation of antioxidant response genes (Adam et al., 2011; Ooi et al., 2011).

There are still many unanswered questions regarding the biology of SDH and FH deficient tumors. In light of the emerging epigenetic effects of 2HG, it is intriguing that succinate has been shown to alter histone demethylase activity in yeast (Smith et al., 2007). Perhaps elevated succinate and fumarate resulting from SDH and FH mutations can promote tumorigenesis in part through epigenetic modulation.

Despite rapid technological advances in studying cell metabolism, we remain unable to reliably distinguish cytosolic metabolites from those in the mitochondria and other compartments. Current fractionation methods often lead to metabolite leakage. Even within one subcellular compartment, there may be distinct pools of metabolites resulting from channeling between metabolic enzymes. A related challenge lies in the quantitative measurement of metabolic flux; i.e., measuring the movement of carbon, nitrogen, and other atoms through metabolic pathways rather than simply measuring the steady-state levels of individual metabolites. While critical fluxes have been quantified in cultured cancer cells and methods for these analyses continue to improve (DeBerardinis et al., 2007; Mancuso et al., 2004; Yuan et al., 2008), many obstacles remain such as cellular compartmentalization and the reliance of most cell culture on complex, incompletely defined media.

Over the past decade, the study of metabolism has returned to its rightful place at the forefront of cancer research. Although Warburg was wrong about mitochondria, he was prescient in his focus on metabolism. Data now support the concepts that altered metabolism results from active reprogramming by altered oncogenes and tumor suppressors, and that metabolic adaptations can be clonally selected during tumorigenesis. Altered metabolism should now be considered a core hallmark of cancer. There is much work to be done.

2.1.2.8 A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

Schell JC, Olson KA, …, Xie J, Egnatchik RA, Earl EG, DeBerardinis RJ, Rutter J.
Mol Cell. 2014 Nov 6; 56(3):400-13
http://dx.doi.org:/10.1016/j.molcel.2014.09.026

Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that the mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells.

Figure 2. Re-Expressed MPC1 and MPC2 Form a Mitochondrial Complex (A and B) (A) Western blot and (B) qRT-PCR analysis of the indicated colon cancer cell lines with retroviral expression of MPC1 (or MPC1-R97W) and/or MPC2. (C) Western blots of human heart tissue, hematologic cancer cells, and colon cancer cell lines with and without MPC1 and MPC2 re-expression. (D) Fluorescence microscopy of MPC1-GFP and MPC2-GFP overlaid with Mitotracker Red in HCT15 cells. Scale bar: 10 mm. (E) Blue-native PAGE analysis of mitochondria from control and MPC1/2-expressing cells. (F) Western blots of metabolic and mitochondrial proteins across four colon cancer cell lines with or without MPC1/2 expression

Figure 3. MPC Re-Expression Alters Mitochondrial Pyruvate Metabolism (A) OCR at baseline and maximal respiration in HCT15 (n = 7) and HT29 (n = 13) with pyruvate as the sole carbon source (mean ± SEM). (B and C) Schematic and citrate mass isotopomer quantification in cells cultured with D-[U-13C]glucose and unlabeled glutamine for 6 hr (mean ± SD, n = 2). (D) Glucose uptake and lactate secretion normalized to protein concentration (mean ± SD, n = 3). (E–G) (E) Western blots of PDH, phospho-PDH, and PDK1; (F) PDH activity assay and (G) CS activity assay with or without MPC1 and MPC2 expression (mean ± SD, n = 4). (H and I) Effects of MPC1/2 re-expression on mitochondrial membrane potential and ROS production (mean ± SD, n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. MPC Re-Expression Alters Growth under Low-Attachment Conditions (A) Cell number of control and MPC1/2 re-expressing cell lines in adherent culture (mean ± SD, n = 7). (B) Cell viability determined by trypan blue exclusion and Annexin V/PI staining (mean ± SD, n = 3). (C–F) (C) EdU incorporation of MPC re-expressing cell lines at 3 hr post EdU pulse. Growth in 3D culture evaluated by (D) soft agar colony formation (mean ± SD, n = 12, see also Table S1) and by ([E] and [F]) spheroid formation ± MPC inhibitor UK5099 (mean ± SEM, n = 12). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7. MPC Re-Expression Alters the Cancer Initiating Cell Population (A) Western blot quantification of ALDHA and Lin28A from control or MPC re-expressing HT29 xenografts (mean ± SEM, n = 10). (B and C) Percentage of ALDHhi (n = 3) and CD44hi (n = 5) cells as determined by flow cytometry (mean ± SEM). (D) Western blot analysis of stem cell markers in control and MPC re-expressing cell lines. (E) Relative MPC1 and MPC2 mRNA levels in ALDH sorted HCT15 cells (n = 4,mean ± SEM). 2D growth of (F) whole-population HCT15 cells and (G) ALDH sorted cells. Area determined by ImageJ after crystal violet staining (mean ± SD, n = 6). (H and I) (H) Adherent and (I) spheroid growth of main population (MP) versus side population (SP) HCT15 cells. (mean ± SD, n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Our demonstration that the MPC is lost or underexpressed in many cancers might provide clarifying context for earlier attempts to exploit metabolic regulation for cancer therapeutics. The PDH kinase inhibitor dichloroacetate, which impairs PDH phosphorylation and increases pyruvate oxidation, has been explored extensively as a cancer therapy (Bonnet et al., 2007; Olszewski et al., 2010). It has met with mixed results, however, and has typically failed to dramatically decrease tumor burden as a monotherapy (Garon et al., 2014;
Sanchez-Arago et al., 2010; Shahrzadetal.,2010). Is one possible reason for these failures that the MPC has been lost or inactivated, thereby limiting the metabolic effects of PDH activity? The inclusion of the MPC adds additional complexity to targeting cancer metabolism for therapy but has the potential to explain why treatments may be more effective in some studies than in others (Fulda et al., 2010; Hamanaka and Chandel, 2012; Tennant et al., 2010; Vander Heiden, 2011). The redundant measures to limit pyruvate oxidation make it easy to understand why expression of the MPC leads to relatively modest metabolic changes in cells grown in adherent culture conditions. While subtle, we observed a number of changes in metabolic parameters, all of which are consistent with enhanced mitochondrial pyruvate entry and oxidation. There are at least two possible explanations for the discrepancy that we observed between the impact on adherent and nonadherent cell proliferation. One hypothesis is that the stress of nutrient deprivation and detachment combines with these subtle metabolic effects to impair survival and proliferation.

2.1.2.9  ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2

Lee KM, Nam K, Oh S, Lim J, Lee T, Shin I.
Cell Signal. 2015 Feb; 27(2):228-35
http://dx.doi.org:/10.1016/j.cellsig.2014.11.004

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

Fig. 1.ECM1 induces a metabolic shift toward promoting Warburg effect. (A) The levels of glucose uptake were examined with a cell-based assay. (B) Levels of lactate production were measured using a lactate assay kit. (C) Cellular ATP content was determined with a Cell Titer-Glo luminescent cell viability assay. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, ***p b 0.0005).

Fig.2. ECM1 up-regulates expression of gene sassociated with the Warburg effect. (A) Cell lysates were analyzed by western blotting using antibodies specific for ECM1, LDHA, GLUT1,and actin (as a loading control). The intensities of the bands were quantified using 1D Scan software and plotted. (BandC) mRNA levels of each gene were determined by real-time PCR using specific primers. (D) HIF-1α-dependent transcriptional activities were examined using a hypoxia response element (HRE) reporter indual luciferase assays. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, **p b 0.005, ***p b 0.0005).

Fig.3. ECM1-dependent upregulation of gene expression is not mediated byEgr-1.

Fig.4. ECM1 activates PKM2 via EGF-mediated ERK activation

Fig. 5. TheWarburg effect is attenuated by silencing of PKM2 in breast cancer cells

Recently, a non-glycolytic function of PKM2 was reported. Phosphorylated PKM2 at Ser37 is translocated into the nucleus after EGFR and ERK activation and regulates the expression of cyclin D1, c-Myc, LDHA, and GLUT1[19,37]. Here, we showed that ECM1 regulates the phosphorylation level and translocation of PKM2 via the EGFR/ ERK pathway. As we previously showed that ECM1 enhances the EGF response and increases EGFR expression through MUC1-dependent stabilization [17], it seemed likely that activation of the EGFR/ERK pathway by ECM1 is linked to PKM2 phosphorylation. Indeed, we show here that ECM1 regulates the phosphorylation of PKM2 at Ser37 and enhances the Warburg effect through the EGFR/ERK pathway. HIF-1α is known to be responsible for alterations in cancer cell metabolism [38] and our current studies showed that the expression level of HIF-1α is up-regulated by ECM1 (Fig. 2C and D). To determine the mechanism by which ECM1 upregulated HIF-1α expression, we focused on the induction of Egr-1 by EGFR/ERK signaling [39]. However, although Egr-1 expression was regulated by ECM1 we failed to find evidence that Egr-1 affected the expression of genes involved in the Warburg effect (Fig. 3C). Moreover, ERK-dependent PKM2 activation did not regulate HIF-1α expression in BT-474 cells (Fig. 4D and5B). These results suggested that the upregulation of HIF-1α by ECM1 is not mediated by the EGFR/ERK pathway.

Conclusions

In the current study we showed that ECM1 altered metabolic phenotypes of breast cancer cells toward promoting the Warburg effect.

Phosphorylation and nuclear translocation of PKM2 were induced by ECM1 through the EGFR/ERK pathway. Moreover, phosphorylated PKM2 increased the expression of metabolic genes such as LDHA and GLUT1, and promoted glucose uptake and lactate production. These findings provide a new perspective on the distinct functions of ECM1 in cancer cell metabolism. Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.cellsig.2014.11.004

References

[1] R.A. Cairns, I.S. Harris, T.W. Mak, Cancer 11 (2011) 85–95.
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[4] N.C. Denko, Cancer 8 (2008) 705–713.
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[6] J.Lum, T.Bui, M.Gruber, J.D.Gordan, R.J.DeBerardinis,.. ,C.B. Thompson, Genes Dev. 21 (2007) 1037–1049.
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2.1.2.10 Glutamine Oxidation Maintains the TCA Cycle and Cell Survival during impaired Mitochondrial Pyruvate Transport

Chendong Yang, B Ko, CT. Hensley,…, J Rutter, ME. Merritt, RJ. DeBerardinis
Molec Cell  6 Nov 2014; 56(3):414–424
http://dx.doi.org/10.1016/j.molcel.2014.09.025

Highlights

  • Mitochondria produce acetyl-CoA from glutamine during MPC inhibition
    •Alanine synthesis is suppressed during MPC inhibition
    •MPC inhibition activates GDH to supply pools of TCA cycle intermediates
    •GDH supports cell survival during periods of MPC inhibition

Summary

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Yang et al, Graphical Abstract

Yang et al, Graphical Abstract

Graphical abstract

Figure 1. Pyruvate Depletion Redirects Glutamine Metabolism to Produce AcetylCoA and Citrate (A) Top: Anaplerosis supplied by [U-13C]glutamine. Glutamine supplies OAA via a-KG, while acetylCoA is predominantly supplied by other nutrients, particularly glucose. Bottom: Glutamine is converted to acetyl-CoA in the absence of glucosederived pyruvate. Red circles represent carbons arising from [U-13C]glutamine, and gray circles are unlabeled. Reductive carboxylation is indicated by the green dashed line. (B) Fraction of succinate, fumarate, malate, and aspartate containing four 13C carbons after culture of SFxL cells for 6 hr with [U-13C]glutamine in the presence or absence of 10 mM unlabeled glucose (Glc). (C) Mass isotopologues of citrate after culture of SFxL cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate (Pyr). (D) Citrate m+5 and m+6 after culture of HeLa or Huh-7 cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. Isolated Mitochondria Convert Glutamine to Citrate (A) Western blot of whole-cell lysates (Cell) and preparations of isolated mitochondria (Mito) or cytosol from SFxL cells. (B) Oxygen consumption in a representative mitochondrial sample. Rates before and after addition of ADP/GDP are indicated. (C) Mass isotopologues of citrate produced by mitochondria cultured for 30 min with [U-13C] glutamine and with or without pyruvate.

Figure 3. Blockade of Mitochondrial Pyruvate Transport Activates Glutamine-Dependent Citrate Formation (A) Dose-dependent effects of UK5099 on citrate labeling from [U-13C]glucose and [U-13C]glutamine in SFxL cells. (B) Time course of citrate labeling from [U-13C] glutamine with or without 200 mM UK5099. (C) Abundance of total citrate and citrate m+6 in cells cultured in [U-13C]glutamine with or without 200 mM UK5099. (D) Mass isotopologues of citrate in cells cultured for 6 hr in [U-13C]glutamine with or without 10 mM CHC or 200 mM UK5099. (E) Effect of silencing ME2 on citrate m+6 after 6 hr of culture in [U-13C]glutamine. Relative abundances of citrate isotopologues were determined by normalizing total citrate abundance measured by mass spectrometry against cellular protein for each sample then multiplying by the fractional abundance of each isotopologue. (F) Effect of silencing MPC1 or MPC2 on formation of citrate m+6 after 6 hr of culture in [U-13C]glutamine. (G) Citrate isotopologues in primary human fibroblasts of varying MPC1 genotypes after culture in [U-13C]glutamine. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 4. Kinetic Analysis of the Metabolic Effects of Blocking Mitochondrial Pyruvate Transport (A) Summation of 13C spectra acquired over 2 min of exposure of SFxL cells to hyperpolarized [1-13C] pyruvate. Resonances are indicated for [1-13C] pyruvate (Pyr1), the hydrate of [1-13C]pyruvate (Pyr1-Hydr), [1-13C]lactate (Lac1), [1-13C]alanine (Ala1), and H[13C]O3 (Bicarbonate). (B) Time evolution of appearance of Lac1, Ala1, and bicarbonate in control and UK5099-treated cells. (C) Relative 13C NMR signals for Lac1, Ala1, and bicarbonate. Each signal is summed over the entire acquisition and expressed as a fraction of total 13C signal. (D) Quantity of intracellular and secreted alanine in control and UK5099-treated cells. Data are the average and SD of three independent cultures. *p < 0.05; ***p < 0.001. See also Figure S2.

Figure 5. Inhibiting Mitochondrial Pyruvate Transport Enhances the Contribution of Glutamine to Fatty Acid Synthesis (A) Mass isotopologues of palmitate extracted from cells cultured with [U-13C] glucose or [U-13C]glutamine, with or without 200 mM UK5099. For simplicity, only even-labeled isotopologues (m+2, m+4, etc.) are shown. (B) Fraction of lipogenic acetyl-CoA derived from glucose or glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. ***p < 0.001. See also Figure S3.

Figure 6. Blockade of Mitochondrial Pyruvate Transport Induces GDH (A) Two routes by which glutamate can be converted to AKG. Blue and green symbols are the amide (g) and amino (a) nitrogens of glutamine, respectively. (B) Utilization and secretion of glutamine (Gln), glutamate (Glu), and ammonia (NH4+) by SFxL cells with and without 200 mM UK5099. (C) Secretion of 15N-alanine and 15NH4+ derived from [a-15N]glutamine in SFxL cells expressing a control shRNA (shCtrl) or either of two shRNAs directed against GLUD1 (shGLUD1-A and shGLUD1-B). (D) Left: Phosphorylation of AMPK (T172) and acetyl-CoA carboxylase (ACC, S79) during treatment with 200 mM UK5099. Right: Steady-state levels of ATP 24 hr after addition of vehicle or 200 mM UK5099. (E) Fractional contribution of the m+6 isotopologue to total citrate in shCtrl, shGLUD1-A, and shGLUD1-B SFxL cells cultured in [U-13C]glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 7. GDH Sustains Growth and Viability during Suppression of Mitochondrial Pyruvate Transport (A) Relative growth inhibition of shCtrl, shGLUD1A, and shGLUD1-B SFxL cells treated with 50 mM UK5099 for 3 days. (B) Relative growth inhibition of SFxL cells treated with combinations of 50 mM of the GDH inhibitor EGCG, 10 mM of the GLS inhibitor BPTES, and 200 mM UK5099 for 3 days. (C) Relative cell death assessed by trypan blue staining in SFxL cells treated as in (B). (D) Relative cell death assessed by trypan blue staining in SF188 cells treated as in (B) for 2 days. (E) (Left) Growth of A549-derived subcutaneous xenografts treated with vehicle (saline), EGCG, CHC, or EGCG plus CHC (n = 4 for each group). Data are the average and SEM. Right: Lactate abundance in extracts of each tumor harvested at the end of the experiment. Data in (A)–(D) are the average and SD of three independent cultures. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.

Mitochondrial metabolism complements glycolysis as a source of energy and biosynthetic precursors. Precursors for lipids, proteins, and nucleic acids are derived from the TCA cycle. Maintaining pools of these intermediates is essential, even under circumstances of nutrient limitation or impaired supply of glucose-derived pyruvate to the mitochondria. Glutamine’s ability to produce both acetyl-CoA and OAA allows it to support TCA cycle activity as a sole carbon source and imposes a greater cellular dependence on glutamine metabolism when MPC function or pyruvate supply is impaired. Other anaplerotic amino acids could also supply both OAA and acetyl-CoA, providing flexible support for the TCA cycle when glucose is limiting. Although fatty acids are an important fuel in some cancer cells (Caro et al., 2012), and fatty acid oxidation is induced upon MPC inhibition, this pathway produces acetyl-CoA but not OAA. Thus, fatty acids would need to be oxidized along with an anaplerotic nutrient in order to enable the cycle to function as a biosynthetic hub. Notably, enforced MPC overexpression also impairs growth of some tumors (Schell et al., 2014), suggesting that maximal growth may require MPC activity to be maintained within a narrow window. After decades of research on mitochondrial pyruvate transport, molecular components of the MPC were recently reported (Halestrap, 2012; Schell and Rutter, 2013). MPC1 and MPC2 form a heterocomplex in the inner mitochondrial membrane, and loss of either component impairs pyruvate import, leading to citrate depletion (Bricker et al., 2012; Herzig et al., 2012). Mammalian cells lacking functional MPC1 display normal glutamine-supported respiration (Bricker et al., 2012), consistent with our observation that glutamine supplies the TCA cycle in absence of pyruvate import. We also observed that isolated mitochondria produce fully labeled citrate from glutamine, indicating that this pathway operates as a self-contained mechanism to maintain TCA cycle function. Recently, two well-known classes of drugs have unexpectedly been shown to inhibit MPC. First, thiazolidinediones, commonly used as insulin sensitizers, impair MPC function in myoblasts (Divakaruni et al.,2013). Second, the phosphodiesterase inhibitor Zaprinast inhibits MPC in the retina and brain (Du et al., 2013b). Zaprinast also induced accumulation of aspartate, suggesting that depletion of acetyl-CoA impaired the ability of a new turn of the TCA cycle to be initiated from OAA; as a consequence, OAA was transaminated to aspartate. We noted a similar phenomenon in cancer cells, suggesting that UK5099 elicits a state in which acetyl-CoA supply is insufficient to avoid OAA accumulation. Unlike UK5099, Zaprinast did not induce glutamine-dependent acetyl-CoA formation. This may be related to the reliance of isolated retinas on glucose rather than glutamine to supply TCA cycle intermediates or the exquisite system used by retinas to protect glutamate from oxidation (Du et al., 2013a). Zaprinast was also recently shown to inhibit glutaminase (Elhammali et al., 2014), which would further reduce the contribution of glutamine to the acetyl-CoA pool.

Comment by reader –

The results from these studies served as a good
reason to attempt the vaccination of patients using p53-
derived peptides, and a several clinical trials are currently
in progress. The most advanced work used a long
synthetic peptide mixture derived from p53 (p53-SLP; ISA
Pharmaceuticals, Bilthoven, the Netherlands) (Speetjens
et al., 2009; Shangary et al., 2008; Van der Burg et al.,
2001). The vaccine is delivered in the adjuvant setting
and induces T helper type cells.

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Pancreatic Islets

Writer and Curator: Larry H. Bernstein, MD, FCAP 

Part I. Endocrine Pancreas

The eclipse and rehabilitation of JJR Macleod, Scotland’s insulin laureate

Bliss, M
Journal of the Royal College of Physicians of Edinburgh  2013;  43(4): 1-8

John JR Macleod (1876-1935,) an Aberdonian Scot who had emigrated to North America, shared the 1923 Nobel Prize with Frederick Banting for their discovery of insulin at the University of Toronto in 1921-22. Macleod finished his career as Regius Professor of Physiology at the University of Aberdeen from 1928 to 1935.Macleod’s posthumous reputation was deeply tarnished by the campaigns against him carried out by his fellow laureate, Banting, and by Banting’s student assistant during the insulin research, Charles Best. Banting’s denigration of Macleod was based on their almost total personality conflict; Best’s was based on a hunger for personal recognition. New research indicates how scarred both men were in their obsessions.The rehabilitation of Macleod’s reputation, begun in 1982 with my book, The Discovery of Insulin, has continued in both scholarly and popular circles. By 2012, the ninetieth anniversary of the discovery of insulin, it had become complete both at the University of Toronto and in Canada.

Almost famous: E. Clark Noble, the common thread in the discovery of insulin and vinblastine

Wright Jr., J.R.
CMAJ 2002; 167 (12), pp. 1391-1396

CLARK NOBLE WAS ONE OF THE FIRST members of the University of Toronto insulin team and came within a coin toss of replacing Charles Best as Frederick Banting’s assistant during the summer of 1921. Noble performed important early studies helping to characterize insulin’s action, and he co-authored many of the original papers describing insulin. Because mass production of insulin from livestock pancreata had proved elusive throughout 1922, J.J.R. Macleod hired Noble during the summer of 1923 to help him test and develop a new method for producing commercial quantities of insulin that Macleod believed would revolutionize insulin production. However, commercial production of insulin from fish proved impractical and was dropped by 1924, as methods to produce large quantities of mammalian insulin had improved very rapidly. Noble later played a small but critical role in the most important Canadian contribution to cancer chemotherapy research: the discovery of vinca alkaloids by his brother Robert Laing Noble. Although one might expect that a physician involved in 2 of Canada’s most important medical discoveries during the 20th century must be famous, such was not Clark Noble’s fate. He died without so much as an obituary in CMAJ.

The Pathophysiology of Diabetes and Cardiovascular Disease

Larry H. Bernstein, MD, FCAP, Reviewer and Curator
and Aviva Lev-Ari, PhD, RN, Curator

https://pharmaceuticalintelligence.com/2014/01/15/pathophysiological-effects-of-diabetes-on-ischemic-cardiovascular-disease-and-on-chronic-obstructive-pulmonary-disease-copd/

This is a multipart article that develops the pathological effects of type-2 diabetes in the progression of a systemic inflammatory disease with a development of neuropathy, and fully developing into cardiovascular disease.  It also identifies a systemic relationship to the development of chronic obstructive pulmonary disease. In medical school we were taught that syphilis is the great masquerader. The more we learn about diabetes, we learn about its generalized systemic effects.

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis

This article is an abstract only of a related publication of the pathogenesis of autonomic neuropathy in diabetics leading to ischemic heart disease.

The role of autonomic cardiovascular neuropathy in pathogenesis of ischemic heart disease in patients with diabetes mellitus

Subjects: Medicine (General), Medicine, Medicine (General), Health Sciences
Authors: Popović-Pejičić Snježana, Todorović-Đilas Ljiljana, Pantelinac Pavle
Publisher: Društvo lekara Vojvodine Srpskog lekarskog društva
Publication: Medicinski Pregled 2006; 59(3-4): Pp 118-123 (2006)
http://dx.doi.org/10.2298/MPNS0604118P

http://www.doiserbia.nb.rs/img/doi/0025-8105/2006/0025-81050604118P.pdf

Keywords: diabetes mellitus, autonomic nervous system diseases, heart diseases, myocardial ischemia, comorbidity

Introduction. Diabetes is strongly associated with macrovascular complications, among which ischemic heart disease is the major cause of mortality. Autonomic neuropathy increases the risk of complications, which calls for an early diagnosis. The aim of this study was to determine both presence and extent of cardiac autonomic neuropathy, in regard to the type of diabetes mellitus, as well as its correlation with coronary disease and major cardiovascular risk factors. Material and methods. We have examined 90 subjects, classified into three groups, with 30 patients each: those with type 1 diabetes, type 2 diabetes and control group of healthy subjects. All patients underwent cardiovascular tests (Valsalva maneuver, deep breathing test, response to standing, blood pressure response to standing sustained, handgrip test), electrocardiogram, treadmill exercise test and filled out a questionnaire referring to major cardiovascular risk factors: smoking, obesity, hypertension, and dyslipidemia. Results. Our results showed that cardiovascular autonomic neuropathy was more frequent in type 2 diabetes, manifesting as autonomic neuropathy. In patients with autonomic neuropathy, regardless of the type of diabetes, the treadmill test was positive, i.e. strongly correlating with coronary disease. In regard to coronary disease risk factors, the most frequent correlation was found for obesity and hypertension. Discussion.  Cardiovascular autonomic neuropathy is considered to be the principal cause of arteriosclerosis and coronary disease. Our results showed that the occurrence of cardiovascular autonomic neuropathy increases the risk of coronary disease due to dysfunction of autonomic nervous system. Conclusions. Cardiovascular autonomic neuropathy is a common complication of diabetes that significantly correlates with coronary disease. Early diagnosis of cardiovascular autonomic neuropathy points to increased cardiovascular risk, providing a basis for preventive and therapeutic measures.

Part 2. a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes

Protocol for ADDITION-PRO: a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes recruited from Danish primary care

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Johansen Nanna B, Hansen Anne-Louise S, Jensen Troels M, Philipsen Annelotte, Rasmussen Signe S, Jørgensen Marit E, Simmons Rebecca K, Lauritzen Torsten, Sandbæk Annelli, Witte Daniel R
Publisher: BioMed Central Date of publication: 2012 December
Published in: BMC Public Health 2012; 12(1): 1078
ISSN(s): 1471-2458   Added to DOAJ: 2013-03-12 http://dx.doi.org/10.1186/1471-2458-12-1078 http://www.biomedcentral.com/1471-2458/12/1078

Keywords: Diabetes, Cardiovascular disease, Primary care, Complications, Microvascular, Impaired fasting glucose, Impaired glucose intolerance, Aortic stiffness, Physical activity, Body composition.

Background: Screening programmes for type 2 diabetes inevitably find more individuals at high risk for diabetes than people with undiagnosed prevalent disease. While well established guidelines for the treatment of diabetes exist, less is known about treatment or prevention strategies for individuals found at high risk following screening. In order to make better use of the opportunities for primary prevention of diabetes and its complications among this high risk group, it is important to quantify diabetes progression rates and to examine the development of early markers of cardiovascular disease and microvascular diabetic complications. We also require a better understanding of the mechanisms that underlie and drive early changes in cardiometabolic physiology. The ADDITION-PRO study was designed to address these issues among individuals at different levels of diabetes risk recruited from Danish primary care. Methods/Design: ADDITION-PRO is a population-based, longitudinal cohort study of individuals at high risk for diabetes. 16,136 eligible individuals were identified at high risk following participation in a stepwise screening programme in Danish general practice between 2001 and 2006. All individuals with impaired glucose regulation at screening, those who developed diabetes following screening, and a random sub-sample of those at lower levels of diabetes risk were invited to attend a follow-up health assessment in 2009–2011 (n = 4,188), of whom 2,082 (50%) attended. The health assessment included detailed measurement of anthropometry, body composition, biochemistry, physical activity and cardiovascular risk factors including aortic stiffness and central blood pressure. All ADDITION-PRO participants are being followed for incident cardiovascular disease and death. Discussion: The ADDITION-PRO study is designed to increase understanding of cardiovascular risk and its underlying mechanisms among individuals at high risk of diabetes. Key features of this study include (i) a carefully characterised cohort at different levels of diabetes risk; (ii) detailed measurement of cardiovascular and metabolic risk factors; (iii) objective measurement of physical activity behaviour; and (iv) long-term follow-up of hard clinical outcomes including mortality and cardiovascular disease. Results will inform policy recommendations concerning cardiovascular risk reduction and treatment among individuals at high risk for diabetes. The detailed phenotyping of this cohort will also allow a number of research questions concerning early changes in cardiometabolic physiology to be addressed.

Part 3.  Clinical significance of cardiovascular dysmetabolic syndrome

This third part is a description of a longitudinal cohort study of individuals at high-risk for diabetes.  Unlike the SSA study, the study is not focused on protein-energy malnutrition. This study also addresses the issue of diabetes insulin resistance leading to cardiovascular dysmetabolic syndrome.

Subjects: Diseases of the circulatory (Cardiovascular) system, Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Deedwania Prakash C
Publisher: BioMed Central Date of publication: 2002 January
Published in: Trials 2002; 3: 1(2)
ISSN(s): 1468-6708
Added to DOAJ: 2004-06-03
http://dx.doi.org/10.1186/1468-6708-3-2
Full text: http://cvm.controlled-trials.com/content/3/1/2

Keywords: cardiovascular dysmetabolic syndrome, coronary heart disease, diabetes mellitus, hyperinsulinemia, insulin resistance

Although diabetes mellitus is predominantly a metabolic disorder, recent data suggest that it is as much a vascular disorder. Cardiovascular complications are the leading cause of death and disability in patients with diabetes mellitus. A number of recent reports have emphasized that many patients already have atherosclerosis in progression by the time they are diagnosed with clinical evidence of diabetes mellitus. The increased risk of atherosclerosis and cardiovascular complications in diabetic patients is related to the frequently associated dyslipidemia, hypertension, hyperglycemia, hyperinsulinemia, and endothelial dysfunction.

The evolving knowledge regarding the variety of metabolic, hormonal, and hemodynamic abnormalities in patients with diabetes mellitus has led to efforts designed for early identification of individuals at risk of subsequent disease. It has been suggested that insulin resistance, the key abnormality in type II diabetes, often precedes clinical features of diabetes by 5–6 years.

Careful attention to the criteria described for the cardiovascular dysmetabolic syndrome should help identify those at risk at an early stage. The application of nonpharmacologic as well as newer emerging pharmacologic therapies can have beneficial effects in individuals with cardiovascular dysmetabolic syndrome and/or diabetes mellitus by improving insulin sensitivity and related abnormalities. Early identification and implementation of appropriate therapeutic strategies would be necessary to contain the emerging new epidemic of cardiovascular disease related to diabetes.

Part 4.   Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences Authors: Siren Reijo, Eriksson Johan G, Vanhanen Hannu
Publisher: BioMed Central Date of publication: 2012 August
Published in: BMC Public Health 2012; 12: 1(631)
ISSN(s): 1471-2458
Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1471-2458-12-631
http://www.biomedcentral.com/1471-2458/12/631

Keywords: Waist circumference, Type 2 diabetes, Cardiovascular disease, Middle-aged men.

Background: Abdominal obesity is a more important risk factor than overall obesity in predicting the development of type 2 diabetes and cardiovascular disease. From a preventive and public health point of view it is crucial that risk factors are identified at an early stage, in order to change and modify behaviour and lifestyle in high risk individuals. Methods: Data from a community based study was used to assess the risk for type 2 diabetes, cardiovascular disease and prevalence of metabolic syndrome in middle-aged men. In order to identify those with increased risk for type 2 diabetes and/or cardiovascular disease sensitivity and specificity analysis were performed, including calculation of positive and negative predictive values, and corresponding 95% CI for eleven different cut-off points, with 1 cm intervals (92 to 102 cm), for waist circumference. Results: A waist circumference ≥94 cm in middle-aged men, identified those with increased risk for type 2 diabetes and/or for cardiovascular disease with a sensitivity of 84.4% (95% CI 76.4% to 90.0%), and a specificity of 78.2% (95% CI 68.4% to 85.5%). The positive predictive value was 82.9% (95% CI 74.8% to 88.8%), and negative predictive value 80.0%, respectively (95% CI 70.3% to 87.1%). Conclusions: Measurement of waist circumference in middle-aged men is a reliable test to identify individuals at increased risk for type 2 diabetes and cardiovascular disease. This measurement should be used more frequently in daily practice in primary care in order to identify individuals at risk and when planning health counselling and interventions.

Part 5.  Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

Subjects: Diseases of the circulatory (Cardiovascular) system, Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Mirrakhimov Aibek E
Publisher: BioMed Central
Date of publication: Oct 2012
ISSN(s): 1475-2840
ADDED to DOAJ: 2013-03-12
Published in: Cardiovascular Diabetology 2012; 11(1):132
Journal Language(s): English Country of publication: United Kingdom
http://dx.doi.org:/10.1186/1475-2840-11-132
Full text: http://www.cardiab.com/content/11/1/132

Keywords: COPD, Dysglycemia, Insulin resistance, Obesity, Metabolic syndrome, Diabetes mellitus endothelial dysfunction, Vasculopathy

Chronic obstructive pulmonary disease, metabolic syndrome and diabetes mellitus are common and underdiagnosed medical conditions. It was predicted that chronic obstructive pulmonary disease will be the third leading cause of death worldwide by 2020. The healthcare burden of this disease is even greater if we consider the significant impact of chronic obstructive pulmonary disease on the cardiovascular morbidity and mortality.

Chronic obstructive pulmonary disease may be considered as a novel risk factor for new onset type 2 diabetes mellitus via multiple pathophysiological alterations such as: inflammation and oxidative stress, insulin resistance, weight gain and alterations in metabolism of adipokines.

On the other hand, diabetes may act as an independent factor, negatively affecting pulmonary structure and function. Diabetes is associated with an increased risk of pulmonary infections, disease exacerbations and worsened COPD outcomes. On the top of that, coexistent OSA may increase the risk for type 2 DM in some individuals.

The current scientific data necessitate a greater outlook on chronic obstructive pulmonary disease and chronic obstructive pulmonary disease may be viewed as a risk factor for the new onset type 2 diabetes mellitus. Conversely, both types of diabetes mellitus should be viewed as strong contributing factors for the development of obstructive lung disease. Such approach can potentially improve the outcomes and medical control for both conditions, and, thus, decrease the healthcare burden of these major medical problems.

The Economic Costs of Diabetes: Is It Time for a New Treatment Paradigm?

Commentary: William H. Herman
Diabetes Care Apr 2013; 36: 775-776

In a series of rigorous and exhaustive descriptive cost analyses conducted over the past decade, the American Diabetes Association (ADA) has documented an inexorable increase in the cost of diabetes in the U.S. and its detrimental impact on productivity. For the 2012 study, the ADA estimated that there were 22.3 million Americans diagnosed with diabetes. These patients incurred $306 billion in direct medical costs, more than 1 of 5 dollars spent on medical care in the U.S. The direct medical costs attributed to diabetes, that is, the costs of medical care for people with diabetes in excess of those that would be expected in the absence of diabetes, were $176 billion or approximately 1 of 8 dollars spent on medical care in the U.S. Americans with diagnosed diabetes have annual medical expenditures that are $7,900 or approximately 2.3 times higher than they would be in the absence of diabetes ($13,700 vs. $5,800). Americans with diabetes also incur $69 billion in costs related to absenteeism, reduced productivity while at work or at home, diabetes-related disability, and premature mortality. The increasing economic burden of diabetes is due in large part to the increase in the number of people with diagnosed diabetes.

Randomized controlled clinical trials have demonstrated that intensive glycemic management can delay the onset of microvascular, neuropathic, and cardiovascular complications in people with both type 1 and type 2 diabetes, and that the benefits of early intensive treatment persist over time. Randomized controlled clinical trials have also demonstrated that blood pressure management (target blood pressure 135/80 mmHg) and lipid management using statin medications can delay or prevent the development of adverse cardiovascular outcomes.

The growing economic and societal burden of diabetes as documented by the ADA in this issue of Diabetes Care highlights the urgent need to implement interventions to delay the development of type 2 diabetes. Both intensive lifestyle and pharmacologic interventions are proven effective and cost-effective. Health policy should support their implementation.

Complimentary societal interventions to delay the onset of type 2 diabetes include school-based health promotion programs and interventions that address advertising, food availability and price, the built and workplace environment, and even tax policy. In addition, early aggressive management of glycemia and cardiovascular risk factors must be implemented for persons diagnosed with diabetes. Increasing access to care, including self management education and nutritional counseling, and ensuring access to necessary treatments and supplies are critical, especially in light of the proven value of early intensive treatment in preventing chronic complications. The cost estimates provided by the ADA from 2002, 2007, and 2012 show that the economic and societal burden of diabetes is growing in the U.S. This trend underscores the importance of prevention and interventions to mitigate the complications of diabetes.

Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells

Liew, C.W., Assmann, A., Templin, A.T., (…), Urano, F., Kulkarni, R.N
2014 Proc National Academy of Sciences  USA  111 (22), pp. E2319-E2328

Insulin resistance, hyperinsulinemia, and hyperproinsulinemia occur early in the pathogenesis of type 2 diabetes (T2D). Elevated levels of proinsulin and proinsulin intermediates are markers of β-cell dysfunction and are strongly associated with development of T2D in humans. However, the mechanism(s) underlying β-cell dysfunction leading to hyperproinsulinemia is poorly understood. Here, we show that disruption of insulin receptor (IR) expression in β cells has a direct impact on the expression of the convertase enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic translation initiation factor 4 gamma 1 translation initiation complex scaffolding protein that is mediated by the key transcription factors pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1, together leading to poor proinsulin processing. Reexpression of IR or restoring CPE expression each independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic insight into the development of hyperproinsulinemia in insulin-resistant states.

Disruption of growth factor receptor-binding protein 10 in the pancreas enhances β-cell proliferation and protects mice from streptozotocin-induced β-cell apoptosis

Zhang, J., Zhang, N., Liu, M., (…), Lu, X.-Y., Liu, F.
2014 Environmental Science and Technology 48 (9), pp. 5179-5186

It has been reported that organotin compounds such as triphenyltin or tributyltin (TBT) induce diabetes and insulin resistance. However, histopathological effects of organotin compounds on the Islets of Langerhans and exocrine pancreas are still unclear. In the present study, male KM mice were orally administered with TBT (0.5, 5, and 50 µg/kg) once every 3 days. The fasting plasma glucose levels significantly elevated, and the levels of serum insulin or glucagon decreased in the animals treated with TBT for 60 days. In animals treated for 45 days, the number of apoptotic cells in the islets and exocrine pancreas was elevated in a dose-dependent manner. The percentage of proliferating (PCNA-positive) cells was decreased in the islets, while it was increased in exocrine acinar cells. Immunohistochemistry analysis showed that estrogen receptor (ER) and androgen receptor (AR) were present in vascular endothelium, ductal cells, and islet cells, but absent from pancreatic exocrine cells. TBT exposure decreased the production of estradiol and triiodothyronine and elevated the concentration of testosterone, and resulted in a decrease of ERβ expression and an elevation of AR in the pancreas measured by Western blotting. The results suggested that TBT inhibited the proliferation and induced the apoptosis of islet cells via multipathways, causing a decrease of relative islet area in the animals treated for 60 days, which could result in a disruption of glucose homeostasis. The different presence of ERs and AR between the islets and exocrine pancreas might be one of reasons causing different effects on cell proliferation

Pancreatic alpha-cell dysfunction contributes to the disruption of glucose homeostasis and compensatory insulin hypersecretion in glucocorticoid-treated rats

Rafacho, A., Gonçalves-Neto, L.M., Santos-Silva, J.C., (…), Nadal, A., Quesada, I.
2014 Journal of Biological Chemistry 289 (9), pp. 6028-604

In α-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (KATP channels). PIP2 binds the Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP 2 on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP 2 dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1-(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat α-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP2 dose-dependent activation of KATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP2 actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP 2-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP2, consequently reducing PIP2 activation of KATP channels. Next, Syn-1A·SUR1 complex modulation of KATP channels could be observed at a physiologically low PIP 2 concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP2 concentrations acting directly on Kir6.2. These effects were specific to PIP2 and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP 2 with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of KATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP2 affects islet β-cell KATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.

Aging and sleep deprivation induce the unfolded protein response in the pancreas: Implications for metabolism

Naidoo, N., Davis, J.G., Zhu, J., (…), Agarwal, B., Baur, J.A.
2014 Aging Cell 13 (1), pp. 131-141

Sleep disruption has detrimental effects on glucose metabolism through pathways that remain poorly defined. Although numerous studies have examined the consequences of sleep deprivation (SD) in the brain, few have directly tested its effects on peripheral organs. We examined several tissues in mice for induction of the unfolded protein response (UPR) following acute SD. In young animals, we found a robust induction of BiP in the pancreas, indicating an active UPR. At baseline, pancreata from aged animals exhibited a marked increase in a pro-apoptotic transcription factor, CHOP, that was amplified by SD, whereas BiP induction was not observed, suggesting a maladaptive response to cellular stress with age. Acute SD increased plasma glucose levels in both young and old animals. However, this change was not overtly related to stress in the pancreatic beta cells, as plasma insulin levels were not lower following acute SD. Accordingly, animals subjected to acute SD remained tolerant to a glucose challenge. In a chronic SD experiment, young mice were found to be sensitized to insulin and have improved glycemic control, whereas aged animals became hyperglycemic and failed to maintain appropriate plasma insulin concentrations. Our results show that both age and SD cooperate to induce the UPR in pancreatic tissue. While changes in insulin secretion are unlikely to play a major role in the acute effects of SD, CHOP induction in pancreatic tissues suggests that chronic SD may contribute to the loss or dysfunction of endocrine cells and that these effects may be exacerbated by normal aging

Bayesian total internal reflection fluorescence correlation spectroscopy reveals hIAPP-induced plasma membrane domain organization in live cells

Guo, S.-M., Bag, N., Mishra, A., Wohland, T., Bathe, M.
2014 Biophysical Journal 106 (1), pp. 190-200

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K + channel activity

Nomura, M., Zhu, H.-L., Wang, L., (…), Takayanagi, R., Teramoto, N.
2014 Diabetologia 57 (1), pp. 157-166

Aims/hypothesis: The TGF-β superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-β family signalling pathway is required for essential islet functions in the adult pancreas. Methods: To identify distinct roles for the downstream components of the canonical TGF-β signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-β signals, in pancreatic beta cells (i.e. Smad2-β- knockout [KO] mice). The activity of ATP-sensitive K+ channels (KATP channels) was recorded in mutant beta cells using patch-clamp techniques. Results: The Smad2-β-KO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in β-cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of KATP channels was decreased in mutant β-cells. Conclusions/interpretation: These results suggest that in the adult pancreas, TGF-β signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of KATP channel activity.

Disruption of protein-tyrosine phosphatase 1B expression in the pancreas affects β-cell function

Liu, S., Xi, Y., Bettaieb, A., (…), Kulkarni, R.N., Haj, F.G.
2014 Endocrinology 155 (9), pp. 3329-3338

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and energy balance. However, the role of PTP1B in pancreatic endocrine function remains largely unknown. To investigate the metabolic role of pancreatic PTP1B, we generated mice with pancreas PTP1B deletion (panc-PTP1B KO). Mice were fed regular chow or a high-fat diet, and metabolic parameters, insulin secretion and glucose tolerance were determined. On regular chow, panc-PTP1B KO and control mice exhibited comparable glucose tolerance whereas aged panc-PTP1B KO exhibited mild glucose intolerance. Furthermore, high-fat feeding promoted earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion in panc-PTP1B KO mice. The secretory defect in glucose-stimulated insulin secretion was recapitulated in primary islets ex vivo, suggesting that the effects were likely cell-autonomous. At the molecular level, PTP1B deficiency in vivo enhanced basal and glucose-stimulated tyrosyl phosphorylation of EphA5 in islets. Consistently, PTP1B overexpression in the glucose-responsive MIN6 β-cell line attenuated EphA5 tyrosyl phosphorylation, and substrate trapping identified EphA5 as a PTP1B substrate. In summary, these studies identify a novel role forPTP1Bin pancreatic endocrine function.

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

Farnsworth, N.L., Hemmati, A., Pozzoli, M., Benninger, R.K.P.
2014 Journal of Physiology 592 (20), pp. 4431-4446

Key points: Gap junctions provide electrical coupling that is critical to the function of pancreatic islets. Disruptions to connexin36 (Cx36) have been suggested to occur in diabetes. No accurate and non-invasive technique has yet been established to quantify changes in Cx36 gap junction coupling in the intact islet. This study developed fluorescence recovery after photobleaching (FRAP) as a non-invasive technique for quantifying Cx36 gap junction coupling in living islets. The study identified treatments that modulate gap junction coupling, confirmed that the cellular distribution of coupling throughout the islet is highly heterogeneous and confirmed that β-cells and β-cells do not form functional Cx36 gap junctions. This technique will enable future studies to examine the regulation of Cx36 gap junction coupling and its disruption in diabetes, and to uncover potential novel therapeutic targets associated with gap junction coupling. The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β-cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β-cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β-cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent  modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between β-cells and β-cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Glucocorticoid treatment and endocrine pancreas function: Implications for glucose homeostasis, insulin resistance and diabetes

Rafacho, A., Ortsäter, H., Nadal, A., Quesada, I.
2014 Journal of Endocrinology 223 (3), pp. R49-R62

Glucocorticoids (GCs) are broadly prescribed for numerous pathological conditions because of their anti-inflammatory, antiallergic and immunosuppressive effects, among other actions. Nevertheless, GCs can produce undesired diabetogenic side effects through interactions with the regulation of glucose homeostasis. Under conditions of excess and/or long-term treatment, GCs can induce peripheral insulin resistance (IR) by impairing insulin signalling, which results in reduced glucose disposal and augmented endogenous glucose production. In addition, GCs can promote abdominal obesity, elevate plasma fatty acids and triglycerides, and suppress osteocalcin synthesis in bone tissue. In response to GC-induced peripheral IR and in an attempt to maintain normoglycaemia, pancreatic β-cells undergo several morphofunctional adaptations that result in hyperinsulinaemia. Failure of β-cells to compensate for this situation favours glucose homeostasis disruption, which can result in hyperglycaemia, particularly in susceptible individuals. GC treatment does not only alter pancreatic β-cell function but also affect them by their actions that can lead to hyperglucagonaemia, further contributing to glucose homeostasis imbalance and hyperglycaemia. In addition, the release of other islet hormones, such as somatostatin, amylin and ghrelin, is also affected by GC administration. These undesired GC actions merit further consideration for the design of improved GC therapies without diabetogenic effects. In summary, in this review, we consider the implication of GC treatment on peripheral IR, islet function and glucose homeostasis.

β-Cell failure in type 2 diabetes: Postulated mechanisms and prospects for prevention and treatment

Halban, P.A., Polonsky, K.S., Bowden, D.W., (…), Sussel, L., Weir, G.C.
2014 Journal of Clinical Endocrinology and Metabolism 99 (6), pp. 1983-1992

OBJECTIVE: This article examines the foundation of β-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of β-cell failure, natural history of β-cell failure, and impact of therapeutic interventions. CONCLUSIONS: β-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased β-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of β-cell failure; 2) identify and characterize genetic loci for T2D; 3) target β-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of β-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to β-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and β-cells.

Metabolic effects of sleep disruption, links to obesity and diabetes

Nedeltcheva, A.V., Scheer, F.A.J.L
2014 Current Opinion in Endocrinology, Diabetes and Obesity 21 (4), pp. 293-298

Purpose of Review: To highlight the adverse metabolic effects of sleep disruption and to open ground for research aimed at preventive measures. This area of research is especially relevant given the increasing prevalence of voluntary sleep curtailment, sleep disorders, diabetes, and obesity. Recent Findings: Epidemiological studies have established an association between decreased self-reported sleep duration and an increased incidence of type 2 diabetes (T2D), obesity, and cardiovascular disease. Experimental laboratory studies have demonstrated that decreasing either the amount or quality of sleep decreases insulin sensitivity and decreases glucose tolerance. Experimental sleep restriction also causes physiological and behavioral changes that promote a positive energy balance. Although sleep restriction increases energy expenditure because of increased wakefulness, it can lead to a disproportionate increase in food intake, decrease in physical activity, and weight gain. SUMMARY: Sleep disruption has detrimental effects on metabolic health. These insights may help in the development of new preventive and therapeutic approaches against obesity and T2D based on increasing the quality and/or quantity of sleep. Video abstract http://links.lww.com/COE/A6.

Impaired proteostasis: Role in the pathogenesis of diabetes mellitus

Jaisson, S., Gillery, P.
2014 Diabetologia 57 (8), pp. 1517-1527

In living organisms, proteins are regularly exposed to ‘molecular ageing’, which corresponds to a set of non-enzymatic modifications that progressively cause irreversible damage to proteins. This phenomenon is greatly amplified under pathological conditions, such as diabetes mellitus. For their survival and optimal functioning, cells have to maintain protein homeostasis, also called ‘proteostasis’. This process acts to maintain a high proportion of functional and undamaged proteins. Different mechanisms are involved in proteostasis, among them degradation systems (the main intracellular proteolytic systems being proteasome and lysosomes), folding systems (including molecular chaperones), and enzymatic mechanisms of protein repair. There is growing evidence that the disruption of proteostasis may constitute a determining event in pathophysiology. The aim of this review is to demonstrate how such a dysregulation may be involved in the pathogenesis of diabetes mellitus and in the onset of its long-term complications.

Influence of miRNA in insulin signaling pathway and insulin resistance: Micro-molecules with a major role in type-2 diabetes

Chakraborty, C., Doss, C.G.P., Bandyopadhyay, S., Agoramoorthy, G.
2014 Wiley Interdisciplinary Reviews: RNA 5 (5), pp. 697-712

The prevalence of type-2 diabetes (T2D) is increasing significantly throughout the globe since the last decade. This heterogeneous and multifactorial disease, also known as insulin resistance, is caused by the disruption of the insulin signaling pathway. In this review, we discuss the existence of various miRNAs involved in regulating the main protein cascades in the insulin signaling pathway that affect insulin resistance. The influence of miRNAs (miR-7, miR-124α, miR-9, miR-96, miR-15α/β, miR-34α, miR-195, miR-376, miR-103, miR-107, and miR-146) in insulin secretion and beta (β) cell development has been well discussed. Here, we highlight the role of miRNAs in different significant protein cascades within the insulin signaling pathway such as miR-320, miR-383, miR-181β with IGF-1, and its receptor (IGF1R); miR-128α, miR-96, miR-126 with insulin receptor substrate (IRS) proteins; miR-29, miR-384-5p, miR-1 with phosphatidylinositol 3-kinase (PI3K); miR-143, miR-145, miR-29, miR-383, miR-33α/β miR-21 with AKT/protein kinase B (PKB) and miR-133α/β, miR-223, miR-143 with glucose transporter 4 (GLUT4). Insulin resistance, obesity, and hyperlipidemia (high lipid levels in the blood) have a strong connection with T2D and several miRNAs influence these clinical outcomes such as miR-143, miR-103, and miR-107, miR-29α, and miR-27β. We also corroborate from previous evidence how these interactions are related to insulin resistance and T2D. The insights highlighted in this review will provide a better understanding on the impact of miRNA in the insulin signaling pathway and insulin resistance-associated diagnostics and therapeutics for T2D

Genetic disruption of sod1 gene causes glucose intolerance and impairs β-cell function

Muscogiuri, G., Salmon, A.B., Aguayo-Mazzucato, C., (…), Van Remmen, H., Musi, N.
2013 Diabetes 62 (12), pp. 4201-4207

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased b-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow-fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction.

VHL-mediated disruption of Sox9 activity compromises β-cell identity and results in diabetes mellitus

Puri, S., Akiyama, H., Hebrok, M.
2013 Genes and Development 27 (23), pp. 2563-2575

Precise functioning of the pancreatic β cell is paramount to whole-body glucose homeostasis, and β-cell dysfunction contributes significantly to diabetes mellitus. Using transgenic mouse models, we demonstrate that deletion of the von Hippel-Lindau (Vhlh) gene (encoding an E3 ubiquitin ligase implicated in, among other functions, oxygen sensing in pancreatic β cells) is deleterious to canonical β-cell gene expression. This triggers erroneous expression of factors normally active in progenitor cells, including effectors of the Notch, Wnt, and Hedgehog signaling cascades. Significantly, an up-regulation of the transcription factor Sox9, normally excluded from functional β cells, occurs upon deletion of Vhlh. Sox9 plays important roles during pancreas development but does not have a described role in the adult β cell. β-Cell-specific ectopic expression of Sox9 results in diabetes mellitus from similar perturbations in β-cell identity. These findings reveal that assaults on the β cell that impact the differentiation state of the cell have clear implications toward our understanding of diabetes mellitus

Second generation antipsychotic-induced type 2 diabetes: A role for the muscarinic M3 receptor

Weston-Green, K., Huang, X.-F., Deng, C.
2013 CNS Drugs 27 (12), pp. 1069-1080

Second generation antipsychotics (SGAs) are widely prescribed to treat various disorders, most notably schizophrenia and bipolar disorder; however, SGAs can cause abnormal glucose metabolism that can lead to insulin-resistance and type 2 diabetes mellitus side-effects by largely unknown mechanisms. This review explores the potential candidature of the acetylcholine (ACh) muscarinic M3 receptor (M3R) as a prime mechanistic and possible therapeutic target of interest in SGA-induced insulin dysregulation. Studies have identified that SGA binding affinity to the M3R is a predictor of diabetes risk; indeed, olanzapine and clozapine, SGAs with the highest clinical incidence of diabetes side-effects, are potent M3R antagonists. Pancreatic M3Rs regulate the glucose-stimulated cholinergic pathway of insulin secretion; their activation on β-cells stimulates insulin secretion, while M3R blockade decreases insulin secretion. Genetic modification of M3Rs causes robust alterations in insulin levels and glucose tolerance in mice. Olanzapine alters M3R density in discrete nuclei of the hypothalamus and caudal brainstem, regions that regulate glucose homeostasis and insulin secretion through vagal innervation of the pancreas. Furthermore, studies have demonstrated a dynamic sensitivity of hypothalamic and brainstem M3Rs to altered glucometabolic status of the body. Therefore, the M3R is in a prime position to influence glucose homeostasis through direct effects on pancreatic β-cells and by potentially altering signaling in the hypothalamus and brainstem. SGA-induced insulin dysregulation may be partly due to blockade of central and peripheral M3Rs, causing an initial disruption to insulin secretion and glucose homeostasis that can progressively lead to insulin resistance and diabetes during chronic treatment.

Islet amyloid polypeptide toxicity and membrane interactions

Cao, P., Abedini, A., Wang, H., (…), Schmidt, A.M., Raleigh, D.P.
2013 Proc National Academy of Sciences USA  110 (48), pp. 19279-19284

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth.  Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of α-sheet or α-helical structures.  A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

Diabetes and beta cell function: From mechanisms to evaluation and clinical implications

Cernea, S., Dobreanu, M.
2013 Biochemia Medica 23 (3), pp. 266-280

Diabetes is a complex, heterogeneous condition that has beta cell dysfunction at its core. Many factors (e.g. hyperglycemia/glucotoxicity, lipotoxicity, autoimmunity, inflammation, adipokines, islet amyloid, incretins and insulin resistance) influence the function of pancreatic beta cells. Chronic hyperglycemia may result in detrimental effects on insulin synthesis/secretion, cell survival and insulin sensitivity through multiple mechanisms: gradual loss of insulin gene expression and other beta-cell specific genes; chronic endoplasmic reticulum stress and oxidative stress; changes in mitochondrial number, morphology and function; disruption in calcium homeostasis. In the presence of hyperglycemia, prolonged exposure to increased free fatty acids result in accumulation of toxic metabolites in the cells (“lipotoxicity”), finally causing decreased insulin gene expression and impairment of insulin secretion. The rest of the factors/mechanisms which impact on the course of the disease are also discusses in detail. The correct assessment of beta cell function requires a concomitant quantification of insulin secretion and insulin sensitivity, because the two variables are closely interrelated. In order to better understand the fundamental pathogenetic mechanisms that contribute to disease development in a certain individual with diabetes, additional markers could be used, apart from those that evaluate beta cell function. The aim of the paper was to overview the relevant mechanisms/factors that influence beta cell function and to discuss the available methods of its assessment. In addition, clinical considerations are made regarding the therapeutical options that have potential protective effects on beta cell function/mass by targeting various underlying factors and mechanisms with a role in disease progression.

The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human β-cells and its targeted inactivation impairs glucose tolerance

Prevost, G., Arabo, A., Jian, L., (…), Pattou, F., Anouar, Y
2013 Endocrinology 154 (10), pp. 3796-3806

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in β-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic α- and β-cells and is involved in the control of glucose homeostasis

SIRT1 deacetylates FOXA2 and is critical for Pdx1 transcription and β-cell formation

Wang, R.-H., Xu, X., Kim, H.-S., Xiao, Z., Deng, C.-X.
2013 International Journal of Biological Sciences 9 (9), pp. 934-946

Pancreas duodenum homeobox 1 (PDX1) is essential for pancreas development and β-cell formation; however more studies are needed to clearly illustrate the precise mechanism regarding spatiotemporal regulation of Pdx1 expression during β-cell formation and development. Here, we demonstrate that SIRT1, FOXA2 and a number of proteins form a protein complex on the promoter of the Pdx1 gene. SIRT1 and PDX1 are expressed in the same set of cells during β-cell differentiation and maturation. Pancreas-specific disruption of SIRT1 diminished PDX1 expression and impaired islet development. Consequently, SIRT1 mutant mice develop progressive hyperglycemia, glucose intolerance, and insulin insufficiency, which directly correlate with the extent of SIRT1 deletion. We further show that SIRT1 interacts with and deacetylates FOXA2 on the promoter of the Pdx1gene, and positively regulates its transcription. These results uncover an essential role of SIRT1 in β-cell formation by maintaining expression of PDX1 and its downstream genes, and identify pancreas-specific SIRT1 mutant mice as a relevant model for studying insulin insufficiency.

NOX, NOX who is there? The contribution of NADPH oxidase one to beta cell dysfunction

Taylor-Fishwick, D.A.
2013 Frontiers in Endocrinology 4 (APR), Article 40

Predictions of diabetes prevalence over the next decades warrant the aggressive discovery of new approaches to stop or reverse loss of functional beta cell mass. Beta cells are recognized to have a relatively high sensitivity to reactive oxygen species (ROS) and become dysfunctional under oxidative stress conditions. New discoveries have identified NADPH oxidases in beta cells as contributors to elevated cellular ROS. Reviewed are recent reports that evidence a role for NADPH oxidase-1 (NOX-1) in β-cell dysfunction. NOX-1 is stimulated by inflammatory cytokines that are elevated in diabetes. First, regulation of cytokine-stimulated NOX-1 expression has been linked to inflammatory lipid mediators derived from 12-lipoxygenase activity. For the first time in beta cells these data integrate distinct pathways associated with beta cell dysfunction. Second, regulation of NOX-1 in
β-cells involves feed-forward control linked to elevated ROS and Src-kinase activation. This potentially results in unbridled ROS generation and identifies candidate targets for pharmacologic intervention. Third, consideration is provided of new, first-in-class, selective inhibitors of NOX-1. These compounds could have an important role in assessing a disruption of NOX-1/ROS signaling as a new approach to preserve and protect beta cell mass in diabetes.

Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and β-cell fate

Cai, E.P., Wu, X., Schroer, S.A., (…), Zacksenhaus, E., Woo, M.
2013 Proc National Academy of Sciences USA 110 (36), pp. 14723-14728

Pancreatic endocrine cells expand rapidly during embryogenesis by neogenesis and proliferation, but during adulthood, islet cells have a very slow turnover. Disruption of murine retinoblastoma tumor suppressor protein (Rb) in mature pancreatic β-cells has a limited effect on cell proliferation. Here we show that deletion of Rb during embryogenesis in islet progenitors leads to an increase in the neurogenin 3-expressing precursor cell population, which persists in the postnatal period and is associated with increased β-cell mass in adults. In contrast, Rb-deficient islet precursors, through repression of the cell fate factor aristaless related homeobox, result in decreased β-cell mass. The opposing effect on survival of Rb-deficient β- and β-cells was a result of opposing effects on p53 in these cell types. As a consequence, loss of Rb in islet precursors led to a reduced α- to β-cell ratio, leading to improved glucose homeostasis and protection against diabetes.

Statin therapy and new-onset diabetes: Molecular mechanisms and clinical relevance

Banach, M., Malodobra-Mazur, M., Gluba, A., (…), Rysz, J., Dobrzyn, A.
2013 Current Pharmaceutical Design 19 (27), pp. 4904-4912

Despite positive effects on the plasma lipid profile and vascular events, statin use is associated with various side effects. Among these, statins might cause a disruption of a number of regulatory pathways including insulin signaling. This may affect insulin sensitivity, pancreatic beta-cell function and adipokine secretion. The statin-associated risk of new-onset diabetes (NOD) appears to be a dose-dependent class effect. It still remains unclear whether statin treatment is associated with increased risk of NOD in the general population or if there are groups of individuals at particular risk. However, according to the available data it seems that cardiovascular (CV) benefits in high-risk individuals strongly favor statin therapy since it outweighs other risks. Whether statins should be used for primary prevention among patients with a relatively low baseline CV risk is still questionable, however the results of primary prevention trials have shown reductions in mortality in this population. Thus, there is a need for randomized, placebo-controlled statin studies with carefully selected groups of patients and NOD as a key end point in order to resolve queries concerning this issue.

Basement membrane extract preserves islet viability and activity in vitro by up-regulating α3 integrin and its signal

Miao, G., Zhao, Y., Li, Y., (…), Li, J., Wei, J
2013 Pancreas 42 (6), pp. 971-976

OBJECTIVE: Survival of transplanted islets is limited partly because of the disruption of the islet basement membrane (BM) occurring during isolation. We hypothesized that the embedment of BM extract (BME) could induce a viable cell mass and prolong islet functionality before transplantation. METHODS: A special reconstituted BME that solidifies into a gel at 37 C was used to embed isolated islets in this study. The strategy was used to re-establish the interaction between the islets and peri-islet BM. RESULTS: Islets embedded in BME showed lower caspase-3 levels and higher Akt activity than those in suspension. Moreover, we found for the first time that the expression of β3 integrin and focal adhesion kinase (FAK) and FAK activity was up-regulated in islets after BME embedment. The reverse effect was observed on islet apoptosis when islets rescued from a 24-hour suspension culture were embedded in BME for the next 24 hours. In addition, expression of pancreatic duodenal homeobox factor-1 and phospho-extracellular signal-regulated kinase 1/2 was partially preserved, suggesting the positive effect of BME on islet development. CONCLUSIONS: These results indicate that BME embedment of islets can up-regulate the expression of β3 integrin and its signal transduction, which may improve islet viability.

Involvement of the Clock Gene Rev-erb alpha in the Regulation of Glucagon Secretion in Pancreatic Alpha-Cells

Vieira, E., Marroquí, L., Figueroa, A.C., (…), Gomis, R., Quesada, I.
2013 PLoS ONE 8 (7), e6993

Disruption of pancreatic clock genes impairs pancreatic β-cell function, leading to the onset of diabetes. Despite the importance of pancreatic α-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erbα as a new intracellular regulator of glucagon secretion. Rev-erbα down-regulation by siRNA (60-70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erbα agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in αTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erbα  antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erbα expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.

Bmal1 and β-cell clock are required for adaptation to circadian disruption, and their loss of function leads to oxidative stress- induced β-cell failure in mice

Lee, J., Moulik, M., Fang, Z., (…), Moore, D.D., Yechoor, V.K.
2013 Molecular and Cellular Biology 33 (11), pp. 2327-2338

Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in β-cell dysfunction and diabetes. But  it is unknown if this is due to loss of cell-autonomous function of Bmal1 in β cells. To address this, we generated mice with β-cell clock disruption by deleting Bmal1 in β cells (β-Bmal1-/-).  β-Bmal1-/- mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in β-Bmal1-/- islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets.

Thus, the cell-autonomous function of Bmal1 is required for normal β-cell function by mitigating oxidative stress and serves to preserve β-cell function in the face of circadian misalignment.

A common landscape for membraneactive peptides

Last, N.B., Schlamadinger, D.E., Miranker, A.D.
2013 Protein Science 22 (7), pp. 870-882

Three families of membrane-active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer’s to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell-penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane-active families of peptides simply represent different facets of what is a shared energy landscape.

Membrane disordering is not sufficient for membrane permeabilization by islet amyloid polypeptide: Studies of IAPP(20-29) fragments

Brender, J.R., Heyl, D.L., Samisetti, S., (…), Pesaru, R.R., Ramamoorthy, A.
2013 Physical Chemistry Chemical Physics 15 (23), pp. 8908-8915

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and a later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected.
Downregulation of Fas activity rescues early onset of diabetes in c-KitWv/+ mice

Feng, Z.-C., Riopel, M., Li, J., Donnelly, L., Wang, R.
2013 American Journal of Physiology – Endocrinology and Metabolism 304 (6), pp. E557-E565

c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-KitWv/++ mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-KitWv/++;Faslpr/lpr (Wv-/-), mice to study the physiological and functional role of Fas with respect to β-cell function in c-KitWv/++ mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-KitWv/++ mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv-/- mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv+/+ mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-?B pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.
Study Suggests Genetic Susceptibility to T2D May Have Shifted with Human Migration

May 24, 2013  By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – The apparent genetic risk for type 2 diabetes seems to vary between human populations from different parts of the world, new research suggests, with populations in Africa and East Asia showing particularly pronounced differences in T2D susceptibility.

A pair of papers appearing online — both led by investigators at Stanford University — outline the approaches and analyses used to reach that conclusion.

For the first study, published in PLOS Genetics, researchers trolled through data on more than 1,000 individuals from around the world who’d been genotyped for the Human Genome Diversity Panel project. Patterns in that data revealed geography or population-related differentiation in the genetic risk associated with certain diseases.

“We demonstrated that differences in genetic risk for multiple diseases go well beyond what is expected by genetic drift,” the study authors noted. “In addition, using a human population phylogenetic tree allowed us to elucidate a substructure of worldwide relationships.”

In the East Asian population, for instance, the team saw diminished genetic risk for both T2D and pancreatic cancer. On the other hand, individuals of African ancestry appeared to be more apt to carry T2D risk alleles, results of the analysis suggest, pointing to possible migration-related shifts in genetic susceptibility to T2D.

For their PLOS Genetics analysis, the researchers used data for 1,043 individuals genotyped for the HGDP to delve into the genetic risk associated with more than 100 diseases, including T2D.

Because the individuals hailed from 51 different populations around the world, the group was able to get a glimpse at relationships between these genetic risk contributors and human migration and population patterns.

From that data, investigators saw at least 11 conditions for which risk variant profiles differed across human populations, researchers reported, including ulcerative colitis, bladder cancer, lupus, and inflammatory bowel disease.

For T2D, that genetic differentiation appeared to correspond with population patterns stemming from human migrations out of Africa and into other parts of the world. For instance, the analysis indicated that genetic risk for T2D dips in East Asian populations but tends to be elevated in populations from Africa — particularly the Mandinka population, which appeared to be at highest genetic risk of T2D.

“East Asians definitely get diabetes,” Stanford University’s Atul Butte, senior author on the study, said in a statement.

Nevertheless, he added, it’s possible that there are population-specific differences in the risk alleles and genetic pathways involved, potentially producing somewhat distinct forms of the disease.

Those involved in the study noted that additional, follow-up research is needed, including whole-genome sequencing analysis, which can offer a look at larger structural variants contributing to disease risk in different populations, for instance.

But if findings from the current analysis hold in future studies, that may ultimately prompt a shift in researchers’ understanding of T2D and the factors contributing to it.

“Other fields of medicine have undergone a radical rethinking in disease taxonomy,” Butte said in a statement, “but this has not happened yet for diabetes, one of the world’s public health menaces.”

“If these are separate diseases at a molecular level, we need to try to understand that,” he added.

A related study in the journal Diabetes Care, also by Stanford’s Butte and his colleagues, touched on the consequences of such genetic differences. That work highlighted apparent clinical differences in T2D-related traits — particularly in insulin resistance and insulin response — in African, East Asian, and Caucasian populations.

More generally, Butte and his colleagues put together a so-called “Genetic Risk World Map” to tie together the information generated from their study of disease risk genetics in the context of human migration. The resource is available online through a Stanford website.
Use of pioglitazone in the treatment of diabetes: effect on cardiovascular risk

Authors: Zou C, Hu H
Published Date: 25 July 2013; 9: 429 – 433
DOI: http://dx.doi.org/10.2147/VHRM.S34421

Pioglitazone and other thiazolidinediones (TZDs) initially showed great promise as unique receptor-mediated oral therapy for type 2 diabetes, but a host of serious side effects, primarily cardiovascular, have limited their utility. It is crucial at this point to perform a risk–benefit analysis to determine what role pioglitazone should play in our current treatment of type 2 diabetes and where the future of this class of drugs is headed. This review provides a comprehensive overview of the present literature. Clinical data currently available indicate that pioglitazone is an effective and generally well-tolerated treatment option for use in patients with type 2 diabetes. Pioglitazone can still reduce adverse cardiovascular risk.

Glucophage, Glucophage XR

In a US double-blind clinical study of GLUCOPHAGE in patients with type 2 diabetes, a total of 141 patients received GLUCOPHAGE therapy (up to 2550 mg per day) and 145 patients received placebo. Adverse reactions reported in greater than 5% of the GLUCOPHAGE patients, and that were more common in GLUCOPHAGE- than placebo-treated patients are reported.

The following adverse reactions were reported in ≥ 1.0% to ≤ 5.0% of GLUCOPHAGE patients and were more commonly reported with GLUCOPHAGE than placebo:

abnormal stools, – myalgia, – lightheaded, – dyspnea,

the following adverse reactions were reported in ≥ 1.0% to ≤ 5.0% of GLUCOPHAGE XR patients and were more commonly reported with GLUCOPHAGE XR than placebo

dizziness, – More common

Metabolic side effects have included lactic acidosis, which is a potentially fatal metabolic complication. The incidence of lactic acidosis has been about 1.5 cases per 10,000 patient years. The risk of lactic acidosis has been particularly high in patients with underlying renal insufficiency. Cases of lactic acidosis occurring in patients with normal renal function have been rarely reported.

  • Signs and symptoms of severe acidosis may include bradycardia  (lactic acidosis)
  • lactic acid concentration, serum electrolytes, blood pH

High-Fructose Corn Syrup Linked to Diabetes

By Brenda Goodman, MA   WebMD Health News
Reviewed By Louise Chang, MD

In a study published in the journal Global Health, researchers compared the average availability of high-fructose corn syrup to rates of diabetes in 43 countries.

About half the countries in the study had little or no high-fructose corn syrup in their food supply. In the other 20 countries, high-fructose corn syrup in foods ranged from about a pound a year per person in Germany to about 55 pounds each year per person in the United States.

The researchers found that countries using high-fructose corn syrup had rates of diabetes that were about 20% higher than countries that didn’t mix the sweetener into foods. Those differences remained even after researchers took into account data for differences in body size, population, and wealth.

But couldn’t that mean that people in countries that used more high-fructose corn syrup were just eating more sugar or more total calories?

The researchers say no: There were no overall differences in total sugars or total calories between countries that did and didn’t use high-fructose corn syrup, suggesting that there’s an independent relationship between high-fructose corn syrup and diabetes.

“It raises a lot of questions about fructose,” says researcher Michael I. Goran, PhD, co-director of the Diabetes and Obesity Research Institute at the Keck School of Medicine at the University of Southern California, in Los Angeles. Although the study found an association, it doesn’t establish a cause/effect relationship.
Genetic association of ADIPOQ gene variants with type 2 diabetes, obesity and serum adiponectin levels in south Indian population.

Ramya K; Ayyappa KA; Ghosh S; Mohan V; Radha V
Gene 2013 Dec 15;532(2):253-62    (ISSN: 1879-0038)

OBJECTIVE: To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population. METHODS: The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin
levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies. RESULTS: Of the 8 variants, four SNPs namely, +276 G/T (rs1501299), -4522 C/T (rs822393), -11365 C/G (rs266729), and +712 G/A (rs3774261) were significantly associated with T2DM in our study population. The -3971 A/G (rs822396) and -11391 G/A (rs17300539) SNPs’ association with T2DM diabetes was mediated through obesity (where  the association with  type 2 diabetes was lost after adjusting for BMI). There was an independent
association of +276 G/T (rs1501299) and -3971 A/G (rs822396) SNPs with generalized obesity and +349 A/G (rs2241767) with central obesity. Four SNPs, -3971 A/G (rs822396), +276 G/T (rs1501299), -4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population. CONCLUSION: The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indianpopulation. [ 2013.].

Association of family history of type 2 diabetes mellitus with markers of endothelial dysfunction in South Indian population.

Dhananjayan R; Malati T; Brindha G; Kutala VK
Indian J Biochem Biophys 2013 Apr;50(2):93-8    (ISSN: 0301-1208)

Studies indicate that risk for type 2 diabetes mellitus (T2D) or cardiovascular disease is detectable in childhood, though these disorders may not emerge until adulthood. This study was aimed to assess the markers of endothelial dysfunction in patients with the family history of T2D from South Indian population. A total of 450 subjects were included in the study comprising Group I (n = 200) of T2D, Group II (n = 200) of age- and sex-matched healthy controls, Group III (n = 25) of children of T2D patients and Group IV (n = 25) of children of healthy controls. Results showed that intimal medial thickening (IMT) was significantly higher in T2D patients, compared with control subjects with no family history of diabetes. The fasting plasma glucose, glycated hemoglobin, serum total cholesterol, triglyceride, LDL-cholesterol, apolipoprotein B (ApoB) and high-sensitive C-reactive protein (hsCRP) levels were significantly increased, whereas HDL-cholesterol and serum nitrite levels were significantly decreased in T2D patients. However, children of T2D patients who were not diabetic did not show significant increase in the IMT, as compared to those of healthy controls. In conclusion, the present study demonstrate that IMT was significantly higher in the T2D patients and increased with age and family history. The increased levels of lipids, hsCRP, IMT and decreased nitrite levels might contribute to the risk of endothelial dysfunction in patients with T2D. However, further studies are warranted with other biomarkers of endothelial dysfunction in T2D patients with increased sample size.

Hemoglobin A1c variability as an independent correlate of cardiovascular disease in patients with type 2 diabetes: a cross-sectional analysis of the renal insufficiency and cardiovascular events (RIACE) Italian multicenter study.

Penno G; Solini A; Zoppini G; Orsi E; Fondelli C; Zerbini G; Morano S; and
Renal Insufficiency and Cardiovascular Events (RIACE) Study Group.
Cardiovasc Diabetol 2013;12:98    (ISSN: 1475-2840)

BACKGROUND: Previous reports have clearly indicated a significant relationship between hemoglobin (Hb) A1c change from one visit to the next and microvascular complications, especially nephropathy (albuminuria and albuminuric chronic kidney disease, CKD). In contrast, data on macrovascular disease are less clear. This study was aimed at examining the association of HbA1c variability with cardiovascular disease (CVD) in the large cohort of subjects with type 2 diabetes from the Renal Insufficiency and Cardiovascular Events (RIACE) Italian Multicenter Study. METHODS: Serial (3-5) HbA1c values obtained during the 2-year period preceding recruitment, including that obtained at the enrolment, were available from 8,290 subjects from 9 centers (out of 15,773 patients from 19 centers). Average HbA1c and HbA1c variability were calculated as the intra-individual mean (HbA1c-MEAN) and standard deviation (HbA1c-SD), respectively, of 4.52 0.76 values. Prevalent CVD, total and by vascular bed, was assessed from medical history by recording previous documented major acute events. Diabetic retinopathy (DR) was assessed by dilated fundoscopy. CKD was defined based on albuminuria, as measured by immunonephelometry or immunoturbidimetry, and estimated glomerular filtration rate, as calculated from serum creatinine. RESULTS: HbA1c-MEAN, but not HbA1c-SD, was significantly higher (P <0.0001) in subjects with history of any CVD (n. 2,133, 25.7%) than in those without CVD (n. 6,157, 74.3%). Median and interquartile range were 7.78 (7.04-8.56) and 7.49 (6.81-8.31), respectively, for HbA1c-MEAN, and 0.47 (0.29-0.75) and 0.46 (0.28-0.73), respectively, for HbA1c-SD. Logistic regression analyses showed that HbA1c-MEAN, but not HbA1c-SD (and independent of it), was a significant correlate of any CVD. Similar findings were observed in subjects with versus those without any coronary or cerebrovascular event or myocardial infarction. Conversely, none of these measures were associated with stroke, whereas both correlated with any lower limb vascular event and HbA1c-SD alone with ulceration/gangrene. All these associations were independent of known CVD risk factors and microvascular complications (DR and CKD). CONCLUSIONS: In patients with type 2 diabetes, HbA1c variability has not a major impact on macrovascular complications, at variance with average HbA1c, an opposite finding as compared with microvascular disease, and particularly nephropathy. TRIAL REGISTRATION: ClinicalTrials.Gov NCT00715481.

Genetic association of adiponectin gene polymorphisms (+45T/G and +10211T/G) with type 2 diabetes in North Indians.

Saxena M; Srivastava N; Banerjee M
Diabetes Metab Syndr 2012 Apr-Jun;6(2):65-9    (ISSN: 1878-0334)

Adiponectin (ADIPOQ) is an abundant protein hormone which belongs to a family of so-called adipokines. It is expressed mostly by adipocytes and is an important regulator of lipid and glucose metabolism. It was shown that decreased serum adiponectin concentration indicated insulin resistance and type 2 diabetes (T2DM) with the risk of cardiovascular complications. The fact that adiponectin is an insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties, we proposed to study the association of ADIPOQ gene polymorphisms in subjects with T2DM. DNA was isolated from venous blood samples, quantified and subjected to Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using suitable primers and restriction endonucleases. Adiponectin levels were measured in serum using ELISA. The genotypic, allelic and carriage rate frequencies distribution in patients and controls were analyzed by PSAW software (ver. 17.0). Odd ratios (OR) with 95% confidence interval (CI) were determined to describe the strength of association by logistic regression model. Out of the two polymorphisms studied, +10211T/G showed significant association (P=0.042), the ‘G’ allele association being highly significant (P=0.022). Further analysis showed that individuals with ‘GG’ haplotype were at increased risk of T2DM up to 15.5 times [P=0.015, OR (95% CI); 15.558 (1.690-143.174)]. The present study showed that the ‘G’ allele of ADIPOQ gene (+10211T/G) plays a prominent role with respect to T2DM susceptibility in North-Indian population. [Copyright 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.].

Association of RAGE gene polymorphism with vascular complications in Indian type 2 diabetes mellitus patients [In Process Citation]

Tripathi AK; Chawla D; Bansal S; Banerjee BD; Madhu SV; Kalra OP
Diabetes Res Clin Pract 2014 Mar;103(3):474-81    (ISSN: 1872-8227)

AIMS: The study was designed to evaluate the association of -374T/A and -429T/C polymorphism in the promoter region and Gly82Ser polymorphism in exon 3 region of RAGE gene with diabetic vascular complications in Indian population. METHODS: We screened 603 subjects which includes 176 healthy controls, 140 type 2 diabetes mellitus (T2DM) subjects without any vascular complications (DM), 152 T2DM subjects with microvascular complications (DM-micro) and 135 T2DM subjects with macrovascular complications (DM-macro) for -374T/A, -429T/C and Gly82Ser polymorphisms of RAGE gene. DNA isolated from the enrolled subjects were genotyped by PCR-RFLP. Logistic regression analysis was used to evaluate the association of single nucleotide polymorphisms (SNPs). RESULTS: The -429 T/C and Gly82Ser RAGE polymorphisms were found to be significantly associated with the development of macrovascular and microvascular complications, respectively, in T2DM subjects while -374A allele showed reduced risk towards the development of macrovascular complications. Further, -429T/C, -374T/A and Gly82Ser haplotype analysis revealed association of CTG haplotype with development of macrovascular complications while haplotype TAG was observed to be significantly protective towards development of macrovascular complications in T2DM subjects (OR=0.617, p=0.0202). CONCLUSIONS: Our data indicates significant association of RAGE SNPs and haplotypes with vascular complications in North Indian T2DM subjects.
Clinical profile and complications of childhood- and adolescent-onset type 2 diabetes seen at a diabetes center in south India.

Amutha A; Datta M; Unnikrishnan R; Anjana RM; Mohan V
Diabetes Technol Ther 2012 Jun;14(6):497-504    (ISSN: 1557-8593)

OBJECTIVE: This study describes the clinical characteristics of childhood- and adolescent-onset type 2 diabetes mellitus (CAT2DM) seen at a diabetes center in southern India. RESEARCH DESIGN AND METHODS: Between January 1992 and December 2009, 368 CAT2DM patients were registered. Anthropometric measurements were done using standardized techniques. Biochemical investigations included C-peptide measurements and glutamic acid decarboxylase antibody assay wherever feasible. Retinopathy was diagnosed by retinal photography; microalbuminuria, if urinary albumin excretion was between 30 and 299vmg/1/4g of creatinine; nephropathy, if urinary albumin excretion was (yen)300vmg/1/4g; and neuropathy, if vibration perception threshold on biothesiometry was (yen)20vV. RESULTS: The proportion of CAT2DM patients, expressed as percentage of total patients registered at our center, rose from 0.01% in 1992 to 0.35% in 2009 (P <0.001). Among the 368 cases of CAT2DM, 96 (26%) were diagnosed before the age of 15 years. The mean age at first visit and age at diagnosis of the CAT2DM subjects were 22.29.7 and 16.12.5 years, respectively. Using World Health Organization growth reference charts, 56% of boys and 50.4% of girls were > 85(th) percentile of body mass index for age. Prevalence rates of retinopathy, microalbuminuria, nephropathy, and neuropathy were 26.7%, 14.7%, 8.4%, and 14.2%, respectively. Regression analysis revealed female gender, body mass index > 85(th) percentile, parental history of diabetes, serum cholesterol, and blood pressure to be associated with earlier age at onset of CAT2DM. CONCLUSIONS: CAT2DM appears to be increasing in urban India, and the prevalence of microvascular complications is high. Female predominance is seen at younger ages.

Variants of the adiponectin gene and diabetic microvascular complications in patients with type 2 diabetes.

Choe EY; Wang HJ; Kwon O; Kim KJ; Kim BS; Lee BW; Ahn CW;  et al.
Metabolism 2013 May;62(5):677-85    (ISSN: 1532-8600)

OBJECTIVE: The aim of this study was to examine the association between common polymorphisms of the adiponectin gene (ADIPOQ) and microvascular complications in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: Rs2241766 and rs1501299 of ADIPOQ were genotyped in 708 patients with T2DM. Fundus photography, nerve conducting velocity, and urine analysis were performed to check for the presence of microvascular complications including diabetic nephropathy, retinopathy and neuropathy. RESULTS: The prevalence of diabetic nephropathy tended to be different according to rs2241766 genotype (p=0.057) and the GG genotype of rs2241766 was associated with diabetic nephropathy [urine albumin/creatinine ratio (UACR) greater than 30 mg/g] after adjusting for age, sex, body mass index, duration of diabetes, HDL-cholesterol, smoking status, and blood pressure (odds ratio=1.96; 95% confidence interval=1.01-3.82, p=0.049). Also, the G allele of rs2241766 demonstrated a trend to be associated with an increase in UACR (p=0.087). Rs2241766 genotype was not associated with diabetic retinopathy (p=0.955) and neuropathy (p=0.104) or any diabetic microvascular complications (p=0.104). There was no significant association between the rs1501299 genotype of ADIPOQ and the prevalence of diabetic retinopathy and neuropathy or any diabetic microvascular complications even after adjustment. CONCLUSION: These data suggest that the GG genotype at rs2241766 is implicated in the pathogenesis of risk for diabetic nephropathy defined as UACR greater than 30 mg/day in patients with T2DM. [Copyright 2013 Elsevier Inc. All rights reserved.].

The prevalence of presarcopenia in Asian Indian individuals with and without type 2 diabetes.

Anbalagan VP; Venkataraman V; Pradeepa R; Deepa M; Anjana RM; Mohan V
Diabetes Technol Ther 2013 Sep;15(9):768-75    (ISSN: 1557-8593)

OBJECTIVE: This study compared the skeletal muscle mass and prevalence of presarcopenia between Asian Indian individuals with and without type 2 diabetes. SUBJECTS AND METHODS: Participants with type 2 diabetes (n=76) and age- and sex-matched controls without diabetes (n=76) were drawn from the Chennai Urban Rural Epidemiological Study (CURES), which was carried out on a representative sample of Chennai City in South India. Skeletal muscle mass was estimated by dual-energy X-ray absorptiometry, and skeletal muscle mass index (SMI) was calculated by dividing the appendicular skeletal muscle mass by the square of the individual’s height in meters and expressed as kg/m. Presarcopenia was defined as an SMI of 7.26 kg/m2 for males and  5.5 kg/m2 for females. Biochemical and anthropometric measurements were done using standardized procedures. RESULTS: The 152 participants included 68 women (44.7%). Mean age was 449 years (range, 28-67 years), and the mean body mass index (BMI) was 25.73.8 kg/m2. The prevalence rates of presarcopenia among individuals with and without diabetes were 39.5% and 15.8%, respectively (P=0.001). The mean SMI values were significantly lower in those with diabetes (6.841.02 kg/m2 compared with participants without diabetes (7.281.01 kg/m2) (P=0.009). SMI showed a positive correlation with BMI and waist circumference but a negative correlation with age, fasting plasma glucose, glycated hemoglobin, and low-density lipoprotien cholesterol in the total study population. Logistic regression analysis showed that diabetes was independently associated with presarcopenia (P=0.001). CONCLUSIONS: Prevalence of presarcopenia is higher among Asian Indian subjects with type 2 diabetes compared with age- and sex-matched participants without diabetes.

Increased risk of type 2 diabetes with ascending social class in urban South Indians is explained by obesity: The Chennai urban rural epidemiology study (CURES-116).

Skar M; Villumsen AB; Christensen DL; Petersen JH; Deepa M; Anjana RM; et al.
Indian J Endocrinol Metab 2013 Nov;17(6):1084-9    (ISSN: 2230-8210)

AIM: The aim of this study is to determine the factors responsible for differences in the prevalence of diabetes mellitus (DM) in subjects of different social class in an urban South Indian population. MATERIALS AND METHODS: Analyses were based on the cross-sectional data from the Chennai Urban Rural Epidemiology Study of 1989 individuals, aged (yen)20 years. Entered in the analyses were information obtained by self-report on (1) household income; (2) family history of diabetes; (3) physical activity; (4) smoking status; (5) alcohol consumption. Biochemical, clinical and anthropometrical measurements were performed and included in the analyses. Social class was classified based on income as low (Rs. <2000) intermediate (Rs. 2000-5000`) and high (Rs. 5000-20000). RESULTS: The prevalence rates of DM were 12.0%, 18.4% and 21.7% in low, intermediate and high social class, respectively (P < 0.001). A significant increase in the risk of diabetes was found with ascending social class (Intermediate class: Odds ratio [OR], 1.7 [confidence interval [CI], 1.2-2.3]; High class: OR, 2.0 [CI-1.4-2.9]). The multivariable adjusted logistic regression analysis revealed that the effect of social class on the risk of diabetes remained significant (P = 0.016) when age, family history of diabetesand blood pressure were included. However, with the inclusion of abdominal obesity in the model, the significant effect of social class disappeared (P = 0.087). CONCLUSION: An increased prevalence of DM was found in the higher social class in this urban South Indian population, which is explained by obesity.

Prevalence of inflammatory markers (high-sensitivity C-reactive protein, nuclear factor-(ordM)B, and adiponectin) in Indian patients with type 2 diabetes mellitus with and without macrovascular complications.

Misra DP; Das S; Sahu PK
Metab Syndr Relat Disord 2012 Jun;10(3):209-13    (ISSN: 1557-8518)

BACKGROUND: Atherosclerosis is more prevalent in subjects with diabetes mellitus. Recent evidence suggests that diabetic atherosclerosis is not simply a disease of hyperlipidemia, but is also an inflammatory disorder. Our aim was to study the prevalence of inflammatory markers such as high-sensitivity C-reactive protein (hsCRP), adiponectin, and nuclear factor-(ordM)B (NF-(ordM)B) expression, in peripheral blood mononuclear cells in Indian patients with type 2 diabetes mellitus (T2DM) with and without macrovascular disease (MVD). METHODS: A total of 29 consecutive cases of T2DM with proven MVD (group A), 28 matched cases without MVD (group B), and 14 healthy controls (group C) were evaluated for the clinical parameters fasting blood glucose (FBG), 2-h postprandial blood glucose (PPBG), glycosylated hemoglobin (HbA1c), lipid profile, and the above-mentioned inflammatory markers. RESULTS: Diabetic subjects with T2DM had higher hsCRP and NF-(ordM)B expression and lower values of adiponectin compared to healthy controls. Group A had significantly higher serum hsCRP than group B (P=0.0001) despite comparable values of BMI, FBG, 2-h PPBG, HbA1c, and lipid parameters. Group A had significantly higher serum hsCRP and NF-(ordM)B expression and significantly lower levels of adiponectin than group C (P=0.0001, 0.007, and 0.02, respectively). In Group A, serum adiponectin negatively correlated with NF-(ordM)B expression. In Group B, adiponectin values correlated negatively with both FBG and 2-h PPBG. CONCLUSIONS: Indian subjects with T2DM with or without MVD had higher hsCRP and lower adiponectin values as compared to healthy controls, whereas hsCRP was significantly higher in those with MVD, suggesting that our patients with T2DM were in a proinflammatory state.

Adiponectin G276T gene polymorphism is associated with cardiovascular disease in Japanese patients with type 2 diabetes.

Katakami N; Kaneto H; Matsuoka TA; Takahara M; Maeda N; Shimizu I; et al.
Atherosclerosis 2012 Feb;220(2):437-42    (ISSN: 1879-1484)

OBJECTIVE: Adiponectin has anti-atherogenic properties and reduced serum adiponectin levels are associated with cardiovascular disease (CVD). In this study, we examined the relationship between CVD and adiponectin (ADIPOQ) gene G276T polymorphism that is associated with serum adiponectin level in a large cohort of type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We enrolled 2637 Japanese type 2 diabetic subjects (males, 61.1%; age, 54.97.9 years old), determined their genotypes regarding ADIPOQ G276T polymorphisms, and evaluated the association between this polymorphism and the prevalence of CVD (myocardial infarction and/or cerebral infarction). RESULTS: The prevalence of CVD tended to be higher as the number of G alleles increased [GG (9.5%), GT (6.8%), TT (5.6%), p value for trend=0.0059] and was significantly higher in the subjects with GG genotype compared to those with GT or TT genotype (9.5% vs. 6.6%, p=0.0060). Multiple logistic regression analyses revealed that the number of G alleles (Odds ratio (OR)=1.49 with 95%CI 1.09-2.05, p=0.0125) and GG genotype (OR=1.66 with 95%CI 1.13-2.43, p=0.0098) were significantly associated with CVD even after adjustment for conventional risk factors. Interestingly, the presence of obesity further and significantly increased the risk of CVD in the subjects with GG genotype (OR=1.67 with 95%CI 1.14-2.44, p=0.0090) but not in the subjects with TT or GT genotype (OR=1.17 with 95%CI 0.73-1.89, NS). CONCLUSIONS: It is likely that the G allele of the ADIPOQ G276T polymorphism is a susceptibility allele for CVD in Japanese type 2 diabetic patients, especially when they accompany obesity. [Copyright 2011 Elsevier Ireland Ltd. All rights reserved.].

A comprehensive investigation of variants in genes encoding adiponectin (ADIPOQ) and its receptors (ADIPOR1/R2), and their association with serum adiponectin, type 2 diabetes, insulin resistance and the metabolic syndrome.

Peters KE; Beilby J; Cadby G; Warrington NM; Bruce DG; Davis WA; et al.
BMC Med Genet 2013;14:15    (ISSN: 1471-2350)

BACKGROUND: Low levels of serum adiponectin have been linked to central obesity, insulin resistance, metabolic syndrome, and type 2 diabetes. Variants in ADIPOQ, the gene encoding adiponectin, have been shown to influence serum adiponectin concentration, and along with variants in theadiponectin receptors (ADIPOR1 and ADIPOR2) have been implicated in metabolic syndrome and type 2 diabetes. This study aimed to comprehensively investigate the association of common variants in ADIPOQ, ADIPOR1 and ADIPOR2 with serum adiponectin and insulin resistance syndromes in a large cohort of European-Australian individuals. METHODS: Sixty-four tagging single nucleotide polymorphisms in ADIPOQ, ADIPOR1 and ADIPOR2 were genotyped in two general population cohorts consisting of 2,355 subjects, and one cohort of 967 subjects with type 2 diabetes. The association of tagSNPs with outcomes were evaluated using linear or logistic modelling. Meta-analysis of the three cohorts was performed by random-effects modelling. RESULTS: Meta-analysis revealed nine genotyped tagSNPs in ADIPOQ significantly associated with serum adiponectinacross all cohorts after adjustment for age, gender and BMI, including rs10937273, rs12637534, rs1648707, rs16861209, rs822395, rs17366568, rs3774261, rs6444175 and rs17373414. The results of haplotype-based analyses were also consistent. Overall, the variants in the ADIPOQ gene explained <5% of the variance in serum adiponectin concentration. None of the ADIPOR1/R2 tagSNPs were associated with serum adiponectin. There was no association between any of the genetic variants and insulin resistance or metabolic syndrome. A multi-SNP genotypic risk score for ADIPOQ alleles revealed an association with 3 independent SNPs, rs12637534, rs16861209, rs17366568 and type 2 diabetes after adjusting foradiponectin levels (OR=0.86, 95% CI=(0.75, 0.99), P=0.0134). CONCLUSIONS: Genetic variation in ADIPOQ, but not its receptors, was associated with altered serum adiponectin. However, genetic variation in ADIPOQ and its receptors does not appear to contribute to the risk of insulin resistance or metabolic syndrome but did for type 2
diabetes in a European-Australian population.
Autophagy: Protection Against T2D?

By Salynn Boyles, Contributing Writer,
MedPage Today  Published: Jul 27, 2014 | Updated: Jul 28, 2014

The cellular regulatory system known as autophagy appeared to play a key role in preventing type 2 diabetes by protecting insulin-secreting beta cells from the accumulation of toxic amylin oligomers, researchers reported.

Findings from three independent research teams, published online in the Journal of Clinical Investigation, suggested autophagy boosting therapies could prove to be a novel approach for type 2 diabetes prevention.

Autophagy — derived from the Greek words for “self” (auto) and “to eat” (phagein) — describes the controlled disposal of damaged organelles within the cell. This cell-cleaning process is increasingly being recognized as a potential protective mechanism against many diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease.

Earlier studies found autophagy to be important for normal beta-cell functionand autophage activity to be increased in beta cells from patients with type 2 diabetes.

The studies provide new insight into how beta cells are normally protected against amylin (IAPP) toxic oligomers, wrote Dhananjay Gupta, PhD, and Jack L. Leahy, MD, of the University of Vermont in Burlington in an accompanying editorial.

Action Points:

  • Autophagy appeared to play a key role in preventing type 2 diabetes by protecting insulin-secreting beta cells from the accumulation of toxic amylin oligomers.
  • Note that the studies suggest that autophagy — controlled disposal of damaged organelles within the cell — boosting therapies could prove to be a novel approach for type 2 diabetes prevention.

Autophagy – continued

IAPP: Co-Expressed With Insulin

Type 2 diabetes is characterized by loss of beta-cell, beta-cell dysfunction, and increased beta-cell apoptosis. Islet pathology in type 2 diabetes is also characterized by accumulation of extracellular islet amyloid derived from islet amyloid polypeptide (IAPP).

“IAPP is a 37-amino acid protein co-expressed and secreted by pancreatic [beta cells] along with insulin,” wrote Peter Butler, MD, from the University of California Los Angeles, and colleagues. “While the extracellular islet amyloid is relatively inert, intracellular membrane-permeant toxic oligomers of IAPP that form within [beta cells in type 2 diabetes] are thought to induce [beta-cell dysfunction and apoptosis].”

In contrast to the human form of IAPP (h-IAPP), which forms toxic membrane-permeant oligomers, the rodent form of IAPP (r-IAPP) is nonamyloidogenic and nontoxic due to proline substitutions. Transgenic expression of h-IAPP in [beta cells] of rodents may lead to development of diabetes as a consequence of [beta-cell] apoptosis and formation of intracellular IAPP oligomers comparable to those found in humans with type 2 diabetes.

In earlier in vitro studies, the authors reported that enhancement of autophagy was protective while attenuated lysosomal degradation rendered beta cells more vulnerable to h-IAPP-induced apoptosis.

In the current study, the researchers determined that beta-cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation.

“Induction of high levels of human IAPP in mouse [beta cells] resulted in accumulation of this amyloidogenic protein as relatively inert fibrils with cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers,” they wrote.

Mice hemizygous for transgenic expression of human IAPP did not develop diabetes. But the loss of beta cell-specific autophagy in the mice induced diabetes as a result of the accumulation of toxic human IAPP oligomers and loss of beta-cell mass, the researchers noted.

“In human IAPP-expressing mice that lack [beta-cell] autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased [beta- cell] apoptosis,” they wrote. “These findings indicate that autophagy/lysosomal degradation defends [beta cells] against proteotoxicity induced by oligomerization-prone human IAPP.”

‘Enhance the Toxic Potential of h-IAPP’

In a separate study, Yoshio Fujitani, PhD, of Juntendo University, Tokyo, and colleagues, examined the pathogenic role of human-IAPP and its relation to autophagy in h-IAPP-knock-in mice.

In animals fed a standard diet, h-IAPP had no toxic effects on beta-cell function. However, h-IAPP-knock-in mice did not exhibit a high-fat diet-induced compensatory increase in beta-cell mass, which was due to limited beta-cell proliferation and enhanced beta-cell apoptosis, the researchers wrote.

Expression of h-IAPP in mice with a beta-cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions.

“Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of h-IAPP and enhance [beta-cell] dysfunction and progression of type 2 diabetes,” the researchers noted.

Autophagy Enhancers

In the third paper, Myung-Shik Lee, MD, PhD, of the Sungkyunkwan University School of Medicine in Seoul, and colleagues, studied transgenic mice with beta cell-specific expression of h-IAPP to evaluate the contribution of autophagy in type 2 diabetes-associated accumulation of h-IAPP.

In mice with beta-cell-specific expression of h-IAPP, a deficiency in autophagy resulted in development of overt diabetes, which was not observed in mice expressing h-IAPP alone or lacking autophagy alone. Lack of autophagy in h-IAPP-expressing animals also resulted in h-IAPP oligomer and amyloid accumulation in pancreatic islets, leading to increased death and decreased mass of beta cells.

“Expression of h-IAPP in purified monkey islet cells or a murine [beta cell] line resulted in pro-h-IAPP dimer formation, while dimer formation was absent or reduced dramatically in cells expressing either nonamyloidogenic mouse-IAPP or nonfibrillar mutant h-IAPP,” the researchers wrote. “In autophagy-deficient cells, accumulation of pro-h-IAPP dimers increased markedly, and pro-h-IAPP trimers were detected in the detergent-insoluble fraction.”

Enhancement of autophagy also improved the metabolic profile of h-IAPP-expressing mice fed a high-fat diet.

“These results suggest that autophagy promotes clearance of amyloidogenic h-IAPP, autophagy deficiency exacerbates pathogenesis of human [type 2 diabetes], and autophagy enhancers have therapeutic potential for islet amyloid accumulation-associated human [type 2 diabetes],” the researchers concluded.

Building on Previous Work

Gupta and Leahy noted that all three research teams generated human IAPP-expressing mice with a beta-cell-specific deficiency of the autophagy indicator ATG7, and all three found that autophagy-dependent packaging of monomeric or unprocessed IAPP dimers or trimers into p62-associated vacuoles allowed autophagosomes to dispose of these molecules, keeping them nontoxic.

Each team showed the activity of this detoxification system to be increased when a high-fat diet was fed to the mice with hyperexpression of h-IAPP.

The studies build on previous work and the findings that don’t discern – “how and when during the course of type 2 diabetes development this autophagy-dependent detoxification system might be overcome, allowing toxic IAPP oligomers to form.”

“There are many additional mechanisms that have been proposed for [beta-cell] dysfunction and death in type 2 diabetes, including ER stress, oxidative stress, and autoimmune damage, all of which have been linked to IAPP toxicity,” they wrote. “While it is tempting to try and connect the dots through a single, unified mechanism, all of these proposed pathways of [beta-cell] dysfunction have been recapitulated and extensively studied in rodent models of diabetogenic systems, such as high-fat feeding and partial pancreatectomy, or through genetic modification.”

Given the absence of rodent IAPP oligomerization, these mechanisms of reduced beta-cell function clearly do not require IAPP activation, they noted.

These papers have implications for the study of target therapies for type 2 diabetes based on the common link to T2D and IAPP oligomerization.

“Patients with type 2 diabetes have an increased risk of Alzheimer’s disease, suggesting a common pathogenesis,” they wrote. Disordered neuronal autophagy, described in Alzheimer’s, with alteration in the clearance of amyloidogenic proteins may be a tie between these two diseases

They concluded that acceptance of the hypothesis that IAPP oligomer formation and subsequent plaque development are a major cause of type 2 diabetes will require a better understanding of

  • when this mechanism is activated and
  • what modulates its destructive potential.

“These current studies may shift the focus away from

  • the biology of how IAPP oligomers cause [beta cell] destruction
  • to probing for defects within the protective system against the formation of toxic IAPP oligomers,” they wrote.

Part 2. Pancreatic Islet Cell Dysfunction
N-terminal fragment of probrain natriuretic peptide is associated with diabetes microvascular complications in type 2 diabetes

Kumiko Hamano, Ikue Nakadaira, Jun Suzuki, Megumi Gonai
Vascular Health and Risk Management 2014:10 585–589
http://dx.doi.org/10.2147/VHRM.S67753

Aim/introduction: Circulating levels of N-terminal fragment of probrain natriuretic peptide (NT-proBNP) are established as a risk factor for cardiovascular disease and mortality in patients with diabetes, as well as in the general population. We sought to examine the possibility of NT-proBNP as a biomarker of microvascular complications in patients with type 2 diabetes.  Materials and methods: In total, 277 outpatients with type 2 diabetes were consecutively enrolled as a hospital cohort. Two hundred and seventeen of these patients (132 males; mean age, 63.4 years) were designated as cases with any of the diabetic complications (retinopathy, neuropathy, nephropathy, ischemic heart disease, strokes, peripheral artery disease), and 60 (42 males; mean age, 54.1 years) were set as controls without clinical evidence of diabetic complications. Diabetic complications were evaluated by medical record and routine laboratory examinations. NT-proBNP was measured and investigated with regard to the associations with diabetic complications. Results: Mean NT-proBNP levels were significantly higher in patients with any of the diabetic complications (59 versus 33 pg/mL; P,0.0001). In logistic regression analysis, NT-proBNP levels .79 pg/mL, which was the highest tertile, were independently associated with a 5.04 fold increased risk of all complications (P,0.0051) compared to the lowest tertile (NT-proBNP levels ,31 pg/mL). Odd ratios of cardiovascular disease and nephropathy, neuropathy, and retinopathy were 9.33, 6.23, 6.6 and 13.78 respectively, in patients with NT-proBNP values in the highest tertile (.79 pg/mL), independently of age, sex, duration of diabetes or other risk factors, such as body mass index or hemoglobin A1c. In addition, NT-proBNP levels were associated with surrogate markers of atherosclerosis, such as brachial-ankle pulse wave velocity (r=0.449, P,0.0001) and left ventricular hypertrophy (r=0.212, P,0.001). Conclusion: In this hospital-based cohort of type 2 diabetes, the NT-proBNP levels were associated with systemic atherosclerosis and comorbid diabetic microvascular as well as macrovascular complications. It is useful to stratify high-risk diabetic patients by measuring NT-proBNP and to start comprehensive care for preventing the progression of diabetic complications. It is necessary to elucidate the underlying mechanism for the progression of diabetic complications represented by an elevation of NT-proBNP and to demonstrate the ability of NT-proBNP as a predictive global biomarker for diabetic complications in Japanese type 2 diabetic patients.
How are patients with type 2 diabetes and renal disease monitored and managed? Insights from the observational OREDIA study

Alfred Penfornis, J F Blicklé, B Fiquet, S Quéré, S Dejager
Vascular Health and Risk Management 2014:10 341–352
http://dx.doi.org/10.2147/VHRM.S60312

Background and aim: Chronic kidney disease (CKD) is frequent in type 2 diabetes mellitus (T2DM), and therapeutic management of diabetes is more challenging in patients with renal impairment (RI). The place of metformin is of particular interest since most scientific societies now recommend using half the dosage in moderate RI and abstaining from use in severe RI, while the classic contraindication with RI has not been removed from the label. This study aimed to assess the therapeutic management, in particular the use of metformin, of T2DM patients with CKD in real life. Methods: This was a French cross-sectional observational study: 3,704 patients with T2DM diagnosed for over 1 year and pharmacologically treated were recruited in two cohorts (two-thirds were considered to have renal disease [CKD patients] and one-third were not [non-CKD patients]) by 968 physicians (81% general practitioners) in 2012. Results: CKD versus non-CKD patients were significantly older with longer diabetes history, more diabetic complications, and less strict glycemic control (mean glycated hemoglobin [HbA1c] 7.5% versus 7.1%; 25% of CKD patients had HbA1c $8% versus 15% of non-CKD patients). Fifteen percent of CKD patients had severe RI, and 66% moderate RI. Therapeutic management of T2DM was clearly distinct in CKD, with less use of metformin (62% versus 86%) but at similar mean daily doses (∼2 g/d). Of patients with severe RI, 33% were still treated with metformin, at similar doses. For other oral anti-diabetics, a distinct pattern of use was seen across renal function (RF): use of sulfonylureas (32%, 31%, and 20% in normal RF, moderate RI, and severe RI, respectively) and DPP4-i (dipeptidyl peptidase-4 inhibitors) (41%, 36%, and 25%, respectively) decreased with RF, while that of glinides increased (8%, 14%, and 18%, respectively). CKD patients were more frequently treated with insulin (40% versus 16% of non-CKD patients), and use of insulin increased with deterioration of RF (19%, 39%, and 61% of patients with normal RF, moderate RI, and severe RI, respectively). Treatment was modified at the end of the study-visit in 34% of CKD patients, primarily to stop or reduce metformin. However, metformin was stopped in only 40% of the severe RI patients.   Conclusion: Despite a fairly good detection of CKD in patients with T2DM, RI was insufficiently taken into account for adjusting anti-diabetic treatment.

Efficacy and safety of insulin glargine added to a fixed-dose combination of metformin and a dipeptidyl peptidase-4 inhibitor: results of the GOLD observational study

Jochen Seufert, Katrin Pegelow, Peter Bramlage
Vascular Health and Risk Management 2013:9 711–717
http://dx.doi.org/10.2147/VHRM.S54362

Background: For patients with type 2 diabetes who are uncontrolled on a combination of two oral antidiabetic agents, addition of the long-acting basal insulin glargine is a well established treatment option. However, data on the efficacy and safety of a combination of metformin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, and insulin glargine are limited in real-world settings. Therefore, the aim of this study was to analyze blood glucose control, rates of hypoglycemia and body weight in a large cohort of patients with type 2 diabetes treated with this combination therapy in real practice. Methods: This noninterventional, multicenter, prospective, observational trial with a follow-up of 20 weeks enrolled insulin-naïve patients who had been on a stable fixed dose of metformin and a DPP-4 inhibitor for at least 3 months, and had a glycosylated hemoglobin (HbA1c) between 7.5% and 10%. Patients were selected at the investigators’ discretion for initiation of insulin glargine at baseline. A total of 1,483 patients were included, of whom 1,262 were considered to be the efficacy set. Primary efficacy parameters were HbA1c and fasting plasma glucose. Secondary outcome measures included achievement of glycemic targets, body weight, rates of hypoglycemia, and other safety parameters, as well as resource consumption. Results: Upon initiation of insulin glargine, mean HbA1c decreased from 8.51% to 7.36% (−1.15%±0.91%; 95% confidence interval [CI] −1.20 to −1.10). An HbA1c level ,6.5% was achieved in 8.2% of patients and a level ,7.0% in 31.5%. Mean fasting plasma glucose decreased from 174±47 mg/dL to 127±31 mg/dL (−47.3±44.1 mg/dL; 95% CI −49.8 to −44.8). In 11.9% of patients, a fasting plasma glucose level ,100 mg/dL was achieved. Bodyweight decreased on average by 0.98±3.90 kg (95% CI 1.19–0.76). Hypoglycemia (blood glucose #70 mg/dL) was observed in 29 patients (2.30%), of whom six (0.48%) had nocturnal hypoglycemia and four (0.32%) had documented severe events (blood glucose ,56 mg/dL). Conclusion: The results of this observational study show that insulin glargine, when added to a fixed-dose combination of metformin and a DPP-4 inhibitor, resulted in a significant and clinically relevant improvement of glycemic control. Importantly, this intervention did not interfere with the action of the DPP-4 inhibitors, resulting in neutral effects on weight and low rates of hypoglycemia. We conclude that this treatment intensification approach may be useful, efficient, and safe in daily clinical practice for patients with type 2 diabetes.

Long-term insulin glargine therapy in type 2 diabetes mellitus: a focus on cardiovascular outcomes

Joshua J Joseph, Thomas W Donner
Vascular Health and Risk Management 2015:11 107–116
http://dx.doi.org/10.2147/VHRM.S50286

Cardiovascular disease is the leading cause of mortality in type 2 diabetes mellitus. Hyperinsulinemia is associated with increased cardiovascular risk, but the effects of exogenous insulin on cardiovascular disease progression have been less well studied. Insulin has been shown to have both cardioprotective and atherosclerosis-promoting effects in laboratory animal studies. Long-term clinical trials using insulin to attain improved diabetes control in younger type 1 and type 2 diabetes patients have shown improved cardiovascular outcomes. Shorter trials of intensive diabetes control with high insulin use in higher risk patients with type 2 diabetes have shown either no cardiovascular benefit or increased all cause and cardiovascular mortality. Glargine insulin is a basal insulin analog widely used to treat patients with type 1 and type 2 diabetes. This review focuses on the effects of glargine on cardiovascular outcomes. Glargine lowers triglycerides, leads to a modest weight gain, causes less hypoglycemia when compared with intermediate-acting insulin, and has a neutral effect on blood pressure. The Outcome Reduction With Initial Glargine Intervention (ORIGIN trial), a 6.2 year dedicated cardiovascular outcomes trial of glargine demonstrated no increased cardiovascular risk.

Visceral obesity is not an independent risk factor of mortality in subjects over 65 years

Frédérique Thomas, Bruno Pannier, Athanase Benetos, Ulrich M Vischer
Vascular Health and Risk Management 2013:9 739–745
http://dx.doi.org/10.2147/VHRM.S49922

The aim of the study was to determine the role of obesity evaluated by body mass index (BMI), waist circumference (WC), and their combined effect on all-cause mortality according to age and related risk factors. This study included 119,090 subjects (79,325 men and 39,765 women), aged from 17 years to 85 years, who had a general health checkup at the Centre d’Investigations Préventives et Cliniques, Paris, France. The mean follow-up was 5.6±2.4 years. The prevalence of obesity, defined by WC and BMI categories, was determined according to age groups (< 55, 55–65, > 65 years). All-cause mortality according to obesity and age was determined using Cox regression analysis, adjusted for related risk factors and previous cardiovascular events.
For the entire population, WC adjusted for BMI, an index of central obesity, was strongly associated with mortality, even after adjustment for hypertension, dyslipidemia, and diabetes. The prevalence of obesity increased with age, notably when defined by WC. Nonetheless, the association between WC adjusted for BMI and mortality was not observed in subjects .65 years old (hazard ratio [HR] =1.010, P=NS) but was found in subjects  < 55 (HR =1.030,
P < 0.0001) and 55–65 years old (HR =1.023, P,0.05). By contrast, hypertension
(HR =1.31, P < 0.05), previous cardiovascular events (HR =1.98, P < 0.05), and smoking (HR =1.33, P < 0.05) remained associated with mortality even after
age 65.
In conclusion, WC adjusted for BMI is strongly and independently associated with all-cause mortality before 65 years of age, after taking into account the associated risk factors. This relationship disappears in subjects
> 65 years of age, suggesting a differential impact of visceral fat deposition according to age.

Insulin degludec/insulin aspart combination for the treatment of type 1 and type 2 diabetes

Angela Dardano, Cristina Bianchi, Stefano Del Prato, Roberto Miccoli
Vascular Health and Risk Management 2014:10 465–475
http://dx.doi.org/10.2147/VHRM.S40097

Glycemic control remains the major therapeutic objective to prevent or delay the onset and progression of complications related to diabetes mellitus. Insulin therapy represents a cornerstone in the treatment of diabetes and has been used widely for achieving glycemic goals. Nevertheless, a large portion of the population with diabetes does not meet the internationally agreed glycemic targets. Moreover, insulin treatment, especially if intensive, may be associated with emergency room visits and hospitalization due to hypoglycemic events. Therefore, fear of hypoglycemia or hypoglycemic events represents the main barriers to the attainment of glycemic targets. The burden associated with multiple daily injections also remains a significant obstacle to initiating and maintaining insulin therapy. The most attractive insulin treatment approach should meet the patients’ preference, rather than demanding patients to change or adapt their lifestyle. Insulin degludec/insulin aspart (IDegAsp) is a new combination, formulated with ultra-long-acting insulin degludec and rapid-acting insulin aspart, with peculiar pharmacological features, clinical efficacy, safety, and tolerability. IDegAsp provides similar, noninferior glycemic control to a standard basal–bolus regimen in patients with type 1 diabetes mellitus, with additional benefits of significantly lower episodes of hypoglycemia (particularly nocturnal) and fewer daily insulin injections. Moreover, although treatment strategy and patients’ viewpoint are different in type 1 and type 2 diabetes, trial results suggest that IDegAsp may be an appropriate and reasonable option for initiating insulin therapy in patients with type 2 diabetes inadequately controlled on maximal doses of conventional oral agents. This paper will discuss the role of IDegAsp combination as a novel treatment option in diabetic patients.

UCP2 Regulates the Glucagon Response to Fasting and Starvation

Emma M. Allister, Christine A. Robson-Doucette, Kacey J. Prentice, et al.
Diabetes  Feb 22, 2013; p 1-11.  http://dx.doi.org:/10.2337/db12-0981
http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0981/-/DC1

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP22/2) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created a-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted a-cells have higher levels of intracellular reactive oxygen species (ROS), due to enhanced  mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, a-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal a-cell glucose sensing and the maintenance of euglycemia.

Main points:

  • UCP2 is efficiently deleted specifically from islet a-cells of UCP2AKO mice.
  • α-Cell UCP2 deletion reduces glucagon secretion in vivo
  • UCP2AKO mice display normal glucose tolerance and GLP-1 secretion
  • α-Cell UCP2 deletion reduces the gluconeogenic response of the liver and switches fatty acid usage during a prolonged fast
  • UCP2 expression is increased after nutrient depletion and glucagon secretion from UCP2AKO islets was impaired.
  • UCP2AKO α-cells display enhanced hyperpolarization of ΔψCm and increased superoxide levels
  • UCP2AKO α-cells have more depolarized plasma membranes and reduced intracellular calcium
  • UCP2 is required for normal glucagon secretion in response to hypoglycemia

Management of Diabetes Mellitus: Could Simultaneous Targeting of Hyperglycemia and Oxidative Stress Be a Better Panacea?

Omotayo O. Erejuwa

Int. J. Mol. Sci. 2012, 13, 2965-2972; http://dx.doi.org:/10.3390/ijms13032965

Oxidative stress is defined as an “imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage”. It is implicated in the pathogenesis and complications of diabetes mellitus. The role of oxidative stress is more definite in the pathogenesis of type 2 diabetes mellitus than in type 1 diabetes mellitus. In regard to diabetic complications, there is compelling evidence in support of the role of oxidative stress in both types of diabetes mellitus. Evidence suggests that elevated reactive oxygen species (ROS), which causes oxidative stress, accumulate in certain micro milieu or tissues (such as retina and kidney) where they cause damage or toxicity. In diabetes mellitus, oxidative stress is enhanced through various sources such as hyperglycemia, dyslipidemia, hyperinsulinemia, insulin resistance, impaired antioxidant defense network, uncoupling of ROS-generating enzymes, elevated level of leptin and sedentary lifestyle.

A number of mechanisms or pathways by which hyperglycemia, the major contributing factor of increased ROS production, causes tissue damage or diabetic complications have been identified. These include: hyperglycemia-enhanced polyol pathway; hyperglycemia-enhanced formation of advanced glycation endproducts (AGEs); hyperglycemia-activated protein kinase C (PKC) pathway; hyperglycemia-enhanced hexosamine pathway; and hyperglycemia-activated Poly-ADP ribose polymerase (PARP) pathway. These pathways are activated or enhanced by hyperglycemia-driven mitochondrial superoxide overproduction. Even though oxidative stress plays an important role in its pathogenesis and complications, unlike other diseases characterized by oxidative stress, diabetes mellitus is unique. Its cure (restoration of euglycemia, e.g., via pancreas transplants) does not prevent oxidative stress and diabetic complications. This is very important because hyperglycemia exacerbates oxidative stress which is linked to diabetic complications]. Theoretically, restoration of euglycemia should prevent oxidative stress and diabetic complications. However, this is not the case.

The primary aim of the current management of diabetes mellitus is to achieve and/or maintain a glycated hemoglobin level of ≤6.5%. However, recent evidence indicates that intensive treatment of hyperglycemia is characterized by increased weight gain, severe hypoglycemia and higher mortality. Besides, evidence suggests that it is difficult to achieve and/or maintain optimal glycemic control in many diabetic patients; and that the benefits of intensively-treated hyperglycemia are restricted to microvascular complications only. In view of these adverse effects and limitations of intensive treatment of hyperglycemia in preventing diabetic complications, which is linked to oxidative stress, this commentary proposes a hypothesis that “simultaneous targeting of hyperglycemia and oxidative stress” could be more effective than “intensive treatment of hyperglycemia” in the management of diabetes mellitus.

 

The Relationship between Inflammation, Oxidative Stress, and Metabolic Risk Factors in Type 2 Diabetic Patients

Fatemeh Azizi Soleiman, N Pahlavani, H Rasad, O Sadeghi, MR Gohari
Iranian Journal Of Diabetes And Obesity 2013; 5(4): 151-156

Increased production of free radicals due to the imbalance between free radicals and antioxidants load may reduce antioxidants levels, partial clearing of free radicals, and cause oxidation of lipids, sugars, proteins and nucleic acids which eventually leads to widespread pathological consequences of diabetes. One of the factors that facilitate formation of atherosclerosis in diabetes is oxidative stress.

Objective: Globally, 3-5.2 percent of people suffer from diabetes which is one of the most serious metabolic disorders resulting in an increase in inflammatory biomarkers e.g. interleukin-6, tumor necrosis factor-alpha, and C-reactive protein. The aim of this study was to investigate the relationship between inflammation, oxidative stress and fasting blood glucose, lipid profile and anthropometric parameters in patients with type 2 diabetes. Material and methods: This study was conducted as a cross sectional study in Tehran through 2009-2010 on 45 men and women aged 35-65 years old with type 2 diabetes. Blood glucose, lipid profile, C-reactive protein, and malonedialdehyde were measured. Independent sample T-test and linear regression analysis were used. Results: Fasting blood glucose, malonedialdehyde, total cholesterol and body mass index were higher in women than in men; but there was no difference between two sexes in other factors. Malonedialdehyde, neither directly or after adjustment for sex was related to fasting blood glucose, total cholesterol, triglycerides and anthropometric indices (weight, body mass index, and body fat mass). Conclusion: This study showed that oxidative stress had no relationship with blood glucose, lipid profile, and anthropometric index, but inflammation was related to glycemia, body mass index, and fat mass. Control of inflammation and oxidative stress is necessary for accelerating treatment process and preventing complications due to them.

This study showed that in diabetic patients, oxidative stress which was measured by MDA, was not significantly associated with fasting blood glucose, lipid profile and anthropometric parameters. However, fasting plasma glucose, body mass index and body fat mass were significant predictors of the inflammatory factor, CRP.

Oxidative Stress as an Underlying Contributor in the Development of Chronic Complications in Diabetes Mellitus

Suziy de M. Bandeira, Lucas José S. da Fonseca, Glaucevane da S. Guedes, et al.
Int. J. Mol. Sci. 2013, 14, 3265-3284; http://dx.doi.doi:/10.3390/ijms14023265

The high prevalence of diabetes mellitus and its increasing incidence worldwide, coupled with several complications observed in its carriers, have become a public health issue of great relevance. Chronic hyperglycemia is the main feature of such a disease, being considered the responsible for the establishment of micro and macrovascular complications observed in diabetes. Several efforts have been directed in order to better comprehend the pathophysiological mechanisms involved in the course of this endocrine disease. Recently, numerous authors have suggested that excess generation of highly reactive oxygen and nitrogen species is a key component in the development of complications invoked by hyperglycemia. Overproduction and/or insufficient removal of these reactive species result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids, leading different research groups to search for biomarkers which would be capable of a proper and accurate measurement of the oxidative stress (OS) in diabetic patients, especially in the presence of chronic complications.
In the face of this scenario, the present review briefly addresses the role of hyperglycemia in OS, considering basic mechanisms and their effects in diabetes mellitus, describes some of the more commonly used biomarkers of oxidative/nitrosative damage and includes selected examples of studies which evaluated OS biomarkers in patients with diabetes, pointing to the relevance of such biological components in general oxidative stress status of diabetes mellitus carriers.
The role of FOXO1 in βcell failure and type 2 diabetes mellitus

Tadahiro Kitamura
Nat. Rev. Endocrinol. 2013; 9, 615–623
http://dx.doi.org:/10.1038/nrendo.2013.157

Over the past two decades, insulin resistance has been considered essential to the etiology of type 2 diabetes mellitus (T2DM). However, insulin resistance does not lead to T2DM unless it is accompanied by pancreatic β‑cell dysfunction, because healthy β cells can compensate for insulin resistance by increasing in number and functional output. Furthermore, β‑cell mass is decreased in patients with diabetes mellitus, suggesting a primary role for β‑cell dysfunction in the pathogenesis of T2DM. The dysfunction of β cells can develop through various mechanisms, including oxidative, endoplasmic reticulum or hypoxic stress, as well as via induction of cytokines; these processes lead to apoptosis, uncontrolled autophagy and failure to proliferate. Transdifferentiation between β cells and α cells occurs under certain pathological conditions, and emerging evidence suggests that β‑cell dedifferentiation or transdifferentiation might account for the reduction in β‑cell mass observed in patients with severe T2DM. FOXO1, a key transcription factor in insulin signaling, is implicated in these mechanisms. This Review discusses advances in our understanding of the contribution of FOXO1 signaling to the development of β‑cell failure in T2DM.

Selective peroxisome proliferator-activated receptor g (PPARg) modulation as a strategy for safer therapeutic PPARg activation

Linda Slanec Higgins and Alex M DePaoli
Am J Clin Nutr 2010;91(suppl):267S–72S.
http://dx.doi.org:/10.3945/ajcn.2009.28449E

Peroxisome proliferator-activated receptor c (PPARc) is a clinically validated target for treatment of insulin resistance. PPARc activation by full agonists such as thiazolidinediones has shown potent and durable glucose-lowering activity in patients with type 2 diabetes without the concern for hypoglycemia or gastrointestinal toxicities associated with some other medications used to treat this disease. However, thiazolidinediones are linked to safety and tolerability issues such as weight gain, fluid retention, edema, congestive heart failure, and bone fracture. Distinctive properties of PPARc provide the opportunity for selective modulation of the receptor such that desirable therapeutic effects may be attained without the unwanted effects of full activation. PPARc is a nuclear receptor that forms a complex with coreceptor RXR and a cell type– and cell state– specific array of coregulators to control gene transcription. PPARc affinity for these components, and hence transcriptional response, is determined by the conformational changes induced by ligand binding within a complex pocket with multiple interaction points. This molecular mechanism thereby offers the opportunity for selective modulation. A desirable selective PPARc modulator profile would include high-affinity interaction with the PPARc-binding pocket in a manner that leads to retention of the insulin-sensitizing activity that is characteristic of full agonists as well as mitigation of the effects leading to increased adiposity, fluid retention, congestive heart failure, and bone fracture. Examples of endogenous and synthetic selective PPARc modulator (SPPARM) ligands have been identified. SPPARM drug candidates are being tested clinically and provide support for this strategy.

Predicting response to incretin-based therapy

Sanjay Kalra, Bharti Kalra, Rakesh Sahay, Navneet Agrawal
Research and Reports in Endocrine Disorders 2011:1 11–19
http://dx.doi.org:/10.2147/RRED.S16282

There are two important incretin hormones, glucose-dependent insulin tropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The biological activities of GLP-1 include stimulation of glucose-dependent insulin secretion and insulin biosynthesis, inhibition of glucagon secretion and gastric emptying, and inhibition of food intake. GLP-1 appears to have a number of additional effects in the gastrointestinal tract and central nervous system. Incretin based therapy includes GLP-1 receptor agonists like human GLP-1 analogs (liraglutide) and exendin-4 based molecules (exenatide), as well as DPP-4 inhibitors like sitagliptin, vildagliptin and saxagliptin. Most of the published studies showed a significant reduction in HbA1c using these drugs. A critical analysis of reported data shows that the response rate in terms of target achievers of these drugs is average. One of the first actions identified for GLP-1 was the glucose-dependent stimulation of insulin secretion from islet cell lines. Following the detection of GLP-1 receptors on islet beta cells, a large body of evidence has accumulated illustrating that GLP-1 exerts multiple actions on various signaling pathways and gene products in the β cell. GLP-1 controls glucose homeostasis through well-defined actions on the islet β cell via stimulation of insulin secretion and preservation and expansion of β cell mass. In summary, there are several factors determining the response rate to incretin therapy. Currently minimal clinical data is available to make a conclusion. Key factors appear to be duration of diabetes, obesity, presence of autonomic neuropathy, resting energy expenditure, plasma glucagon levels and plasma free fatty acid levels. More clinical evidence is required to identify the factors affecting response rate to incretin therapy.

Regulation of Large Conductance Ca2+-activated K+ (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels

Fu Yi, Huan Wang, Qiang Chai, Xiaoli Wang, et al.
J. Biol. Chem. 2014, 289: 10853-10864
http://dx.doi.org:/10.1074/jbc.M113.520940

Background: Impaired BK channel function in diabetic vessels is associated with decreased BK channel[1]1 subunit (BK-β1) expression. Results: Muscle RING finger protein 1 (MuRF1) physically interacts with BK-β1 and accelerates BK-β1 proteolysis. Conclusion: Increased MuRF1 expression is a novel mechanism underlying diabetic BK channelopathy and vasculopathy. Significance: MuRF1 is a potential therapeutic target of BK channel dysfunction and vascular complications in diabetes.

The large conductance Ca2+-activated K+ (BK) channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human

coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK channelopathy and vasculopathy in diabetes.
The origin of circulating CD36 in type 2 diabetes

MJ Alkhatatbeh, AK Enjeti, S Acharya, RF Thorne, and LF Lincz
Nutrition and Diabetes (2013) 3, e59; http://dx.doi.org:/10.1038/nutd.2013.1

Objective: Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36 + MPs in persons with T2DM. Design: An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI) =39.9±6.4 kgm2; age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI =23.6±1.8 kgm2 and 33.5±5.9 kgm2, respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235α, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations. Results: CD36 + MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235α + = 35.8±14.6%); although this did not correlate with hemoglobin A1c. By contrast, the main source of CD36 + MPs in non-T2DM individuals was endothelial cells (CD105 + = 40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 µgml-1 and was positively correlated with CD36 +MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36 + MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively). Conclusions: Both the levels and cellular profile of CD36 + MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.
A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets

Jakob D. Wikstrom, Samuel B. Sereda, Linsey Stiles, Alvaro Elorza, et al.
PLoS ONE 7(5): e33023. http://dx.doi.org:/10.1371/journal.pone.0033023

Background: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. Methodology/Principal Findings: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. Conclusions/Significance: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

Refeeding and metabolic syndromes: two sides of the same coin

OA Obeid, DH Hachem and JJ Ayoub
Nutrition & Diabetes (2014) 4, e120; http://dx.doi.org:/10.1038/nutd.2014.21

Refeeding syndrome describes the metabolic and clinical changes attributed to aggressive rehabilitation of malnourished subjects. The metabolic changes of refeeding are related to hypophosphatemia, hypokalemia, hypomagnesemia, sodium retention and hyperglycemia, and these are believed to be mainly the result of increased insulin secretion following high carbohydrate intake. In the past few decades, increased consumption of processed food (refined cereals, oils, sugar and sweeteners, and so on) lowered the intake of several macrominerals (mainly phosphorus, potassium and magnesium). This seems to have compromised the postprandial status of these macrominerals, in a manner that mimics low grade refeeding syndrome status. At the pathophysiological level, this condition favored the development of the different components of the metabolic syndrome. Thus, it is reasonable to postulate that metabolic syndrome is the result of long term exposure to a mild refeeding syndrome.

HSP72 protects against obesity-induced insulin resistance

Jason Chung, Anh-Khoi Nguyen, Darren C. Henstridge, Anna G. Holmes, et al.
PNAS  Feb 5, 2008; 105(5): 1739–1744
http://www.pnas.org/cgi/doi/10.1073/pnas.0705799105

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of B kinase, and tumor necrosis factor-β, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

pH-responsive modulation of insulin aggregation and structural transformation of the aggregates

Ekaterina Smirnova, I Safenkova, V Stein-Margolina, V Shubin, et al.
Biochimie 109 (2015) 49e59
http://dx.doi.org/10.1016/j.biochi.2014.12.006

Over the past two decades, much information has appeared on electrostatically driven molecular mechanisms of protein self-assembly and formation of aggregates of different morphology, varying from soluble amorphous structures to highly-ordered amyloid-like fibrils. Protein aggregation represents a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. This has awakened interest in identification of pH-triggered regulators of transformation of aggregation-prone proteins into structures of higher order. The objective of the present study is to elucidate the effects of low-molecular-weight biogenic agents on aggregation and formation of supramolecular structures of human recombinant insulin, as a model therapeutic protein. Using dynamic light scattering, turbidimetry, circular dichroism, fluorescence spectroscopy, atomic force microscopy, transmission electron microscopy, and nuclear magnetic resonance, we have demonstrated that the amino acid L-arginine (Arg) has the striking potential to influence insulin aggregation propensity. It was shown that modification of the net charge of insulin induced by changes in the pH level of the incubation medium results in dramatic changes in the interaction of the protein with Arg. We have revealed the dual effects of Arg, highly dependent on the pH level of the solution e suppression or acceleration of the aggregation of insulin at pH 7.0 or 8.0, respectively. These effects can be regulated by manipulating the pH of the environment. The results of this study may be of interest for development of appropriate drug formulations and for the more general insight into the functioning of insulin in living systems, as the protein is known to release by exocytosis from pancreatic beta cells in a pH-dependent manner.
Human β-cell proliferation by promoting Wnt signaling

Carol Wilson
Original article Aly, H. et al. A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype. PLoS ONE 2013; 8, e66131
Nature Reviews Endocrinology 2013; 9, 502
http://dx.doi.org:/10.1038/nrendo.2013.130

Islet transplantation for patients with type 1 diabetes mellitus typically requires 2–4 donors for one recipient, whereas use of one donor would minimize the risk of immune rejection. Proliferation of adult β cells in vitro could hold the key to providing one donor for one recipient.

“In previous studies, we found that activation of the Wnt/GSK-3/β-catenin pathway by pharmacologic inhibition of GSK-3 in combination with nutrient activation of mTOR, modestly enhanced human β-cell proliferation in vitro,” says lead researcher Haytham Aly of the Washington University School of Medicine in St. Louis, MO, USA. “However, expansion of human islets was associated with a loss of insulin content and secretory function.”

In the current study, the researchers aimed to engage canonical and noncanonical Wnt signalling at the receptor level to increase the proliferation of human β cells in vitro, without losing the capacity of the cells to produce and secrete insulin.

The researchers treated cadaver-derived intact human islets with a conditioned medium from L cells that constitutively produce Wnt-3a, R-spondin-3 and Noggin. A similar medium had previously enabled successful proliferation of mouse colonic intestinal epithelial cells. The researchers added inhibitors of ROCK and RhoA to this medium to augment cell survival.

The conditioned medium with the inhibitors lead to ~20-fold proliferation of the human β cells above that with glucose alone. Crucially, treatment with this conditioned medium did not impair glucose-stimulated insulin secretion or decrease insulin content of the cells.

“This novel strategy has clear potential for use in the in vitro expansion of human islets and the subsequent treatment of impaired β-cell functional mass in type 1 diabetes mellitus and type 2 diabetes mellitus,” concludes Aly.

Betatrophin—inducing β-cell expansion to treat diabetes mellitus?

Elisabeth Kugelberg
Original article Yi, P. et al. Betatrophin: a hormone that controls pancreatic β cell proliferation. Cell http://dx.doi.org:/10.1016/j.cell.2013.04.008
Nature Reviews Endocrinology 2013; 9, 379; http://dx.doi.org:/10.1038/nrendo.2013.98

Betatrophin, a newly identified hormone, increases the production and expansion of insulin-secreting β cells in mice, research from Harvard University suggests.

When insulin resistance develops, pancreatic β cells undergo an expansion in mass and proliferation to compensate for increasing insulin needs. To date, the mechanisms regulating β-cell replication are unclear.

Yi et al. developed a mouse model of insulin resistance using the insulin receptor antagonist S961. Subcutaneous injections of the S961 peptide into mice led to dose-dependent, instant β-cell proliferation and hyperglycemia.

Microarray analysis revealed that a highly conserved mammalian gene, betatrophin, was upregulated fourfold in liver and threefold in white adipose tissue cells in response to the acute peripheral insulin resistance induced by S961.

Yi and coworkers found that Betatrophin encodes a secreted protein that can be detected in human plasma. Intravenous injection of betatrophin-expressing constructs into mice resulted in a 17-fold higher β-cell proliferation rate compared with control vectors, and ultimately led to increased islet size and insulin content, with improvements in glucose tolerance, in betatrophin-injected animals.

The mechanisms of action of betatrophin are still unknown, and the next step is to test the effects of recombinant betatrophin protein on β-cell mass. The authors conclude that the identification of betatrophin and its control of β-cell proliferation opens a new door to possible diabetes therapy.

Blocking RANKL signaling might prevent T2DM

Carol Wilson
Original article Kiechl, S. et al. Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus. Nat. Med.
http://dx.doi.org:/10.1038/nm.3084

Nature Reviews Endocrinology 2013; 9, 188;
http://dx.doi.org:/10.1038/nrendo.2013.43

Blockade of receptor activator of nuclear factor κB ligand (RANKL) signaling in hepatocytes protects against type 2 diabetes mellitus (T2DM), report researchers.

“It is well known that activation of nuclear factor κB (NF-κB) in the liver is a crucial event in the development of hepatic insulin resistance and T2DM,” explains lead author Stefan Kiechl of the Medical University of Innsbruck, Austria. “RANKL, a member of the tumour necrosis factor superfamily, is a potent activator of NF-κB, and its receptor RANK is expressed on liver cells. We, thus, hypothesized that RANKL is involved in hepatic NF-κB activation, leading to T2DM.”

The researchers studied the association between serum levels of soluble RANKL and osteoprotegerin and subsequent risk of developing T2DM in 844 men and women without T2DM aged 40–79 years. Soluble RANKL was assessed because it has been shown to be functionally active.

During follow-up, between 1990 and 2005, 78 individuals of the cohort developed T2DM. Baseline levels of soluble RANKL between individuals who had and had not developed T2DM differed considerably: risk of T2DM was elevated in the group with the top tertile T2DM of concentrations of soluble RANKL compared with the group with the bottom tertile (OR 4.06, 95% CI 2.01–8.20). Adjustment for lifestyle factors and body composition did not significantly affect the risk association. Interestingly, although concentrations of osteoprotegerin were not elevated preceding T2DM onset, as they were for soluble RANKL, increased levels were found in individuals after disease occurrence.

In a series of mouse models in which RANKL signaling was downregulated systemically or in the liver, the investigators showed that hepatic insulin sensitivity and plasma glucose concentrations improved with blockade of RANKL signaling. In one such experiment, mice with a hepatocyte-specific Rank knockout were fed a high-fat diet for 4 weeks. These mice did not develop insulin resistance, whereas control mice did.

The investigators note that medications for T2DM already available, such as metformin, lower RANKL activity in bone and might also lower RANKL activity in the liver. They speculate that RANKL antagonism could be a yet unknown.

SFRP4—a biomarker for islet dysfunction?

Carol Wilson
Original article Mahdi, T. et al. Secreted frizzled-related protein 4 reduces insulin secretion and is overexpressed in type 2 diabetes. Cell Metab. http://doi.org:/10.1016/j.cmet.2012.10.009

Secreted frizzled-related protein 4 (SFRP4) reduces insulin secretion and is a potential biomarker for islet dysfunction in type 2 diabetes mellitus (T2DM), report researchers.

Mahdi et al. discovered these insights into the pathophysiology of T2DM by the analysis of global gene expression in human pancreatic islets. The researchers identified a group of co-expressed genes (also called a gene co-expression module) associated with T2DM, reduced insulin secretion and elevated HbA1c levels after analysing global microarray expression data from human islets of 48 individuals, including 10 with T2DM. This module was enriched for IL-1-related genes.

The investigators identified SFRP4 as a gene highly expressed in islets from patients with T2DM. The protein encoded by SFRP4 is an extracellular regulator of the Wnt pathway, and has roles in tissue development, cancer and phosphate metabolism. Further study revealed that the expression and release of SFRP4 from islets was stimulated by IL-1β. Furthermore, elevated systemic SFRP4 levels led to reduced glucose tolerance as a result of decreased islet expression of voltage-gated Ca2+ channels and supressed insulin exocytosis.

Interestingly, levels of SFRP4 were elevated in serum of patients a few years before they developed T2DM, which indicates that this protein has potential to be used as a biomarker for T2DM. The researchers also point out that their data suggest that SFRP4 could be a therapeutic target for the treatment of islet dysfunction.

Add-on to metformin in T2DM —linagliptin or glimepiride?

Mikkel Christensen and Filip K. Knop
Nat. Rev. Endocrinol. 2012; 8, 576–578  http://dx.doi.org:/10.1038/nrendo.2012.163

Dipeptidyl peptidase 4 (DPP4) inhibitors, also known as gliptins, are a rapidly expanding class of oral antidiabetic drugs for the treatment of type 2 diabetes mellitus (T2DM). Since 2006, five DPP4 inhibitors have reached the market and, because they can be administered orally and have an almost impeccable safety profile, these drugs have gained widespread use in the treatment of T2DM. The DPP4 inhibitor linagliptin was approved in 2011 by the FDA and the European Medicines Agency (EMA) for use in patients with T2DM as second-line therapy to add on to metformin either alone or in combination with another second-line treatment.

The UK Prospective Diabetes Study trial showed that sulphonylurea treatment was more effective than metformin treatment after 1 year in terms of reducing HbA1c levels; however, after 6 years of treatment, the effectiveness of sulphonylurea treatment declined and metformin treatment was more effective. A decline in the effectiveness of the sulphonylurea treatment over time could be due to sulphonylureas inducing stress and possibly causing apoptosis in β cells. However, in the trial by Gallwitz et al. the sustained efficacies of the add-on treatments with linagliptin and glimepiride were similar after 2 years.

The inhibitors of DPP4 enhance glucose-dependent insulin secretion and could even augment the counter-regulatory glucagon response to hypoglycemia. DPP4 inhibition generally has a neutral effect upon body weight.

The study by Gallwitz et al. included patients whose plasma glucose levels were near-normal whilst they were receiving metformin monotherapy (baseline level 6–7 mmol/l), which could result in increased occurrence of hypoglycemia. Treating patients whose blood glucose levels were, by many standards, already adequately controlled with metformin with a drug known to be associated with inducing hypoglycemia would be expected to increase the frequency of hypoglycemia in this group, inflating the differences in the frequency of this event between the group receiving linagliptin and that receiving glimepiride.

The most groundbreaking findings in the study by Gallwitz et al. are related to cardiovascular outcomes. Although the study was not adequately powered to detect subtle differences in cardiovascular event frequency, significantly fewer patients who received linagliptin than glimepiride experienced major cardiovascular events (12 versus 26 individuals, respectively). This difference was driven by fewer patients experiencing nonfatal myocardial infarctions and nonfatal strokes in the linagliptin-treated group than in the glimepiride-treated group (9 versus 21 individuals, respectively).

Clinicians are responsible for selecting a suitable second-line treatment for patients with type 2 diabetes mellitus when metformin monotherapy fails. New evidence could aid clinicians in deciding between one of the most commonly used second-line agents, glimepiride, and the recently approved dipeptidyl peptidase 4 inhibitor linagliptin.

Relation of Mitochondrial Oxygen Consumption in Peripheral Blood Mononuclear Cells to Vascular Function in Type 2 Diabetes Mellitus

Mor-Li Hartman, Orian S. Shirihai, Monika Holbrook, Guoquan Xu, et al.
Vasc Med. 2014 February ; 19(1): 67–74. http://dx.doi.org:/10.1177/1358863X14521315.

Recent studies have shown mitochondrial dysfunction and increased production of reactive

oxygen species in peripheral blood mononuclear cells (PBMC’s) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to ATP production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMC’s that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMC’s and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192±47 vs. 161±44 pMoles/min, P=0.01), uncoupled (64±16 vs. 53±16 pMoles/min, P=0.007), and maximal (795±87 vs. 715±128 pMoles/min, P=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1±6.6 vs. 15.8±4.8%, P=0.03) and correlated with maximal oxygen consumption (R= −0.64, P=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Musashi expression in b-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

M Szabat, TB Kalynyak, GE Lim, KY Chu, YH Yang, A Asadi, BK Gage, et al.
Cell Death and Disease (2011) 2, e232
http://dx.doi.org:/10.1038/cddis.2011.119

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic b-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates b-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of b-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and b-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and b-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory b-cell proliferation and the loss of b-cell gene expression seen in specific phases of the progression to type 2 diabetes.

Cooperation between brain and islet in glucose homeostasis and diabetes

Michael W. Schwartz, RJ Seeley, MH Tschöp, SC Woods, et al.
Nature  7 Nov 2013; 503: 59–66          http://dx.doi.org/10.1038/nature12709

Although a prominent role for the brain in glucose homeostasis was proposed by scientists in the nineteenth century, research throughout most of the twentieth century focused on evidence that the function of pancreatic islets is both necessary and sufficient to explain glucose homeostasis, and that diabetes results from defects of insulin secretion, action or both. However, insulin-independent mechanisms, referred to as ‘glucose effectiveness’, account for roughly 50% of overall glucose disposal, and reduced glucose effectiveness also contributes importantly to diabetes pathogenesis. Although mechanisms underlying glucose effectiveness are poorly understood, growing evidence suggests that the brain can dynamically regulate this process in ways that improve or even normalize glycaemia in rodent models of diabetes. Here we present evidence of a brain-centred glucoregulatory system (BCGS) that can lower blood glucose levels via both insulin-dependent and -independent mechanisms, and propose a model in which complex and highly coordinated interactions between the BCGS and pancreatic islets promote normal glucose homeostasis. Because activation of either regulatory system can compensate for failure of the other, defects in both may be required for diabetes to develop. Consequently, therapies that target the BCGS in addition to conventional approaches based on enhancing insulin effects may have the potential to induce diabetes remission, whereas targeting just one typically does not.

The traditional view holds that diabetes arises as a consequence of damage to, and ultimately failure of, beta-cell function. We propose a two-component model in which failure of glucose homeostasis can begin after initial impairment.

Schematic illustrations of brain- and islet-centred glucoregulatory systems

Schematic illustrations of brain- and islet-centred glucoregulatory systems

Schematic illustrations of brain- and islet-centred glucoregulatory systems
The BCGS is proposed to regulate tissue glucose metabolism and plasma glucose levels via mechanisms that are both insulin dependent (for example, by regulating tissue insulin sensitivity) and insulin independent

Proposed contributions of defective brain- and islet-centred glucoregulatory systems to T2D pathogenesis

Proposed contributions of defective brain- and islet-centred glucoregulatory systems to T2D pathogenesis

Proposed contributions of defective brain- and islet-centred glucoregulatory systems to T2D pathogenesis

Insulin’s discovery: New insights on its ninetieth birthday

Jesse Roth, Sana Qureshi, Ian Whitford, Mladen Vranic, et al.
Diabetes Metab Res Rev 2012; 28: 293–304
http://dx.doi.org:/10.1002/dmrr.2300

2012 marks the 90th year since the purification of insulin and the miraculous rescue from death of youngsters with type 1 diabetes. In this review, we highlight several previously unappreciated or unknown events surroundingthe discovery.
(i) We remind readers of the essential contributions of each of the four discoverers – Banting, Macleod, Collip, and Best.
(ii) Banting and Best (each with his own inner circle) worked not only to accrue credit for himself but also to minimize credit to the other discoverers.
(iii) Banting at the time of the insulin research was very likely suffering from post-traumatic stress disorder (PTSD) that originated during his heroic service as a surgeon in World War I on the Western Front in 1918, including an infected shrapnel wound that threatened amputation of his arm. His war record along with the newly discovered evidence of a suicide threat goes along with his paranoia, combativeness, alcohol excess, and depression, symptoms we associate with PTSD.
(iv) Banting’s eureka idea, ligation of the pancreatic duct to preserve the islets, while it energized the early research, was unnecessary and was bypassed early.
(v) Post discovery,Macleod uncovered many features of insulin action that he summarized in his 1925 Nobel Lecture.Macleod closed by raising the question – what is the mechanism of insulin action in the body? – a challenge that attracted many talented investigators but remained unanswered until the latter third of the 20th century.

Genetic Variants Associated With Glycine Metabolism and Their Role in Insulin Sensitivity and Type 2 Diabetes

Weijia Xie, Andrew R. Wood, Valeriya Lyssenko, Michael N. Weedon, et al.
Diabetes 2013; 62:2141–2150 http://dx.doi.org:/10.2337/db12-0876

Circulating metabolites associated with insulin sensitivity may represent useful biomarkers, but their causal role in insulin sensitivity and diabetes is less certain. We previously identified novel metabolites correlated with insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. The top-ranking metabolites were in the glutathione and glycine biosynthesis pathways. We aimed to identify common genetic variants associated with metabolites in these pathways and test their role in insulin sensitivity and type 2 diabetes. With 1,004 nondiabetic individuals from the RISC study, we performed a genome-wide association study (GWAS) of 14 insulin sensitivity–related metabolites and one metabolite ratio. We replicated our results in the Botnia study (n = 342). We assessed the association of these variants with diabetes-related traits in GWAS meta-analyses (GENESIS [including RISC, EUGENE2, and Stanford], MAGIC, and DIAGRAM). We identified four associations with three metabolites—glycine (rs715 at CPS1), serine (rs478093 at PHGDH), and betaine (rs499368 at SLC6A12; rs17823642 at BHMT)—and one association signal with glycine-to-serine ratio (rs1107366 at ALDH1L1). There was no robust evidence for association between these variants and insulin resistance or diabetes. Genetic variants associated with genes in the glycine biosynthesis pathways do not provide consistent evidence for a role of glycine in diabetes related traits.

Fractalkine (CX3CL1), a new factor protecting b-cells against TNFa

Sabine Rutti, Caroline Arous, Domitille Schvartz, Katharina Timper, et al.
MOLMET164_proof ■ 14 Aug 2014 ■ 1/11
http://dx.doi.org/10.1016/j.molmet.2014.07.007

Objective: We have previously shown the existence of a muscleepancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. Methods: Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1e50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1.   Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in b-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFa on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFa on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.
Heart Failure, Saxagliptin and Diabetes Mellitus: Observations from the SAVOR – TIMI 53 Randomized Trial

Benjamin M. Scirica; Eugene Braunwald; Itamar Raz, and SAVOR-TIMI 53 Steering Committee and Investigators
Circulation. Sept 4, 2014  http://dx.doi.org:/10.1161/CIRCULATIONAHA.114.010389
Background—Diabetes and heart failure frequently coexist. However, few diabetes trials have prospectively evaluated and adjudicated heart failure as an endpoint. Methods and Results—16,492 patients with type 2 diabetes and a history of, or at risk for, cardiovascular events were randomized to saxagliptin or placebo (mean followup-2.1 years). The primary endpoint was the composite of cardiovascular death, myocardial infarction, or ischemic stroke. Hospitalization for heart failure was a predefined component of the secondary endpoint. Baseline NT-proBNP was measured in 12,301 patients. More patients treated with saxagliptin (289, 3.5%) were hospitalized for heart failure compared to placebo (228, 2.8%) (HR 1.27; 95%CI 1.07-1.51; p=0.007). Corresponding rates at 12-months were 1.9% vs.1.3% (HR 1.46, 95%CI 1.15-1.88, p=0.002, with no significant difference thereafter time-varying interaction
p=0.017). Subjects at greatest risk for hospitalization for heart failure had prior heart failure, EGFR < 60 ml/min and/or elevated baseline levels of NT-proBNP. There was no evidence of heterogeneity between NT-proBNP and saxagliptin (p for interaction=0.46), though the absolute risk excess for heart failure with saxagliptin was greatest in the highest NT-proBNP quartile (2.1%). Even in patients at high-risk for hospitalization for heart failure, the risk of the primary and secondary endpoints were similar between treatment groups. Conclusions—In the context of balanced primary and secondary endpoints, saxagliptin treatment was associated with an increased risk for hospitalization for heart failure. This increase in risk was highest among patients with elevated levels of natriuretic peptides, prior heart failure, or chronic kidney disease.
Angiotensin 1–7 improves insulin sensitivity by increasing skeletal muscle glucose uptake in vivo

Omar Echeverría-Rodríguez, Leonardo Del Valle-Mondragón, Enrique Hong
Peptides 51 (2014) 26– 30 http://dx.doi.org/10.1016/j.peptides.2013.10.022

The renin–angiotensin system (RAS) regulates skeletal muscle insulin sensitivity through different mechanisms. The overactivation of the ACE (angiotensin-converting enzyme)/Ang (angiotensin) II/AT1R (Ang IItype 1 receptor) axis has been associated with the development of insulin resistance, whereas the stimulation of the ACE2/Ang 1–7/MasR (Mas receptor) axis improves insulin sensitivity. The in vivo mechanismsby which this axis enhances skeletal muscle insulin sensitivity are scarcely known. In this work, we investigated whether rat soleus muscle expresses the ACE2/Ang 1–7/MasR axis and determined the effect ofAng 1–7 on rat skeletal muscle glucose uptake in vivo. Western blot analysis revealed the expression ofACE2 and MasR, while Ang 1–7 levels were detected in rat soleus muscle by capillary zone electrophoresis. The euglycemic clamp exhibited that Ang 1–7 by itself did not promote glucose transport, but itincreased insulin-stimulated glucose disposal in the rat. In a similar manner, captopril (an ACE inhibitor) enhanced insulin-induced glucose uptake and this effect was blocked by the MasR antagonist A-779. Our results show for the first time that rat soleus muscle expresses the ACE2/Ang 1–7/MasR axis of the RAS,and Ang 1–7 improves insulin sensitivity by enhancing insulin-stimulated glucose uptake in rat skeletal muscle in vivo. Thus, endogenous (systemic and/or local) Ang 1–7 could regulate insulin-mediated glucose transport in vivo.

Evolving concepts in advanced glycation, diabetic nephropathy, and diabetic vascular disease

George Jerums, S Panagiotopoulos, J Forbes, T Osicka, and Mark Cooper
Archives of Biochemistry and Biophysics 419 (2003) 55–62
http://dx.doi.org:/10.1016/j.abb.2003.08.017

Advanced glycation endproducts (AGEs) have been postulated to play a role in the development of both nephropathy and large vessel disease in diabetes. However, it is still not clear which AGE subtypes play a pathogenetic role and which of several AGE receptors mediate AGE effects on cells. This review summarises the renoprotective effect of inhibitors of AGE formation, including aminoguanidine, and of cross-link breakers, including ALT-711, on experimental diabetic nephropathy and on mesenteric vascular hypertrophy. It also demonstrates similar effects of aminoguanidine and ramipril (an angiotensin converting enzyme inhibitor) on fluorescent and immunoassayable AGE levels, renal protein kinase C activity, nitrotyrosine expression, lysosomal function, and protein handling in experimental diabetes. These findings indicate that inhibition of the renin angiotensin system blocks both upstream and downstream pathways leading to tissue injury. We postulate that the chemical pathways leading to advanced glycation endproduct formation and the renin angiotensin systems may interact through the generation of free radicals, induced both by glucose and angiotensin II. There is also evidence to suggest that AGE-dependent pathways may play a role in the development of tubulointerstitial fibrosis in the diabetic kidney. This effect is mediated through RAGE and is TGF-b and CTGF-dependent.

Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

Zuheng Ma, Noah Moruzzi, Sergiu-Bogdan Catrina, Ingrid Hals, et al.
PLoS ONE 8(7): e67498. http://dx.doi.org:/10.1371/journal.pone.0067498

Objective: Beta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia. Methods and Design: Rat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia. Results: Insulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to nondiabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.
Conclusions:
1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage,
2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors,
3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.

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Prefacing the e-Book Epilogue: Metabolic Genomics and Pharmaceutics

Author and Curator: Larry H. Bernstein, MD, FCAP

 

Adieu, adieu, adieu …

Sound of Music

Snoopy - Charlie happiness

Snoopy – Charlie happiness

This work has been a coming to terms with my scientific and medical end of career balancing in a difficult time after retiring, but it has been rewarding.  In the clinical laboratories, radiology, anesthesiology, and in pharmacy, there has been some significant progress in support of surgical, gynecological, developmental, medical practices, and even neuroscience directed disciplines, as well as epidemiology over a period of half a century.  Even then, cancer and neurological diseases have been most difficult because the scientific basic research has either not yet uncovered a framework, or because that framework has proved to be multidimensional.  In the clinical laboratory sciences, there has been enormous progress in instrumental analysis, with the recent opening of molecular methods not yet prepared for routine clinical use, which will be a very great challenge to the profession, which has seen the development of large sample volume, multianalite, high-throughput, low-cost support emerging for decades.  The capabilities now underway will also enrrich the the capabilities of the anatomic pathology suite and the capabilities of 3-dimensional radiological examination.  In both pathology and radiology, we have seen the division of the fields into major subspecialties.  The development of the electronic health record had to take lessons from the first developments in the separate developments of laboratory, radiology, and pharmacy health record systems, to which were added, full cardiology monitoring systems.  These have been unintegrated.  This made it difficult to bring forth a suitable patient health record because the information needed to support decision-making by practitioners was in separate “silos”.  The mathematical methods that are being applied to the -OMICS sciences, can be brought to bear on the simplification and amplification of the clinicians’ ability to make decisions with near “errorless” discrimination, still allowing for an element of “art” in carrying out the history, physical examination, and knowledge unique to every patient.

We are at this time opening a very large, complex, study of biology in relationship to the human condition.  This will require sufficient resources to be invested in the development of these for a better society, which I suspect, will go on beyond the life of my grandchildren.  Hopefully, the long-term dangers of climate change will be controlled in that time.  As a society, or as a group of interdependent societies, we have no long term interest in continuing self-destructive behaviors that have predominated in the history of mankind.  I now top off these discussions with some further elucidation of what lies before us.

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Douglas B. Kell and Royston Goodacre
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK
Drug Discovery Today Feb 2014;19(2)  http://dx.doi.org/10.1016/j.drudis.2013.07.014

Metabolism represents the ‘sharp end’ of systems biology,

  • because changes in metabolite concentrations
  • are necessarily amplified relative to
  • changes in the transcriptome, proteome and enzyme activities,
  • which can be modulated by drugs.

To understand such behaviour, we therefore need
(and increasingly have)

  • reliable consensus (community) models of the human metabolic network
  • that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters,

  • because drugs bear structural similarities to metabolites known
  • from the network reconstructions and from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic
network reconstruction; it can predict inter alia:

  1. the effects of inborn errors of metabolism;
  2. which metabolites are exometabolites, and
  3. how metabolism varies between tissues and cellular compartments.

Even these qualitative network models are not yet complete. As our
understanding improves so do we recognize more clearly the need for a systems (poly)pharmacology.

Modelling biochemical networks – why we do so
There are at least four types of reasons as to why one would wish to model a biochemical network:

  1. Assessing whether the model is accurate, in the sense that it
    reflects – or can be made to reflect – known experimental facts.
  2. Establishing what changes in the model would improve the
    consistency of its behaviour with experimental observations
    and improved predictability, such as with respect to metabolite
    concentrations or fluxes.
  3. Analyzing the model, typically by some form of sensitivity
    analysis, to understand which parts of the system contribute
    most to some desired functional properties of interest.
  4. Hypothesis generation and testing, enabling one to analyse
    rapidly the effects of manipulating experimental conditions in
    the model without having to perform complex and costly
    experiments (or to restrict the number that are performed).

In particular, it is normally considerably cheaper to perform
studies of metabolic networks in silico before trying a smaller
number of possibilities experimentally; indeed for combinatorial
reasons it is often the only approach possible. Although
our focus here is on drug discovery, similar principles apply to the
modification of biochemical networks for purposes of ‘industrial’
or ‘white’ biotechnology.
Why we choose to model metabolic networks more than

  • transcriptomic or proteomic networks

comes from the recognition – made particularly clear

  • by workers in the field of metabolic control analysis

– that, although changes in the activities of individual enzymes tend to have

  • rather small effects on metabolic fluxes,
  • they can and do have very large effects on metabolite concentrations (i.e. the metabolome).

Modelling biochemical networks – how we do so

Although one could seek to understand the

  1. time-dependent spatial distribution of signalling and metabolic substances within indivi
    dual cellular compartments and
  2. while spatially discriminating analytical methods such as Raman spectroscopy and
    mass spectrometry do exist for the analysis of drugs in situ,
  • the commonest type of modelling, as in the spread of substances in
    ecosystems,
  • assumes ‘fully mixed’ compartments and thus ‘pools’ of metabolites.

Although an approximation, this ‘bulk’ modelling will be necessary for complex ecosystems such as humans where, in addition to the need for tissue- and cell-specific models, microbial communities inhabit this superorganism and the
gut serves as a source for nutrients courtesy of these symbionts.

Topology and stoichiometry of metabolic networks as major constraints on fluxes
Given their topology, which admits a wide range of parameters for
delivering the same output effects and thereby reflects biological
robustness,

  • metabolic networks have two especially important constraints that assist their accurate modelling:

(i) the conservation of mass and charge, and
(ii) stoichiometric and thermodynamic constraints.

These are tighter constraints than apply to signalling networks.

New developments in modelling the human metabolic network
Since 2007, several groups have been developing improved but nonidentical models of the human metabolic network at a generalised level and in tissue-specific forms. Following a similar community-driven strategy in Saccharomyces cerevisiae, surprisingly similar to humans, and in Salmonella typhimurium,

we focus in particular on a recent consensus paper that provides a highly curated and semantically annotated model of the human metabolic network, termed

In this work, a substantial number of the major groups active in this area came together to provide a carefully and manually constructed/curated network, consisting of some 1789 enzyme-encoding genes, 7440 reactions and 2626 unique metabolites distributed over eight cellular compartments.  A variety of dead-end metabolites and blocked reactions remain (essentially orphans and widows). But Recon2 was able to

  • account for some 235 inborn errors of metabolism,
  • a variety of metabolic ‘tasks’ (defined as a non-zero flux through a reaction or through a pathway leading to the production of a metabolite Q from a metabolite P).
  • filtering based on expression profiling allowed the construction of 65 cell-type-specific models.
  • Excreted or exometabolites are an interesting set of metabolites,
  • and Recon2 could predict successfully a substantial fraction of those

Role of transporters in metabolic fluxes

The uptake and excretion of metabolites between cells and their macrocompartments

  • requires specific transporters and in the order of one third of ‘metabolic’ enzymes,
  • and indeed of membrane proteins, are in fact transporters or equivalent.

What is of particular interest (to drug discovery), based on their structural similarities, is the increasing recognition (Fig. 3) that pharmaceutical drugs also

  • get into and out of cells by ‘hitchhiking’ on such transporters, and not –

to any significant extent –

  • by passing through phospholipid bilayer portions
    of cellular membranes.

This makes drug discovery even more a problem of systems biology than of biophysics.

role of solute carriers and other transporters in cellular drug uptake

role of solute carriers and other transporters in cellular drug uptake

Two views of the role of solute carriers and other transporters in cellular drug uptake. (a) A more traditional view in which all so-called ‘passive’drug uptake occurs through any unperturbed bilayer portion of membrane that might be present.
(b) A view in which the overwhelming fraction of drug is taken up via solute transporters or other carriers that are normally used for the uptake of intermediary metabolites. Noting that the protein:lipid ratio of biomembranes is typically 3:1 to 1:1 and that proteins vary in mass and density (a typical density is 1.37 g/ml) as does their extension, for example, normal to the ca. 4.5 nm lipid bilayer region, the figure attempts to portray a section of a membrane with realistic or typical sizes and amounts of proteins and lipids. Typical protein areas when viewed normal to the membrane are 30%, membranes are rather more ‘mosaic’ than ‘fluid’ and there is some evidence that there might be no genuinely ‘free’ bulk lipids (typical phospholipid masses are 750 Da) in biomembranes that are uninfluenced by proteins. Also shown is a typical drug: atorvastatin (LipitorW) – with a molecular mass of 558.64 Da – for size comparison purposes. If proteins are modelled as
cylinders, a cylinder with a diameter of 3.6 nm and a length of 6 nm has a molecular mass of ca. 50 kDa. Note of course that in a ‘static’ picture we cannot show the dynamics of either phospholipid chains or lipid or protein diffusion.

‘Newly discovered’ metabolites and/or their roles

To illustrate the ‘unfinished’ nature even of Recon2, which concentrates on the metabolites created via enzymes encoded in the human genome, and leaving aside the more exotic metabolites of drugs and foodstuffs and the ‘secondary’ metabolites of microorganisms, there are several examples of interesting ‘new’ (i.e. more or less recently recognised) human metabolites or roles thereof that are worth highlighting, often from studies seeking biomarkers of various diseases – for caveats of biomarker discovery, which is not a topic that we are covering here, and the need for appropriate experimental design. In addition, classes of metabolites not well represented in Recon2 are oxidised molecules such as those caused by nonenzymatic reaction of metabolites with free radicals such as the hydroxyl radical generated by unliganded iron. There is also significant interest in using methods of determining small molecules such as those in the
metabolome (inter alia) for assessing the ‘exposome’, in other words all the potentially polluting agents to which an
individual has been exposed.

Recently discovered effects of metabolites on enzymes 

Another combinatorial problem reflects the fact that in molecular enzymology it is not normally realistic to assess every possible metabolite to determine whether it is an effector (i.e.activator or inhibitor) of the enzyme under study. Typical proteins are highly promiscuous and there is increasing evidence for the comparative promiscuity of metabolites
and pharmaceutical drugs. Certainly the contribution of individual small effects of multiple parameter changes can have substantial effects on the potential flux through an overall pathway, which makes ‘bottom up’ modelling an inexact science. Even merely mimicking the vivo (in Escherichia coli) concentrations of K+, Na+, Mg2+, phosphate, glutamate, sulphate and Cl significantly modulated the activities of several enzymes tested relative to the ‘usual’ assay conditions. Consequently, we need to be alive to the possibility of many (potentially major) interactions of which we are as yet ignorant. One class of example relates to the effects of the very widespread post-translational modification on metabolic
enzyme activities.

A recent and important discovery (Fig. 4) is that a single transcriptome experiment, serving as a surrogate for fluxes through individual steps, provides a huge constraint on possible models, and predicts in a numerically tractable way and
with much improved accuracy the fluxes to exometabolites without the need for such a variable ‘biomass’ term. Other recent and related strategies that exploit modern advances in ‘omics and network biology to limit the search space in constraint-based metabolic modelling.

Fig 4. Workflow for expression-profile-constrained metabolic flux estimation

  1. Genome-scale metabolic model with gene-protein-reaction relationships
  2. Map absolute gene expression levels to reactions
  3. Maximise correlation between absolute gene expression and metabolic flux
  4. Predict fluxes to exometabolites
  5. Compare predicted with experimental fluxes to exometabolites

Drug Discovery Today

The steps in a workflow that uses constraints based on (i) metabolic network stoichiometry and chemical reaction properties (both encoded in the model) plus, and (ii) absolute (RNA-Seq) transcript expression profiles to enable the
accurate modelling of pathway and exometabolite fluxes. .

Concluding remarks – the role of metabolomics in systems pharmacology

What is becoming increasingly clear, as we recognize that to understand living organisms in health and disease we must treat them as systems, is that we must bring together our knowledge of the topologies and kinetics of metabolic networks with our knowledge of the metabolite concentrations (i.e. metabolomes) and fluxes. Because of the huge constraints imposed on metabolism by reaction stoichiometries, mass conservation and thermodynamics, comparatively few well-chosen ‘omics measurements might be needed to do this reliably (Fig. 4). Indeed, a similar approach exploiting constraints has come to the fore in denovo protein folding and interaction studies.

What this leads us to in drug discovery is the need to develop and exploit a ‘systems pharmacology’ where multiple binding targets are chosen purposely and simultaneously. Along with other measures such as phenotypic screening, and the integrating of the full suite of e-science approaches, one can anticipate considerable improvements in the rate of discovery of safe and effective drugs.

Metabolomics: the apogee of the omics trilogy
Gary J.!Patti, Oscar Yanes and Gary Siuzdak

Metabolites, the chemical entities that are transformed during metabolism, provide a functional readout of cellular biochemistry. With emerging technologies in mass spectrometry, thousands of metabolites can now be
quantitatively measured from minimal amounts of biological material, which has thereby enabled systems-level analyses. By performing global metabolite profiling, also known as untargeted metabolomics, new discoveries linking cellular pathways to biological mechanism are being revealed and are shaping our understanding of cell biology, physiology and medicine.

Metabolites are small molecules that are chemically transformed during metabolism and, as such, they provide a functional readout of cellular state. Unlike genes and proteins, the functions of which are subject to epigenetic regulation and posttranslational modifications, respectively, metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. In this context, metabolite profiling, or metabolomics, has become a powerful approach that has been widely adopted for clinical diagnostics.

The field of metabolomics has made remarkable progress within the past decade and has implemented new tools that have offered mechanistic insights by allowing for the correlation of biochemical changes with phenotype.

In this Innovation article, we first define and differentiate between the targeted and untargeted approaches to metabolomics. We then highlight the value of untargeted metabolomics in particular and outline a guide to performing such studies. Finally, we describe selected applications of un targeted metabolomics and discuss their potential in cell biology.

  • metabolites serve as direct signatures of biochemical activity
  1. In some instances, it may be of interest to examine a defined set of metabolites by using a targeted approach.
  2. In other cases, an untargeted or global approach may be taken in which as many metabolites as possible are measured and compared between samples without bias.
  3. Ultimately, the number and chemical composition of metabolites to be studied is a defining attribute of any metabolomic experiment and shapes experimental design with respect to sample preparation and choice of instrumentation.

The targeted and untargeted workflow for LC/MS-based metabolomics.

a | In the triple quadrupole (QqQ)-based targeted metabolomic workflow, standard compounds for the metabolites of interest are first used to set up selected reaction monitoring methods. Here, optimal instrument voltages are determined and response curves are generated for absolute quantification. After the targeted methods have been established
on the basis of standard metabolites, metabolites are extracted from tissues, biofluids or cell cultures and analysed. The data output provides quantification only of those metabolites for which standard methods have been built.

b | In the untargeted metabolomic workflow, metabolites are first isolated from biological samples and subsequently analysed by liquid chromatography followed by mass spectrometry (LC/MS). After data acquisition, the results are processed by using bioinformatic software such as XCMS to perform nonlinear retention time alignment and identify peaks that are changing between the groups of samples measured. The m/z value s for the peaks of interest are searched in metabolite databases to obtain putative identifications. Putative identifications are then confirmed
by comparing tandem mass spectrometry (MS/MS) data and retention time data to that of standard compounds. The untargeted workflow is global in scope and outputs data related to comprehensive cellular metabolism.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros1*, Daniel J. Brackett2 and George G. Harrigan3
1UCLA School of Medicine, Harbor-UCLA Research and Education Institute, Torrance, CA. 2Department of Surgery, University of Oklahoma Health Sciences Center & VA Medical Center, Oklahoma City, OK, 3Global High Throughput
Screening (HTS), Pharmacia Corporation, Chesterfield, MO.
Current Cancer Drug Targets, 2003, 3, 447-455.

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype
with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited denovo fatty acid synthesis and TCA cycle glucose oxidation. This primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment. Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique
disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Navigating the HumanMetabolome for Biomarker Identification and Design of Pharmaceutical Molecules

Irene Kouskoumvekaki and Gianni Panagiotou
Department of Systems Biology, Center for Biological Sequence Analysis, Building 208, Technical University of Denmark, Lyngby, Denmark
Hindawi Publishing Corporation  Journal of Biomedicine and Biotechnology 2011, Article ID 525497, 19 pages
http://dx.doi.org:/10.1155/2011/525497

Metabolomics is a rapidly evolving discipline that involves the systematic study of endogenous small molecules that characterize the metabolic pathways of biological systems. The study of metabolism at a global level has the potential to contribute significantly to biomedical research, clinical medical practice, as well as drug discovery. In this paper, we present the most up-to-date metabolite and metabolic pathway resources, and we summarize the statistical, and machine-learning tools used for the analysis of data from clinical metabolomics.

Through specific applications on cancer, diabetes, neurological and other diseases, we demonstrate how these tools can facilitate diagnosis and identification of potential biomarkers for use within disease diagnosis. Additionally, we
discuss the increasing importance of the integration of metabolomics data in drug discovery. On a case-study based on the Human Metabolome Database (HMDB) and the Chinese Natural Product Database (CNPD), we demonstrate the close relatedness of the two data sets of compounds, and we further illustrate how structural similarity with human metabolites could assist in the design of novel pharmaceuticals and the elucidation of the molecular mechanisms of medicinal plants.

Metabolites are the byproducts of metabolism, which is itself the process of converting food energy to mechanical energy
or heat. Experts believe there are at least 3,000 metabolites that are essential for normal growth and development (primary metabolites) and thousands more unidentified (around 20,000, compared to an estimated 30,000 genes and 100,000 proteins) that are not essential for growth and development (secondary metabolites) but could represent prognostic, diagnostic, and surrogate markers for a disease state and a deeper understanding of mechanisms of disease.

Metabolomics, the study of metabolism at the global level, has the potential to contribute significantly to biomedical
research, and ultimately to clinical medical practice. It is a close counterpart to the genome, the transcriptome and the proteome. Metabolomics, genomics, proteomics, and other “-omics” grew out of the Human Genome Project, a massive research effort that began in the mid-1990s and culminated in 2003 with a complete mapping of all the genes in the human body. When discussing the clinical advantages of metabolomics, scientists point to the “real-world” assessment
of patient physiology that the metabolome provides since it can be regarded as the end-point of the “-omics” cascade. Other functional genomics technologies do not necessarily predict drug effects, toxicological response, or disease states at the phenotype but merely indicate the potential cause for phenotypical response. Metabolomics can bridge this information gap. The identification and measurement of metabolite profile dynamics of host changes provides the closest link to the various phenotypic responses. Thus it is clear that the global mapping of metabolic signatures pre- and postdrug treatment is a promising approach to identify possible functional relationships between medication and medical phenotype.

Human Metabolome Database (HMDB). Focusing on quantitative, analytic, or molecular scale information about
metabolites, the enzymes and transporters associated with them, as well as disease related properties the HMDB represents the most complete bioinformatics and chemoinformatics medical information database. It contains records for
thousands of endogenous metabolites identified by literature surveys (PubMed, OMIM, OMMBID, text books), data
mining (KEGG, Metlin, BioCyc) or experimental analyses performed on urine, blood, and cerebrospinal fluid samples.
The annotation effort is aided by chemical parameter calculators and protein annotation tools originally developed for
DrugBank.

A key feature that distinguishes the HMDB from other metabolic resources is its extensive support for higher level database searching and selecting functions. More than 175 hand-drawn-zoomable, fully hyperlinked human
metabolic pathway maps can be found in HMDB and all these maps are quite specific to human metabolism and
explicitly show the subcellular compartments where specific reactions are known to take place. As an equivalent to
BLAST the HMDB contains a structure similarity search tool for chemical structures and users may sketch or
paste a SMILES string of a query compound into the Chem-Query window. Submitting the query launches a
structure similarity search tool that looks for common substructures from the query compound that match the
HMDB’s metabolite database. The wealth of information and especially the extensive linkage to metabolic diseases
to normal and abnormal metabolite concentration ranges, to mutation/SNP data and to the genes, enzymes, reactions
and pathways associated with many diseases of interest makes the HMDB one the most valuable tool in the hands
of clinical chemists, nutritionists, physicians and medical geneticists.

Metabolomics in Drug Discovery and Polypharmacology Studies

Drug molecules generally act on specific targets at the cellular level, and upon binding to the receptors, they exert
a desirable alteration of the cellular activities, regarded as the pharmaceutical effect. Current drug discovery depends
largely on ransom screening, either high-throughput screening (HTS) in vitro, or virtual screening (VS) in silico. Because
the number of available compounds is huge, several druglikeness filters are proposed to reduce the number of compounds that need to be evaluated. The ability to effectively predict if a chemical compound is “drug-like” or “nondruglike” is, thus, a valuable tool in the design, optimization, and selection of drug candidates for development. Druglikeness is a general descriptor of the potential of a small molecule to become a drug. It is not a unified descriptor
but a global property of a compound processing many specific characteristics such as good solubility, membrane
permeability, half-life, and having a pharmacophore pattern to interact specifically with a target protein. These
characteristics can be reflected as molecular descriptors such as molecular weight, log P, the number of hydrogen bond
donors, the number of hydrogen-bond acceptors, the number of rotatable bonds, the number of rigid bonds, the
number of rings in a molecule, and so forth.

Metabolomics for the Study of Polypharmacology of Natural Compounds

Internationally, there is a growing and sustained interest from both pharmaceutical companies and public in medicine
from natural sources. For the public, natural medicine represent a holistic approach to disease treatment, with
potentially less side effects than conventional medicine. For the pharmaceutical companies, bioactive natural products
constitute attractive drug leads, as they have been optimized in a long-term natural selection process for optimal interaction with biomolecules. To promote the ecological survival of plants, structures of secondary products have evolved to interact with molecular targets affecting the cells, tissues and physiological functions in competing microorganisms,
plants, and animals. In this, respect, some plant secondary products may exert their action by resembling endogenous
metabolites, ligands, hormones, signal transduction molecules, or neurotransmitters and thus have beneficial
effects on humans.

Future Perspectives

Metabolomics, the study of metabolism at the global level, is moving to exciting directions.With the development ofmore
sensitive and advanced instrumentation and computational tools for data interpretation in the physiological context,
metabolomics have the potential to impact our understanding of molecular mechanisms of diseases. A state-of-theart
metabolomics study requires knowledge in many areas and especially at the interface of chemistry, biology, and
computer science. High-quality samples, improvements in automated metabolite identification, complete coverage of
the human metabolome, establishment of spectral databases of metabolites and associated biochemical identities, innovative experimental designs to best address a hypothesis, as well as novel computational tools to handle metabolomics data are critical hurdles that must be overcome to drive the inclusion of metabolomics in all steps of drug discovery and drug development. The examples presented above demonstrated that metabolite profiles reflect both environmental and genetic influences in patients and reveal new links between metabolites and diseases providing needed prognostic,diagnostic, and surrogate biomarkers. The integration of these signatures with other omic technologies is of utmost importance to characterize the entire spectrum of malignant phenotype.

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Summary of Proteomics

Author and Curator: Larry H. Bernstein, MD, FCAP 

 

We have completed a series of discussions on proteomics, a scientific endeavor that is essentially 15 years old.   It is quite remarkable what has been accomplished in that time.  The interest is abetted by the understanding of the limitations of the genomic venture that has preceded it.  The thorough, yet incomplete knowledge of the genome, has led to the clarification of its limits.  It is the coding for all that lives, but all that lives has evolved to meet a demanding and changing environment with respect to

  1. availability of nutrients
  2. salinity
  3. temperature
  4. radiation exposure
  5. toxicities in the air, water, and food
  6. stresses – both internal and external

We have seen how both transcription and translation of the code results in a protein, lipoprotein, or other complex than the initial transcript that was modeled from tRNA. What you see in the DNA is not what you get in the functioning cell, organ, or organism.  There are comparabilities as well as significant differences between plants, prokaryotes, and eukaryotes.  There is extensive variation.  The variation goes beyond genomic expression, and includes the functioning cell, organ type, and species.

Here, I return to the introductory discussion.  Proteomics is a goal directed, sophisticated science that uses a combination of methods to find the answers to biological questions. Graves PR and Haystead TAJ.  Molecular Biologist’s Guide to Proteomics.
Microbiol Mol Biol Rev. Mar 2002; 66(1): 39–63.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC120780/

Peptide mass tag searching

Peptide mass tag searching

Peptide mass tag searching. Shown is a schematic of how information from an unknown peptide (top) is matched to a peptide sequence in a database (bottom) for protein identification. The partial amino acid sequence or “tag” obtained by MS/MS is combined with the peptide mass (parent mass), the mass of the peptide at the start of the sequence (mass tag 1), and the mass of the peptide at the end of the sequence (mass tag 2). The specificity of the protease used (trypsin is shown) can also be included in the search.