Biochemistry and Dysmetabolism of Aging and Serious Illness

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Biochemistry and Dysmetabolism of Aging and Serious Illness

November 19, 2015 by larryhbern

Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002

Highlights

•Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
•Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
•Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A₂ sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H₂O₂ production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H₂O₂ production to activation of the cPLA₂-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H₂O₂ activation of cPLA₂-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA₂-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H₂O₂, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA₂-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

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Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H₂O₂ (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H₂O₂ functions as a signaling molecule to activate cPLA₂, the initiating enzyme in the cPLA₂-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA₂ immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA₂-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA₂ (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H₂O₂ production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA₂ by physiologically relevant concentrations of H₂O₂ was confirmed in vitro. Astrocytic involvement in the cPLA₂-sphingomyelinase pathway was also indicated by an increase in cPLA₂ positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA₂immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA₂, neuronal cPLA₂ is activated by an influx of intracellular calcium, whereas astrocytic cPLA₂ is directly activated by excessive generation of H₂O₂ (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA₂ in astrocytes by H₂O₂ and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA₂ is the cellular mediator of the H₂O₂ dependent cPLA₂-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H₂O₂ in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA₂, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA₂-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H₂O₂generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9. http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.

Ricciardi MR¹, Mirabilii S², Allegretti M², Licchetta R², Calarco A³, Torrisi MR¹, Foà R², Nicolai R⁴, Peluso G³, Tafuri A¹. Author information

Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.

Oncogene. 2015 Oct 12. http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]

Tumour-suppression function of KLF12 through regulation of anoikis.

Godin-Heymann N¹, Brabetz S¹, Murillo MM^1,², Saponaro M³, Santos CR⁴, Lobley A⁵, East P⁵, Chakravarty P⁵, Matthews N⁶, Kelly G⁵, Jordan S¹, Castellano E¹, Downward J^1,². Author information

Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.

Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.

Montal ED¹, Dewi R², Bhalla K³, Ou L², Hwang BJ², Ropell AE⁴, Gordon C⁴, Liu WJ³, DeBerardinis RJ⁵, Sudderth J⁵, Twaddel W⁶, Boros LG⁷, Shroyer KR⁴,Duraisamy S⁸, Drapkin R⁸, Powers RS⁶, Rohde JM⁹, Boxer MB⁹, Wong KK¹⁰, Girnun GD¹¹. Author information

Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

Mol Cancer Res. 2015 Oct;13(10):1408-20. http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.

Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.

Kardos GR¹, Wastyk HC², Robertson GP³. Author information

Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway. This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.

J Natl Cancer Inst. 2015 Oct 12;108(1). pii: djv287. http://dx.doi.org:/10.1093/jnci/djv287.

SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.

Saxena N¹, Maio N¹, Crooks DR¹, Ricketts CJ¹, Yang Y¹, Wei MH¹, Fan TW¹, Lane AN¹, Sourbier C¹, Singh A¹, Killian JK¹, Meltzer PS¹, Vocke CD¹, Rouault TA¹, Linehan WM¹. Author information

BACKGROUND: Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.

METHODS: In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

Cancer Res. 2015 Oct 15;75(20):4429-36. http://dx.doi.org:/10.1158/0008-5472.CAN-15-0074.

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Feeley KP¹, Bray AW¹, Westbrook DG¹, Johnson LW², Kesterson RA², Ballinger SW¹, Welch DR³. Author information

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4. http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]

Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Ju R¹, Guo L¹, Li J¹, Zhu L¹, Yu X¹, Chen C¹, Chen W¹, Ye C², Zhang D³. Author information

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

Cancer Immunol Res. 2015 Nov;3(11):1236-47. http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Hossain F¹, Al-Khami AA², Wyczechowska D², Hernandez C², Zheng L², Reiss K³, Valle LD⁴, Trillo-Tinoco J², Maj T⁵, Zou W⁵, Rodriguez PC⁶, Ochoa AC⁷. Author information

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0. http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

Sato PY¹, Chuprun JK¹, Ibetti J¹, Cannavo A¹, Drosatos K¹, Elrod JW¹, Koch WJ². Author information

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]

All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.

Yang L¹, Luo C², Chen C¹, Wang X¹, Shi W¹, Liu J¹. Author information

BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

J Biol Chem. 2015 Oct 23;290(43):25834-46. http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.

Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

Trefely S¹, Khoo PS², Krycer JR³, Chaudhuri R³, Fazakerley DJ³, Parker BL³, Sultani G¹, Lee J⁴, Stephan JP⁴, Torres E⁴, Jung K⁴, Kuijl C⁴, James DE⁵,Junutula JR⁶, Stöckli J⁷. Author information

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

FASEB J. 2015 Oct 19. http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]

Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Cho Y¹, Hazen BC¹, Gandra PG¹, Ward SR¹, Schenk S¹, Russell AP¹, Kralli A². Author information

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Cell Death Dis. 2015 Oct 29;6:e1944. http://dx.doi.org:/10.1038/cddis.2015.306.

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

Mughal W^1,², Nguyen L², Pustylnik S³, da Silva Rosa SC^1,², Piotrowski S^1,², Chapman D^2,⁴, Du M³, Alli NS³, Grigull J^3,⁵, Halayko AJ^6,⁷, Aliani M⁸, Topham MK⁹, Epand RM¹⁰, Hatch GM^2,¹¹, Pereira TJ^2,¹¹, Kereliuk S^2,¹¹, McDermott JC³, Rampitsch C¹², Dolinsky VW^2,¹¹, Gordon JW^1,^2,⁴. Author information

Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.