Blood forming precursors in bone marrow

March 15, 2016 by larryhbern

Blood forming precursors in bone marrow

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Blood stem cells study could pave the way for new cancer therapy

UNIVERSITY OF EDINBURGH

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This image shows the formation of blood stem cells inside the embryonic vessel called dorsal aorta. In green is shown secreted molecule called NOGGIN, which plays an important role in this process. The University of Edinburgh

People with leukaemia could be helped by new research that sheds light on how the body produces its blood supply.

Scientists are a step closer to creating blood stem cells that could reduce the need for bone marrow transplants in patients with cancer or blood disorders.

Enabling scientists to grow the stem cells artificially from pluripotent stem cells could also lead to the development of personalised blood therapies, researchers say.

Blood stem cells are found in bone marrow and produce all blood cells in the body. These cells – known as haematopoietic stem cells (HSCs) – help to restore blood supply in patients who have been treated for leukaemia.

Researchers used a mouse model to pinpoint exactly how HSCs develop in the womb. They showed for the first time how three key molecules interact together to generate the cells, which are later found in adult bone marrow.

The discovery could help scientists to recreate this process in the lab, in the hope that HSCs could one day be developed for clinical use.

Scientists say this fundamental understanding of early development may also have an impact on other diseases that affect blood formation and supply.

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The research has been published in Nature Communications.

Professor Alexander Medvinsky, of the University of Edinburgh’s MRC Centre for Regenerative Medicine said: “There is a pressing need to improve treatments for diseases like leukaemia and this type of research brings us a step closer to that milestone. The more we understand about how embryos develop these blood stem cells, the closer we come to being able to make them in the lab.”

http://www.ed.ac.uk/news/2016/stem-cells-100316

Céline Souilhol, Christèle Gonneau, Javier G. Lendinez, Antoniana Batsivari, Stanislav Rybtsov, Heather Wilson, Lucia Morgado-Palacin, David Hills, Samir Taoudi, Jennifer Antonchuk, Suling Zhao, Alexander Medvinsky. Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nature Communications, 2016; 7: 10784 DOI: 10.1038/ncomms10784

During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta–gonad–mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso–ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs.

Haematopoietic stem cells (HSCs) lie at the foundation of the adult haematopoietic system, and give rise to cells of all blood lineages throughout the lifespan of an organism. An important property of adult (definitive) haematopoietic stem cells (dHSCs) is that they are capable of long-term reconstitution of the haematopoietic system upon transplantation into irradiated recipients. In the mouse, such cells develop by embryonic stages E10–E11 in the aorta–gonad–mesonephros (AGM) region^{1, 2, 3, 4}. An ex vivo approach showed that the AGM region has a robust autonomous capacity to generate dHSCs¹. The AGM region comprises the dorsal aorta flanked on both sides by the urogenital ridges (UGRs), which contain embryonic rudiments of kidney and mesonephros. HSCs develop in a polarized manner, predominantly in the ventral floor of the dorsal aorta (AoV), more rarely in the dorsal domain of the dorsal aorta (AoD), and are absent in the UGRs^{2, 5, 6, 7}. Localization of dHSCs to the AoV in mouse and human embryos was shown by long-term reconstitution experiments^{5, 6}.

Abundant evidence indicates that during development, a specialized embryonic endothelial compartment known as haematogenic (or haemogenic) endothelium gives rise to haematopoietic stem and progenitors cells^{7, 8, 9, 10}. The haematopoietic programme in various vertebrate models is executed predominantly in the AoV, and is recognized by the expression of essential haematopoietic transcription factors, for example, Runx1 and cKit, and the appearance of clusters of haematopoietic cells budding from the endothelium of the dorsal aorta^{6, 8, 9, 11, 12, 13, 14}.

It is broadly accepted that HSCs develop from the haematogenic endothelium within intra-aortic clusters. This transition involves several consecutive maturation steps of HSC precursors: pro-HSCspre-HSC type Ipre-HSC type IIdHSC^{15, 16, 17}. All these precursors express endothelial markers, such as vascular-endothelial cadherin (VC) and CD31, and sequentially upregulate haematopoietic surface markers: CD41 (pro-HSCs), CD43 (pre-HSC type I) and finally CD45 (pre-HSC type II). This maturation process occurs in the dorsal aorta between E9 and E11. Specifically, pro-HSCs emerge at E9, pre-HSCs Type I appear at E10 and pre-HSCs type II predominantly at E11. Unlike dHSCs, pre-HSCs cannot reconstitute the adult haematopoietic system by direct transplantation and require prior maturation in an embryonic or neonatal environment^{15, 16, 17, 18,19}.

A number of signalling pathways (Notch, Wnt, retinoic acid, interleukin-3 and inflammatory) have been implicated in HSC development; however, a coherent picture is yet to be elucidated^{15, 17, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31}. HSC precursors (pro-HSCs, pre-HSCs type I and pre-HSCs type II) express cKit¹⁷ from early developmental stages. A recent study has shown that the cKit ligand, known as stem cell factor (SCF), is a key regulator driving maturation of these HSC precursors into dHSCs in the AGM region¹⁷, which is in agreement with the marked decline of HSC activity in SCF mutant mice^{32, 33}. In the adult, SCF is critically important for HSC maintenance in the bone marrow niche, mainly in the endothelial compartment³². Sonic Hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) pathways are also important mediators; in zebrafish, these two morphogenes are involved in arterial specification and haematopoietic patterning, respectively^34,35. In the mouse, subaortic BMP4 and Shh/Indian Hedgehog derived from gut were also proposed to be responsible for HSC development^{36, 37}.

During development, interactions between spatially segregated compartments are essential for tissue patterning and specification, and are often mediated by gradients of secreted molecules^{38,39, 40}. Molecules secreted by distant tissues, such as somites, can influence HSC development in the AGM region^{41, 42, 43, 44, 45}. Developing HSCs are embedded in the complex AGM microenvironment, suggesting that HSC development may require signals derived from different compartments of the AGM region. We sought to test this hypothesis. However, the analysis of HSC development in vivo is significantly hampered by low accessibility of embryos developing in utero, fast maturation of dHSCs, lack of uniquely specific markers for HSC precursors and their low numbers in the AGM region. Therefore, we employed here a robust ex vivo culture system that models HSC development in the embryo in combination with functional HSC analysis using in vivolong-term reconstitution assay^{15, 16, 17}. Specifically, to study interactions between AGM subregions, we took advantage of the in vitro reaggregation system that enables close juxtaposition of cell types¹⁵.

We show that interactions between three compartments of the AGM, the AoV, the AoD and the UGRs, are necessary for efficient generation of dHSCs. First, we show that dHSC activity in the isolated E10.5 AoV is limited but can be significantly enhanced by co-culture with the AoD, and that this is mediated at least partly by Shh, secreted dorsally in vivo. Second, while HSC activity in isolated E11.5 AoD is limited, co-culture with a competent AoV microenvironment activates dHSC generation in the AoD. We found that this effect is mediated by SCF, which is secreted abundantly by the AoV stroma in vivo as shown here. Third, we show that downregulation of BMP4 signalling by BMP antagonist Noggin, which is present at high levels in the AoV and especially in intra-aortic clusters as revealed here by in vivo observations, is required for HSC development. Fourth, UGRs, which express high levels of SCF, also enhance HSC development in the dorsal aorta.

Our results based on in vivo observations and ex vivo modelling strongly suggest that juxtaposed, anatomically distinct domains within the AGM region create a complex landscape of interactive signals that underpins HSC development.

Pre-HSCs localize preferentially to the AoV

As dHSCs mature from pre-HSCs, we investigated whether the emergence of dHSC predominantly in the AoV⁶ is a result of asymmetric (ventralized) distribution of pre-HSCs. Dorsal aortae were separated from UGRs and bisected into AoV and AoD (including notochord) as described previously⁶ (Supplementary Fig. 1a). The different domains were then directly transplanted into irradiated mice to detect dHSCs. We first confirmed our previous observation that at E11.5 dHSCs appear almost exclusively in the AoV, although some dHSCs were in the AoD and engrafted few recipients at high level (Supplementary Fig. 1b). Limiting dilution analysis showed that dHSCs are approximately four times more frequent in the AoV compared with AoD. UGRs did not contain HSCs in line with previous reports^{2, 6}.

We then investigated the spatial distribution of pre-HSCs type I and pre-HSCs type II in E10.5–E11.5 embryos using the OP9 co-culture system supplemented with Il3+SCF+Flt3 (termed 3GF), which allows pre-HSCs (which do not engraft by direct transplantation) to mature into dHSC that become detectable by long-term repopulation assay as described previously¹⁶. Doses of transplanted cells (expressed in embryo equivalents, e.e.) were chosen based on the requirements of individual experiments (explained in Methods section). In these experiments (Fig. 1), the dose injected was high (1–2 e.e.) to detect potentially low dHSC numbers in AoD and UGRs.

(a) E10.5 AoV, AoD and UGRs were co-aggregated with OP9 and cultured for 5 days, and the formation of dHSCs was then tested by transplantation into irradiated mice (2 e.e. per recipient; AoV: six independent experiments; AoD: four independent experiments; UGRs: two independent experiments). Dashed line indicates the cutoff for high-level engraftment (>70% donor chimaerism). (b) E11.5 aortas and UGRs were transplanted after reaggregate culture (Ao: 0.2 e.e. per recipient and UGRs: 1 e.e. per recipient; two independent experiments). (c,d) Pre-HSCs type I (VC⁺CD45⁻) (c) or type II (VC⁺CD45⁺) (d) sorted from E11.5 AoV and AoD were co-aggregated with OP9 cells and transplanted after culture (1 e.e. per recipient; two independent experiments). (a–d) Levels of engraftment are plotted, and number of repopulated versus total number of transplanted mice are shown in brackets. Number of embryo equivalents (ee) injected in each experiment are indicated on the graphs. (*P<0.05; ***P<0.005; Mann–Whitney U-test). In all these experiments, tissues were cultured with three growth factors (Flt3I, Il3 and SCF). AGM, aorta–gonad–mesonephros region; Ao, dorsal Aorta; AoV, ventral domain of the dorsal aorta; AoD, dorsal domain of the dorsal aorta; UGRs, urogenital ridges.

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We have shown previously that E10.5 AGM region mainly contains type I pre-HSCs, whereas at E11.5, type I and type II pre-HSCs co-exist¹⁶. Dissected E10.5 AGM regions co-cultured with OP9 in 3GF for 5 days were transplanted into adult irradiated recipients. Out of 21 recipients that received cultured AoV, 20 showed high levels (>70%) of donor-derived long-term haematopoietic chimerism (Fig. 1a). In contrast, only 7 out of 16 recipients of cultured AoD were repopulated at high levels (>70%), while the remaining recipients showed lower or no repopulation (7 and 2, respectively). Cultured UGRs did not produce dHSCs (Fig. 1a). Thus, we conclude that the E10.5 AoD does contain pre-HSCs but at significantly lower numbers than the AoV.

We then investigated whether pre-HSCs localization changes in E11.5 embryos and found that pre-HSCs were still exclusively localized to the dorsal aorta; UGRs carefully separated from the lateral mesenchyme adjacent to the dorsal aorta did not give any repopulation after culture (Fig. 1b). To establish the location of pre-HSCs within the E11.5 dorsal aorta, cell populations enriched for pre-HSCs type I (VC⁺CD45^–) and pre-HSCs type II (VC⁺CD45⁺) were sorted from AoV and AoD, and co-cultured with OP9 stromal cells in the presence of 3GF as described previously¹⁶. We again were able to detect pre-HSC activity in AoD although at lower levels than in AoV. After maturation ex vivo, pre-HSCs type I from AoV and AoD repopulated 7 of 11 and 2 of 8 recipients, respectively (Fig. 1c). Similarly, cultured pre-HSCs type II from AoV and AoD repopulated 11 out of 12 and 4 out of 10 recipients, respectively (Fig. 1d). In all cases, multilineage engraftment was confirmed (Supplementary Fig. 2). These data show that pre-HSCs are significantly enriched in AoV.

Reciprocal inductive interactions between AoD and AoV

To explore hypothetical interactions between AoD and AoV, we made use of a dissociation–reaggregation system that recapitulates HSC development ex vivo¹⁵. This system allowed us to integrate AGM domains in a three-dimensional tissue-like organoid¹⁵ and study their interactions in HSC development. To track the origin of dHSCs, AoV and AoD from wild-type (WT) and green fluorescent protein (GFP) embryos with constitutive expression of GFP⁴⁶ were co-aggregated (termed AoV//AoD co-aggregates) and cultured for 5 days in the presence of 3GF before transplantation (Fig. 2a). Mice transplanted with AoV//AoD co-aggregates can be reconstituted by dHSCs coming from AoD and AoV. The presence of GFP allowed the individual contributions of AoV and AoD to the total repopulation level within the same mouse to be assessed (Fig. 2b,c). This is presented in two separate columns in the graph. Namely, while columns 1 and 3 represent the same recipient mice, the former shows exclusively the contribution of the AoD and the latter shows exclusively the contribution of the AoV into each recipient. To assess the influence of AoD and AoV interaction on HSC development, the repopulation by co-aggregated AoD (column 1) or AoV (column 3) can then be compared with repopulation by independently cultured AoD (column 2) or AoV (column 4). All experiments included reciprocal use of WT and GFP tissues in AoV//AoD co-aggregates, and we observed no difference in repopulation properties between WT and GFP embryos. Homotypic AoV//AoV and AoD//AoD co-aggregates were always used as controls. Note that in these experiments, only 0.2 e.e. were injected per recipient, to ensure that the repopulation levels were not saturated and to allow any inductive effects to be revealed.

(a) Experimental design: the ventral domain (AoV) and the dorsal domain (AoD) of the aorta, and the urogenital ridges (UGRs) from wild-type (WT) and GFP+ embryos were subdissected, and chimeric reaggregates from tissues of these two genotypes were generated. Left column: to test interactions between AoV and AoD, chimeric AoV//AoD reaggregates were generated and transplanted into irradiated recipients after 4–5 days of culture (b,c). Right column: to test interactions between Ao and UGRs, chimeric Ao//UGR reaggregates were generated and transplanted into irradiated recipients after 4–5 days culture (d). GFP+ and/or GFP− donor-derived long-term repopulation allowed us to conclude whether dHSCs originated from AoV, AoD or UGRs. Accordingly, the tissue of origin of donor dHSCs is indicated below each graph. (b) E10.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates as depicted schematically above plots. The reciprocal combination of WT and GFP tissues was used to generate AoV//AoD reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2 e.e. per recipient; two independent experiments). (c) E11.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2 e.e. per recipient; two independent experiments). (d) E11.5 aortas (Ao) and UGRs from WT and GFP embryos were used to generate Ao//UGR chimeric reaggregates. As depicted schematically above the plot, the reciprocal combination of WT and GFP tissues was used to generate Ao//UGR reaggregates. The tissue source of dHSCs is shown separately in left (Ao) and right (UGRs) columns as indicated below the plot (0.01 e.e. per recipient; six independent experiments). (e) Reaggregation of WT Ao with UGRs generate more dHSCs than Ao alone (0.05 e.e. per recipient; two independent experiments). (b–e) In all these experiments, tissues were cultured with three growth factors.

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Using this approach, we found that the E10.5 AoV generates more dHSCs when combined with AoD than on its own (Fig. 2b, compare two rightmost columns). One day later, E11.5 AoD had no positive influence on dHSC generation by AoV (Fig. 2c, compare two rightmost columns). Conversely, the E11.5 AoD produced more HSCs when reaggregated with the AoV than on its own (Fig. 2c, compare two leftmost columns). This inductive effect of AoV on AoD was not observed at E10.5 (Fig. 2b, compare two leftmost columns). These ex vivo modelling experiments revealed reciprocal stage-specific effects of AoV and AoD on HSC development, which could be explained by the differential release of factors by the two regions and/or by differences in the competency of the target cells to respond to signals.

UGRs enhance HSC development in the dorsal aorta  

SCF expression is involved in polarized HSC development

Figure 3: Involvement of polarized stem cell factor in HSC development.

(a) qRT–PCR on fresh AoV, AoD and UGRs at E10.5 and E11.5 showed high expression levels of stem cell factor (SCF) in AoV and UGRs, compared with AoD (data are mean±s.e.m; *P<0.05, **P<0.01, t-test; three independent experiments). No significant difference was observed between E10.5 and E11.5 expression level in any of the tissues. (b) Expression of SCF-GFP and CD31 determined by immunostaining on thick section (300 μm) of SCF-GFP-positive E10.5 AGM region and on SCF-GFP-negative control. Bars, 50 μm. (c) Expression of SCF in sorted populations from fresh E10.5–E11.5 AoV (V) and AoD (D) determined by qRT–PCR. Endo, endothelial population (VC⁺CD45⁻CD43⁻); type I, pre-HSCs type I (VC⁺CD45⁻CD43⁺); type II, pre-HSCs type II (VC⁺CD45⁺); stroma, stromal population (VC⁻CD45⁻CD43⁻). (*P<0.05, t-test; five independent experiments). (d) E10.5 AoD were cultured as reaggregates in the presence of Il3 and Flt3L with or without SCF and human SCF antagonist (SCF-Rh). (0.5 e.e. per recipient; three independent experiments). (e,f) E11.5 AoD (two independent experiments) (e) and E10.5 AoV (two independent experiments) (f) were cultured as explants with or without SCF (no other cytokines); (0.2 e.e. per recipient).

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Shh signalling enhances dHSC generation

 

Figure 4: Sonic Hedgehog is a positive modulator of pre-HSC type I.

(a) Expression level of Sonic Hedgehog (Shh) in E10.5 and E11.5 AGM region determined by qRT–PCR. (data are mean±s.e.m; *P<0.05, t-test; E10.5: three independent experiments and E11.5: two independent experiments). (b) Patched1 and Gli1 expression in endothelial cells (endo: VC⁺CD45⁻CD43⁻), pre-HSCs type I (I: VC⁺CD45⁻CD43⁺) and type II (II: VC⁺CD45⁺) sorted from E11.5 AoV and AoD (two independent experiments). (c) E10.5 AoV and AoD explants were cultured in presence of Shh recombinant protein before transplantation (AoV: 0.1 e.e. per recipient; two independent experiments and AoD: 0.2 e.e. per recipient; three independent experiments). (d) E10.5 AoV and doxycyline-inducible OP9-Shh were co-aggregated and cultured in presence or absence of doxycycline and/or Hedgehog (Hh) antagonist (200 nM) before transplantation (0.2 e.e. per recipient; two independent experiments). (e) 10.5 AoV and AoD co-aggregated with OP9 were cultured in presence of three growth factors with Hh antagonist before transplantation; (0.2 e.e. per recipient; two independent experiments). (f) E11.5 AoV explants were cultured in presence of Shh recombinant protein before transplantation; (0.2 e.e. per recipient; two independent experiments). (g): E11.5 AGM reaggregates were cultured in presence of Hh antagonist before transplantation; (0.1 e.e. per recipient; two independent experiments). (c,d,f,g) In all these experiments, tissues were cultured without cytokines. Hh anta, Hh antagonist; Dox, doxycycline.

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BMP signalling is downregulated in the dHSC lineage

Figure 5: Bone morphogenetic protein signalling is downregulated in dHSC lineage.

(a) Expression of bone morphogenetic protein 4 (BMP4) at E10.5 determined by qRT–PCR; (data are mean±s.e.m.; *P<0.05, t-test; three independent experiments). (b) Expression of BMP4 in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50 μm. Zoomed image shows the subendothelial localization of BMP4 (arrowheads). Bars, 10 μm. (c) Expression of phosphorylated-Smad (P-Smad) in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50 μm. (d) Id genes expression in endothelial cells, pre-HSCs type I and type II directly isolated from E10.5 and E11.5 AoV determined by qRT–PCR. Endo, endothelial population (VC⁺CD45⁻CD43⁻); type I, pre-HSCs type I (VC⁺CD45⁻CD43⁺); type II, pre-HSCs type II (VC⁺CD45⁺); stroma, stromal population (VC⁻CD45⁻CD43⁻). (Data are mean±s.e.m.; *P<0.05, **P<0.01; t-test; five independent experiments). (e–g) Expression of P-Smad, CD31 and CD45 in the endothelium and haematopoietic clusters of E10.5 dorsal aorta. White arrowheads indicate cells with pre-HSC type II phenotype (CD31⁺CD45⁺); green arrows show (CD31⁺CD45^−/low) cells budding out of the dorsal aorta and expressing P-Smad; asterisks indicate CD31⁺CD45⁻ cells expressing P-Smad within the endothelium. Bars, 10 μm. A positive control showing P-Smad staining in the dorsal part of the neural tube can be found in h.

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Figure 6: Haematopoietic clusters are exposed to low concentration of BMP4 and high levels of Noggin.

(a) Expression of BMP antagonists at E10.5 determined by qRT–PCR (data are mean±s.e.m.; *P<0.05,***P<0.005; t-test; three independent experiments). (b) Expression of Noggin in the E10.5 AGM region determined by immunostaining on frozen sections. Note the expression of Noggin in the notochord (Nt) as expected. Bar, 50 μm. (c) Expression of Noggin and BMP4 in intra-aortic clusters characterized by cKit and CD31 expression. Note that BMP4 is mainly expressed underneath the dorsal aorta (arrowheads), while Noggin is expressed in the cluster (arrows). Bars, 10 μm. (d) Expression of Noggin in isolated populations from E10.5 and E11.5 AoV (V) and AoD (D) determined by qRT–PCR. (*P<0.05, t-test; five independent experiments). (e) Model showing downregulation of BMP activity in dHSC lineage. BMP4 is mainly expressed in the ventral mesenchyme, while Noggin is found in haematopoeitic clusters. Accordingly, BMP activity, assessed by the phosphorylation of Smad1,5 and 8 (P-Smad), is high in mesenchymal cells underneath the aortic endothelium and in some endothelial cells (CD31⁺CD45⁻) of the aortic endothelium and decreases in the haematopoeitic clusters. While some pre-HSC type I cells (CD31⁺CD45^−/low) exhibit BMP signalling at a low level, acquisition of CD45 (shown in red) is accompanied by a complete loss of BMP activity. EC, endothelial cells; MC, mesenchymal cells; I, pre-HSC type I; II, pre-HSC type II.

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BMP signalling inhibits HSC development

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Interactions between SCF, Shh and BMP signalling pathways

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We have shown previously that during murine embryo development definitive HSCs emerge predominantly in the ventral domain of the dorsal aorta (AoV)⁶. This spatially polarized production of HSCs might be explained by different origins of dorsal and ventral endothelium and/or by asymmetric production of key factors involved in HSC development^{37, 52, 53} and we reasoned that directional inductive interactions between AGM compartments could be involved. Great insight into inductive interactions in various organs has previously been obtained through in vitro modelling³⁹. Here we modelled interactions between AGM domains in a co-culture system, which supports HSC development¹⁵. Using this ex vivo system, we demonstrate that at early stages (E10.5) HSC maturation in the AoV region is enhanced by the presence of the AoD. One day later (E11.5), the AoV microenvironment is able to induce HSC development in the AoD, previously thought to be mostly devoid of HSC activity⁶. We also found that UGRs can enhance HSC production from the dorsal aorta, but cannot generate dHSCs themselves, even under influence of the dorsal aorta. Thus, our data strongly suggest that reciprocal stage-specific inductive AoD//AoV interactions and involvement of UGRs are required for execution of the robust development of HSCs in vivo.

Our data indicate that previously established dorso–ventrally polarized HSC development⁶ is defined by two main factors. First, our current data show that although the AoD contains pre-HSCs (both type I and type II), their numbers are lower than in AoV, in line with lower intra-aortic cluster formation previously described in mouse AoD^{6, 13}. Second, as shown here, dHSCs can be induced in the AoD by the AoV, and therefore the dHSCs deficiency in AoD cannot be explained solely by asymmetric pre-HSC distribution, but may also be influenced by differences in the microenvironment.

To study this, we focused on SCF, Shh and BMP4, whose expressions are dorso–ventrally polarized in the AGM region^{36, 47, 49} (and current data). We found that SCF is an inductive signal that is expressed at high levels in the AoV and UGRs, and can stimulate HSC development in isolated AoD, a region which had previously been considered to be mostly devoid of HSC activity. This is in agreement with a key role of SCF in HSC maturation¹⁷. We found that the aortic endothelial compartment expresses high levels of SCF, suggesting its important role in HSC development comparable to the bone marrow microenvironment of adult HSCs³². Importantly, we found that the pre-HSC type I population expresses SCF suggesting a positive-autocrine loop, which could promote HSC development.

Shh signalling in zebrafish is required for aortic angioblast migration and subsequent arterial specification of the dorsal aorta^{34, 54}. We found that in mouse Shh stimulates and a Hh antagonist inhibits the development of HSCs at E10.5 but not at E11.5, in keeping with a previous study³⁷. The induction of dHSCs in AoV by AoD is also limited to the E10.5 stage. Since Shh is secreted by the notochord (which is included in AoD-dissected tissue), this stage specificity is likely defined by the predominant presence of pre-HSCs type I at E10.5, which express higher levels of Shh signalling components (Ptch1 and Gli1) compared with pre-HSCs type II. By E11.5, the pre-HSC population is mainly represented by type II cells¹⁵. Stage-specific loss of sensitivity to Hh signalling was also described in the developing neural tube⁵⁵. Notably, the poor ability of AoD to develop HSCs despite abundant presence of Shh can also be explained by lower levels of Ptch1 and Gli1 detected in AoD- compared with AoV-derived pre-HSC type I. Our ex vivo modelling data indicate that AoD-derived Shh is an active inducer of HSC development in the AGM region. This conclusion does not exclude the possibility that Shh secreted by the gut could also reach the dorsal aorta³⁷, although by E10.5 these sites are separated by an extended mesentery.

BMP4 signalling is a key factor involved during differentiation of ventral mesoderm and its further specification into haematopoietic cells. In zebrafish, BMP signalling is clearly required during the patterning of the dorsal aorta and for the emergence of dHSCs in the ventral wall³⁴. Its role in mouse is less clear due to the early lethality of BMP mutants⁵⁶. Several lines of evidence point to BMP4 as a good candidate regulating HSC development. Indeed, BMP4 is highly expressed in the ventral mesenchyme underneath the dorsal aorta^{34, 36, 49}; some reports suggested its role in controlling dHSC emergence^{36, 57, 58}. However, the in vitro systems used likely assayed the maintenance of dHSCs, rather than their maturation. It was also reported that BMP4 signalling can define their differentiation potential⁵⁹. BMP4 is also involved in the regulation of essential haematopoietic transcription factors such as Scl/Gata2/Fli1 and Runx1 (refs 60, 61). Here we analysed BMP signalling activity in the dHSC lineage in the AGM region. We show that in vivo the pre-HSC type I to type II transition is accompanied by a downregulation of BMP targets (Id genes). This correlates with our data demonstrating that BMP activity is downregulated in intra-aortic clusters and the observations of others that Runx1 expression is attenuating in the developing HSC lineage^{60, 62, 63}. How is this decrease of BMP activity achieved in vivo, despite the presence of BMP4 in AoV? It has previously been noted that in amphibian embryos several BMP inhibitors are also expressed in AoV³⁴. Similarly, our analysis of the embryo showed high expression of a number of BMP antagonists as well as inhibitory Smad6 and Smad7 in mouse AoV that may counteract BMP4 action in HSC lineage. Furthermore, we found that in the AGM region BMP4 and Noggin are spatially segregated: Noggin being present in haematopoietic clusters and BMP4 being mainly expressed underneath the aortic endothelium. Therefore, maturing HSCs in clusters are exposed to low BMP4 concentration and high concentration of the BMP antagonist Noggin. Furthermore, our qRT–PCR analysis shows that the pre-HSC type I population expresses Noggin, which possibly creates a very effective shield that protects them from BMP4. Accordingly, our ex vivo analysis strongly suggests that downregulation of BMP signalling is functionally important for HSC development in the embryo. Indeed, forced BMP signalling activation by the addition of BMP4, strongly inhibits HSC development, and conversely the addition of Noggin stimulates HSC development in E10.5–E11.5 AGM cultures. These results are in line with recent observation that deletion of Smad4, a common transducer for BMP4/TGFβ signalling, markedly augments the formation of intra-aortic clusters⁶⁴. Our data do not exclude the possibility that BMP4 is essential for specification of mouse dHSCs at earlier stages, as described in the zebrafish model, where BMP signalling is required for HSC development at stages closer to mouse E8.5 (ref. 34).

Our analysis indicates that all three signalling pathways studied can cooperate for HSC development (Fig. 8c). Notably, the interplay of Shh and BMP pathways is broadly involved in development. For example, counter gradients of polarized Shh and BMP signalling in the developing spinal cord specify neuronal subsets along the dorso–ventral axis⁶⁵, and the dorsal aorta resembles the neural tube with inverse orientation of Shh- and BMP-secreting centres³⁴. However, we detected an antagonistic relationship between Shh and BMP pathways. At the molecular level, Shh can induce Noggin and Smad6 expression, thus inhibiting BMP4 signalling. In turn, BMP4 suppresses and, accordingly, Noggin enhances Shh signalling. Cooperation between Shh and Noggin has been previously described as critically important for developmental specification of somitic, neural and hair follicle cells^{66, 67, 68}. Our in vitro data suggest that the feed-forward loop ShhNoggin/NogginShh is also involved in HSC development in vivo.

We propose a model where the polarized secreted factors form complex fields of gradients in vivo, which define an effector zone for optimal HSC development in the dorsal aorta and lead to the ventrally shifted appearance of dHSCs (Fig. 8c). Of interest, intra-aortic clusters are abundant in ventro–lateral positions⁶⁹, which may reflect the position of this zone. The dissection close to such a zone could lead to accidental inclusion of powerful dHSCs in AoD samples observed here. Furthermore, it is possible that spatial segregation of co-operating and spatial overlap of antagonizing factors may also be important for adjustment of HSC development in vivo. Indeed, although the pool of pre-HSCs in the AGM region markedly expands during E9.5–11.5 (Rybtsov et al., submitted), complete maturation of the HSC pool is limited: while the majority of cells reach the pre-HSC type II stage, only one or two dHSCs are generated by the end of E11. Such controlled dynamics of HSC development may be needed to prevent a burst of active haematopoiesis in the AGM region. How exactly HSC maturation dynamics depend on overlapping concentrations of factors requires further analysis. Although ex vivo modelling is a powerful tool to dissect mechanisms of HSC development in vivo, there will likely be some variation in details. For example, spatial polarization in the developing HSC niche may define kinetics of HSC development in vivo.While we have demonstrated spatial polarization in vivo of the factors driving HSC development in our model system, it is currently unclear whether any factors become expressed in a polarized manner within the reaggregates and as such, whether polarization is also a pre-requisite for HSC maturation. Alternatively, if polarization is not required, the entire reaggregate may replicate the optimal zone for HSC development, resulting in massive generation of dHSCs. The distinction between these two scenarios will require further investigation.

In summary, our ex vivo modelling experiments suggest that HSC development in the embryo involves stage-dependent interactions between dorsal, ventral and lateral domains of the AGM region, mediated at least partly by the interplay of SCF, Shh, BMP4 and Noggin. Further detailed analysis will be required to better understand the complexity of the AGM signalling landscape in which HSC development takes place. Such knowledge may lead to development of novel protocols for the generation of definitive HSCs in vitro for clinical applications.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

Jie He1, Omar Abdel-Wahab2, Michelle K. Nahas1, Kai Wang1, Raajit K. Rampal3, Andrew M. Intlekofer4, et al.
http://www.bloodjournal.org/content/early/2016/03/10/blood-2015-08-664649March 10, 2016

Key Points

• Novel clinically-available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies.

• Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded (FFPE), blood and bone marrow samples with high accuracy in a clinically relevant timeframe, which is performed in our CLIA-certified CAP-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs and gene fusions, with similar accuracy to lower-throughput assays which focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically-relevant genomic alterations with therapeutic relevance.

Cohesin Ring Rules Blood Stem Cells, Binds Them to Renewal or Expansion

GEN News    http://www.genengnews.com/gen-news-highlights/cohesin-ring-rules-blood-stem-cells-binds-them-to-renewal-or-expansion/81252512/

A genome-wide RNAi screen was used to assess the effects of 15,000 genes on the balance between self-renewal and differentiation of human hematopoietic stem cells (HSCs). The screen identified candidate genes whose knockdown maintained the HSC phenotype during culture. Such findings could lead to better protocols to grow these cells outside the body, potentially making bone marrow transplants more available to patients suffering blood cancers, or even identifying novel genes to target during the treatment of leukemia (left and right panels). Four genes in particular implicated cohesin, a ring-like protein complex that binds to the DNA in all of our cells, in the control of self-renewal versus differentiation in HSCs. Deficiency of cohesin causes an increase in self-renewal and a decrease in differentiation of HSCs. [Cell Reports]

Best known for its ability to regulate the separation of sister chromatids during cell division, the cohesin protein complex, a ring-shaped structure, has shown that it has other powers, such as the facilitation of DNA repair and the modification of transcription. And now, according to scientists based at Lund University, there is evidence that the cohesin complex controls the growth of blood stem cells. More to the point, the cohesin complex determines whether blood stem cells self-renew or differentiate.

The new finding is significant because it can help scientists improve the expansion of blood stem cells outside the body, thus increasing the supply of blood stem cells to patients suffering leukemia or hereditary blood disorders. Besides making bone marrow transplant material more available, the new finding could point scientists to new points of attack for the treatment of blood cancer, which is a disruption between blood stem cell multiplication and maturation.

The Lund University scientists, led by Jonas Larsson, presented their results March 17 in the journal Cell Reports, in an article entitled “Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs.” The article describes how a genome-wide RNA interference (RNAi) screen was performed in primary human CD34⁺ cells. This screen enabled the scientists to identify candidate genes whose knockdown maintained the HSC phenotype during culture.

“A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen,” wrote the authors. “Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro.”

A similar expansion, the authors added, was observed in vivo following transplantation to immunodeficient mice.

“Transcriptome analysis of cohesin-deficient CD34⁺ cells showed an upregulation of HSC-specific genes,” the authors continued. This finding, the authors asserted, demonstrates that when cohesin is deficient, transcription shifts to a more stem cell–like pattern.

“The research is unique as the study of so many genes alongside one another is unprecedented,” said Dr. Larsson. “In addition, we have used human blood stem cells, which is difficult in itself as it is requires the gathering of a large amount of material.”

Of the 15,000 genes that were tested, the Lund team found around 20 candidates with a strong capacity to affect the balance of growth in the blood stem cells. What was striking was that four of these 20 genes were physically connected through cooperation in a protein complex.

“The discovery showed that this protein complex is crucial and has an overarching function in the growth of the blood stem cells,” emphasized Dr. Larsson.

The cohesin complex acts as a sort of brace that holds different parts of the DNA strand together in the cell. The researchers believe that this allows the cohesin complex to control access to the “on/off switches” in DNA and to change the impulses the blood stem cells receive from various genes, thereby affecting cell division. The blood stem cell either multiplies or matures to become a specialized cell with other tasks.

Independently of the Lund researchers’ discovery, other research in the field of blood cancer has recently identified mutations in exactly the same four genes in patients with various forms of blood cancer.

“This is incredibly exciting! Together with the results from our study, this indicates that the cohesin genes are directly and crucially significant in the development of blood cancer,” exclaimed the study’s lead author, Ph.D. candidate Roman Galeev. “Our findings entail a new understanding of how the expansion of blood stem cells is controlled. Eventually, this can lead to new ways of affecting the process, either to prevent the development of cancer or to expand the stem cells for transplant.”

UNPRECEDENTED PRECISION STUDY IDENTIFIES THE FOUR GENES RESPONSIBLE FOR BLOOD STEM CELL DEVELOPMENT.

Posted on March 23, 2016 by Healthinnovations

Unprecedented precision study identifies the four genes responsible for blood stem cell development.

An important element in getting blood stem cells to multiply outside the body is to understand which of the approximately 20 000 genes in the human body control their growth. Now, a study from researchers at Lund University has studied close to 15 000 of these genes alongside each other; a feat which is unprecendented. The team state that they have succeeded in identifying four key genes which, together, govern the growth and multiplication of the stem cells. The opensource study is now being published in the journal Cell Reports.

An important step on the way is to map which of the thousands of human genes affect the regulation of stem cell growth.  The current study used RNA interference, a technique in which individual genes in the stem cells are selectively neutralized, to successfully identify the genes which affect the regulation of blood stem cells.

Results show that the protein complex, consisting of the four genes plus one further gene, is called cohesin and forms a sort of brace which holds different parts of the DNA strand together in the cell. The lab hypothesize that this allows the cohesin complex to control access to the ‘on/off switches’ in the DNA, and to change the impulses the blood stem cells receive from various genes, thereby affecting cell division.  They conclude the blood stem cell either multiplies or matures to become a specialised cell with other tasks.

The group state that clarifying what regulates the balance between multiplication and maturation of blood stem cells could provide the right keys to expanding them outside the body. The go on to add that, in addition, it would enable the identification of new points of attack for the treatment of blood cancer, which is precisely a disruption of the balance between multiplication and maturation.

Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs

Roman Galeev, Aurélie Baudet, Praveen Kumar, …., Anders Kvist, Jonas Larsson

http://dx.doi.org/10.1016/j.celrep.2016.02.082Article Info

Highlights

• •A genome-wide RNAi screen was performed in primary human CD34⁺ cells
• •Several cohesin genes were identified as modifiers of renewal and differentiation
• •Cohesin-deficient HSCs show enhanced reconstitution capacity in vivo
• •Cohesin deficiency induces immediate HSC-specific transcriptional programs

Summary

To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, andSMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34⁺ cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate.

Human hematopoiesis is maintained by a small number of hematopoietic stem cells (HSCs) that are capable of generating all blood cell lineages at an extremely rapid pace for the entire lifespan of a human being (Orkin and Zon, 2008). HSCs have been studied extensively during the last four decades and are probably the best functionally characterized adult stem cells. However, despite this, the regulatory mechanisms that govern different cellular fate options in HSCs have remained incompletely defined. In particular, it has been challenging to understand the molecular basis of the inherent ability of HSCs to self-renew and preserve their undifferentiated state, which has hampered efforts to expand HSCs ex vivo for therapeutic benefit (Dahlberg et al., 2011). Ex vivo expansion of HSCs would allow for critical improvements of bone marrow transplantation procedures in treatment of malignant and inherited hematological diseases (Chou et al., 2010). Defining the genetic and molecular basis of self-renewal of HSCs is thus important to enhance current cell-therapy strategies, but it is also essential in order to better understand mechanisms behind dysregulated hematopoiesis that may cause leukemia. Genes and pathways balancing cell-fate options between renewal and differentiation in stem cells are often key players in cancer development (Orkin and Zon, 2008).

Figure 1

Genome-wide RNAi Screen in Primitive Human Hematopoietic Cells Defines Genes and Pathways Associated with Cancer Progression and Cell Proliferation

(A) Overview of the experimental outline for the primary screen. 60 million cord blood-derived CD34⁺ cells were transduced with a pooled lentiviral library containing 75,000 shRNAs across six transduction replicates in total. A fraction of the cells were isolated after 72 hr, and proviral inserts were deep sequenced to determine the initial library distribution. Following 20 days of culture, CD34⁺ cells were magnetically isolated and proviral inserts were sequenced again to determine the changes in distribution for all shRNAs.

(B) Relative distribution of shRNAs following 20 days of in vitro culture, ranked from the most enriched to the most depleted. The y axis shows the average enrichment value across six replicate screens.

(C) Gene ontology analysis for all genes represented by multiple shRNAs in the most enriched (10%) fraction.

(D) KEGG pathway analysis showing strong enrichment for cancer-associated pathways among the top-scoring genes.

See also Figure S1 and Table S1.

View Large Image | Download PowerPoint Slide

1. A Secondary Screen Targeted to CD34⁺CD38⁻CD90⁺CD45RA⁻Cells Identifies the Cohesin Complex as a Candidate Regulator of HSC Renewal and Differentiation
2. Knockdown of Cohesin Genes Restricts Differentiation and Expands Primitive Cells In Vitro
3. Cohesin-Deficient HSCs Show Delayed Differentiation but Overall Increased Reconstitution Potential following Transplantation to Primary and Secondary Immunodeficient Mice
4. Silencing of Cohesin Genes Results in Immediate Transcriptional Changes with a Shift toward a More HSC-like Gene Expression Signature

We report here on the successful development of a genome-wide RNAi screening approach targeted to primary human hematopoietic stem and progenitor cells to define genes and pathways associated with self-renewal and differentiation. Based on findings from the screen, we implicate the cohesin complex as a crucial regulator of cell-fate decisions influencing self- renewal and differentiation in HSCs both in vitro and in vivo.

These efforts represent a genome-wide RNAi screen targeted to primary human HSPCs. The main limiting factor when performing functional screens in primary human cells is cell number. This obviously becomes even more challenging when rare cell subsets, such as stem and progenitor cells, are studied. Through unique access to cord blood with daily deliveries from several local hospitals, we were able to gather the necessary quantities to perform a screen in enriched primary HSPCs with reasonable coverage (300X).

 

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Posted in Bone marrow derived cells, Clinical & Translational, Hematology, Stem Cells for Regenerative Medicine | Tagged hemopoiesis, stem cells | Leave a Comment »

Brookings Papers on Economic Activity (BPEA): Wealth and Income Concentration is rising by about half as much as previously thought

March 14, 2016 by 2012pharmaceutical

Brookings Papers on Economic Activity (BPEA): Wealth and Income Concentration is rising by about half as much as previously thought

Reporter: Aviva Lev-Ari, PhD, RN

 

 

SOURCE

From: “Janice Eberly and Jim Stock, Brookings Institution” <economicstudies@brookings.edu>

Reply-To: <economicstudies@brookings.edu>

Date: Monday, March 14, 2016 at 1:40 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Subject: Challenging Piketty, a “Shadow Stimulus,” and other BPEA Findings

Read new research from the Brookings Papers on Economic Activity. View this email in your browser here.

Friends and Colleagues, We spent Thursday and Friday of last week sitting at Brookings, discussing six new papers with dozens of the world’s top economists and readying the findings to be published in our economics journal, the Brookings Papers on Economic Activity (BPEA). One of the great things about this twice-yearly conference is that, in the spirit of true academic discussion, it often raises as many questions as it answers. Here are six things we wanted to share from inside our discussion room: 1. “If this is our technological future, economists aren’t sure it adds up to much.” The Washington Post’s Wonkblog has a great summary of important new research on why we can’t blame the ongoing productivity decline on our inability to measure the benefits of the Internet and other technological innovations. 2. A group of Fed economists challenged what renowned economists Piketty, Saez and others say about the richest 1%. The blue line in the chart below represents the new BPEA estimates. The blue line is still rising—but a lot more slowly. The authors ultimately conclude that wealth and income concentration is rising by about half as much as previously thought. 3. We debated whether federal lending programs served as a “shadow stimulus.” (h/t Wall Street Journal on the apt phrasing.) These programs, especially housing and student loans, grew dramatically during the Great Recession. There was a lively discussion around a finding that the government’s housing and education credit programs were nearly as large as the 2009 stimulus act (ARRA) and on average three times more effective per dollar of cost at stimulating the economy. 4. Boys whose mothers did not complete high school are even more likely to drop out of high school themselves—if they live in areas with high income inequality. Typical explanations, like school quality, don’t account for this finding. The boys’ academic attainment explains part of it, though, reinforcing the view that interventions to get kids on track early may break the link between inequality and low mobility. This interactive map illustrates the point well. 5. American workers are moving around the labor market less and less, and it’s not because of disruptive technologies. This decline is pervasive across losing a job, finding a job, dropping out of the labor force, and moving from one job to another. The “fluidity rate” has been declining for at least three decades, but a closer look at the rate in different U.S. states shows that the decline in fluidity was smaller in states with more workers in routine jobs displaced by technological advancements. See the chart here. 6. If you’ve ever lived through hyperinflation, you know that very poor people are by necessity very sophisticated about inflation and exchange rates. That’s an observation from a conference participant, but it helps explain a finding that the average Argentinian wasn’t fooled by the government’s notorious manipulation of inflation data from 2007- 2013. David Wessel has moreon this “interesting question…[of] how much public perceptions matter.” As the Fed worries about inflation expectations going forward, the lesson from Argentina is that the general public can be very sophisticated when the stakes get high.   If you want to know more about BPEA or the new research submitted to this edition of the journal,visit BPEA on the Brookings website. We’ll be back in the fall with more new findings from leading economists.   Sincerely, Janice Eberly and James Stock Co-editors, Brookings Papers on Economic Activity Nonresident Senior Fellows, Economic Studies at Brookings

SOURCE

From: “Janice Eberly and Jim Stock, Brookings Institution” <economicstudies@brookings.edu>

Reply-To: <economicstudies@brookings.edu>

Date: Monday, March 14, 2016 at 1:40 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Subject: Challenging Piketty, a “Shadow Stimulus,” and other BPEA Findings

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Posted in US and Global Economy: Trends and Findings | Tagged a “Shadow Stimulus, ” and other BPEA Findings, Brookings Papers on Economic Activity, Challenging Piketty | Leave a Comment »

Metformin and vitamin B12 deficiency?

March 14, 2016 by larryhbern

Metformin and vitamin B12 deficiency?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Years of taking popular diabetes drug tied to risk of B12 deficiency

BY KATHRYN DOYLE  http://www.reuters.com/article/us-health-metformin-b12-deficiency-idUSKCN0WC211

(Reuters Health) – – People taking metformin, a common type 2 diabetes medication, for several years may be at heightened risk of vitamin B12 deficiency and anemia, according to a new analysis of long-term data.

Metformin helps to control the amount of sugar, or glucose, in the blood by reducing how much glucose is absorbed from food and produced by the liver, and by increasing the body’s response to the hormone insulin, according to the National Institutes of Health.

“Metformin is the most commonly used drug to treat type 2 diabetes, so many millions of people are taking it, usually for a prolonged period (many years),” said senior study author Dr. Jill P Crandall of Albert Einstein College of Medicine in New York City, by email.

“Smaller numbers of people take metformin for prevention of diabetes or treatment of polycystic ovary syndrome,” Crandall told Reuters Health.

The researchers used data from the Diabetes Prevention Program and the Diabetes Prevention Program Outcomes Study, which followed participants at high risk for type 2 diabetes for more than 10 years.

The study began with more than 3,000 people age 25 years and older with high blood sugar. The participants were randomly assigned to receive either 850 milligrams of metformin twice daily, placebo medication or an intensive lifestyle program than did not include medication. For the new analysis only those taking placebo or metformin were considered, and about 50 participants were excluded after having weight-loss surgery, which would affect their diabetes outcomes.

During follow-up, the participants provided blood samples at the five- and 13-year points.

Using these blood samples, the researchers found that at year five, average B12 levels were lower in the metformin group than the placebo group, and B12 deficiency was more common, affecting 4 percent of those on metformin compared to 2 percent of those not taking the drug.

Borderline low B12 levels affected almost 20 percent of those on metformin and 10 percent of those taking placebo.

Average vitamin B12 levels were higher by year 13 than in year five, but B12 deficiency was also more common in both the metformin and placebo groups, as reported in the Journal of Clinical Endocrinology and Metabolism.

Vitamin B12 deficiency may lead to nerve damage which can be severe and may be irreversible, Crandall said.

“Severe and prolonged B12 deficiency has also been linked to impaired cognition and dementia,” she said. “It can also cause anemia (low red blood cell count) – fortunately, this condition is reversible with treatment.”

More people in the metformin group were also anemic at year five than in the placebo group.

There are more than three million cases of vitamin B12 deficiency each year in the U.S., which can be caused by diet or certain medical conditions. Symptoms include fatigue, and numbness or tingling.

Humans do not make vitamin B12 and need to consume it from animal sources or supplements. Vegetarians may get enough from eating eggs and dairy products, but vegans need to rely on supplements or fortified grains.

Doctors who prescribe metformin to patients long-term for type 2 diabetes, gestational diabetes, polycystic ovarian syndrome or other indications should consider routine measurement of vitamin B12 levels, the authors conclude.

“The FDA and organizations such as the American Diabetes Association do not have any formal recommendations for B12 monitoring for people taking metformin,” Crandall said. “That said, our study (and others) suggests that a small but significant number of people may develop deficiency.”

“People who are taking metformin should ask their doctor about measuring their B12 level,” she said.

Restoring healthy B12 levels is easy to accomplish with pills or monthly injections, she added.

“The risk of B12 deficiency should not be considered a reason to avoid taking metformin,” Crandall said.

SOURCE: bit.ly/1SWRxed Journal of Clinical Endocrinology and Metabolism, online February 22, 2016.

 

Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study

 Vanita R ArodaMD¹, Sharon L EdelsteinScM², Ronald B GoldbergMD³, William C KnowlerMD, DrPH⁴, …., Marinella G TemprosaPhD², Neil H WhiteMD⁹, Jill P CrandallMD¹⁰, and Diabetes Prevention Program Research Group

http://dx.doi.org/10.1210/jc.2015-3754#sthash.ievZyXLD.dpuf    The Journal of Clinical Endocrinology & Metabolism    2016 Feb 22; jc20153754

Context:

 Vitamin B12 deficiency may occur with metformin treatment, but few studies have assessed risk with long-term use.

Objective:

 To assess risk of B12 deficiency with metformin use in the DPP/DPPOS

Design:

 Secondary analysis from DPP/DPPOS. Participants were assigned to placebo (n=1082, PLA) or metformin (n=1073, MET) for 3.2 years; MET received open-label metformin for an additional 9 years.

Setting:

27 study centers in the US

Patients:

 DPP eligibility: elevated fasting glucose, impaired glucose tolerance, and overweight/obesity. Analytic population: participants with available stored samples. B12 levels were assessed at 5 (n=857, n=858) and 13 years [n=756, n=764] in PLA and MET, respectively.

Interventions:

 Metformin 850 mg twice daily versus placebo (DPP); open-label metformin in MET (DPPOS)

Main Outcome Measures:

 B12 deficiency, anemia, peripheral neuropathy

Results:

 Low B12 (≤203 pg/ml) occurred more often in MET than PLA at 5 years (4.3% vs 2.3%, p=0.02) but not at 13 years (7.4% vs. 5.4%, p=0.12). Combined low and borderline-low B12 (≤298 pg/ml) was more common in MET at 5 (19.1% vs 9.5%, p<0.01) and 13 years (20.3% vs 15.6%, p=0.02). Years of metformin use was associated with increased risk of B12 deficiency (OR B12 deficiency/year metformin use, 1.13, 95% CI 1.06–1.20). Anemia prevalence was higher in MET, but did not differ by B12 status. Neuropathy prevalence was higher in MET with low B12 levels.

Conclusions:

 Long-term use of metformin in DPPOS was associated with biochemical B12 deficiency and anemia. Routine testing of vitamin B12 levels in metformin-treated patients should be considered.

 

Metformin linked to vitamin B₁₂ deficiency

David Holmes   Nature Reviews Endocrinology(2016)    http://dx.doi.org:/10.1038/nrendo.2016.39

Secondary analysis of data from the Diabetes Prevention Program Outcomes Study (DPPOS), one of the largest and longest studies of metformin treatment in patients at high risk of developing type 2 diabetes mellitus, shows that long-term use of metformin is associated with vitamin B₁₂deficiency.

Aroda, V. R. et al. Long-term metformin use and vitamin B₁₂ deficiency in the Diabetes Prevention Program Outcomes Study. J. Clin. Endocrinol. Metab. http://dx.doi.org/10.1210/jc.2015-3754 (2016)

 

Long-term Follow-up of Diabetes Prevention Program Shows Continued Reduction in Diabetes Development

http://www.diabetes.org/newsroom/press-releases/2014/long-term-follow-up-of-diabetes-prevention-program-shows-reduction-in-diabetes-development.html

San Francisco, California
June 16, 2014

Treatments used to decrease the development of type 2 diabetes continue to be effective an average of 15 years later, according to the latest findings of the Diabetes Prevention Program Outcomes Study, a landmark study funded by the National Institutes of Health (NIH).

The results, presented at the American Diabetes Association’s 74th Scientific Sessions®, come more than a decade after the Diabetes Prevention Program, or DPP, reported its original findings. In 2001, after an average of three years of study, the DPP announced that the study’s two interventions, a lifestyle program designed to reduce weight and increase activity levels and the diabetes medicinemetformin, decreased the development of type 2 diabetes in a diverse group of people, all of whom were at high risk for the disease, by 58 and 31 percent, respectively, compared with a group taking placebo.

The Diabetes Prevention Program Outcomes Study, or DPPOS, was conducted as an extension of the DPP to determine the longer-term effects of the two interventions, including further reduction in diabetes development and whether delaying diabetes would reduce the development of the diabetes complications that can lead to blindness, kidney failure, amputations and heart disease. Funded largely by the NIH’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the new findings show that the lifestyle intervention and metformin treatment have beneficial effects, even years later, but did not reduce microvascular complications.

Delaying Type 2 Diabetes

Participants in the study who were originally assigned to the lifestyle intervention and metformin during DPP continued to have lower rates of type 2 diabetes development than those assigned to placebo, with 27 percent and 17 percent reductions, respectively, after 15 years.

“What we’re finding is that we can prevent or delay the onset of type 2 diabetes, a chronic disease, through lifestyle intervention or with metformin, over a very long period of time,” said David M. Nathan, MD, Chairman of the DPP/DPPOS and Professor of Medicine at Harvard Medical School. “After the initial randomized treatment phase in DPP, all participants were offered lifestyle intervention and the rates of diabetes development fell in the metformin and former placebo groups, leading to a reduction in the treatment group differences over time.  However, the lifestyle intervention and metformin are still quite effective at delaying, if not preventing, type 2 diabetes,” Dr. Nathan said. Currently, an estimated 79 million American adults are at high-risk for developing type 2 diabetes.

Microvascular Complications
The DPPOS investigators followed participants for an additional 12 years after the end of the DPP to determine both the extent of diabetes prevention over time and whether the study treatments would also decrease the small vessel -or microvascular- complications, such as eye, nerve and kidney disease. These long-term results did not demonstrate significant differences among the lifestyle intervention, metformin or placebo groups on the microvascular complications, reported Kieren Mather, MD, Professor of Medicine at Indiana University School of Medicine and a study investigator.

“However, regardless of type of initial treatment, participants who didn’t develop diabetes had a 28 percent lower occurrence of the microvascular complications than those participants who did develop diabetes. These findings show that intervening in the prediabetes phase is important in reducing early stage complications,” Dr. Mather noted. The absence of differences in microvascular complications among the intervention groups may be explained by the small differences in average glucose levels among the groups at this stage of follow-up.

Risk for Cardiovascular Disease

The DPP population was relatively young and healthy at the beginning of the study, and few participants had experienced any severe cardiovascular events, such as heart attack or stroke, 15 years later. The relatively small number of events meant that the DPPOS researchers could not test the effects of interventions on cardiovascular disease. However, the research team did examine whether the study interventions, or a delay in the onset of type 2 diabetes, improved cardiovascular risk factors.

“We found that cardiovascular risk factors, such as hypertension, are generally improved by the lifestyle intervention and somewhat less by metformin,” said Ronald Goldberg, MD, Professor of Medicine at the University of Miami and one of the DPPOS investigators. “We know that people with type 2 diabetes are at much higher risk for heart disease and stroke than those who do not have diabetes, so a delay in risk factor development or improvement in risk factors may prove to be beneficial.”

Long-term Results with Metformin

The DPP/DPPOS is the largest and longest duration study to examine the effects of metformin, an inexpensive, well-known and generally safe diabetes medicine, in people who have not been diagnosed with diabetes. For DPPOS participants, metformin treatment was associated with a modest degree of long-term weight loss. “Other than a small increase in vitamin B-12 deficiency, which is a recognized consequence of metformin therapy, it has been extremely safe and well-tolerated over the 15 years of our study,” said Jill Crandall, MD, Professor of Medicine at Albert Einstein College of Medicine and a DPPOS investigator. “Further study will help show whether metformin has beneficial effects on heart disease and cancer, which are both increased in people with type 2 diabetes.”

Looking to the Future

In addition to the current findings, the DPPOS includes a uniquely valuable population that can help researchers understand the clinical course of type 2 diabetes.  Since the participants did not have diabetes at the beginning of the DPP, for those who have developed diabetes, the data show precisely when they developed the disease, which is rare in previous studies. “The DPP and DPPOS have given us an incredible wealth of information by following a very diverse group of people with regard to race and age as they have progressed from prediabetes to diabetes,” said Judith Fradkin, MD, Director of the NIDDK Division of Diabetes, Endocrinology and Metabolic Diseases. “The study provides us with an opportunity to make crucial discoveries about the clinical course of type 2 diabetes.”

Dr. Fradkin noted that the study population held promise for further analyses because researchers would now be able to examine how developing diabetes at different periods of life may cause the disease to progress differently. “We can look at whether diabetes behaves differently if you develop it before the age of 50 or after the age of 60,” she said. “Thanks to the large and diverse population of DPPOS that has remained very loyal to the study, we will be able to see how and when complications first develop and understand how to intervene most effectively.”

She added that NIDDK had invited the researchers to submit an application for a grant to follow the study population for an additional 10 years.

The Diabetes Prevention Program Outcomes Study was funded under NIH grant U01DK048489 by the NIDDK; National Institute on Aging; National Cancer Institute; National Heart, Lung, and Blood Institute; National Eye Institute; National Center on Minority Health and Health Disparities; and the Office of the NIH Director; Eunice Kennedy Shriver National Institute of Child Health and Human Development; Office of Research on Women’s Health; and Office of Dietary Supplements, all part of the NIH, as well as the Indian Health Service, Centers for Disease Control and Prevention and American Diabetes Association. Funding in the form of supplies was provided by Merck Sante, Merck KGaA and LifeScan.

The American Diabetes Association is leading the fight to Stop Diabetes® and its deadly consequences and fighting for those affected by diabetes. The Association funds research to prevent, cure and manage diabetes; delivers services to hundreds of communities; provides objective and credible information; and gives voice to those denied their rights because of diabetes. Founded in 1940, our mission is to prevent and cure diabetes and to improve the lives of all people affected by diabetes. For more information please call the American Diabetes Association at 1-800-DIABETES (1-800-342-2383) or visit http://www.diabetes.org. Information from both these sources is available in English and Spanish.

Association of Biochemical B₁₂Deficiency With Metformin Therapy and Vitamin B₁₂Supplements  

The National Health and Nutrition Examination Survey, 1999–2006

Lael Reinstatler, Yan Ping Qi, Rebecca S. Williamson, Joshua V. Garn, and Godfrey P. Oakley Jr.
Diabetes Care February 2012 vol. 35 no. 2 327-333      http://dx.doi.org:/10.2337/dc11-1582

OBJECTIVE To describe the prevalence of biochemical B₁₂deficiency in adults with type 2 diabetes taking metformin compared with those not taking metformin and those without diabetes, and explore whether this relationship is modified by vitamin B₁₂supplements.

RESEARCH DESIGN AND METHODS Analysis of data on U.S. adults ≥50 years of age with (n = 1,621) or without type 2 diabetes (n = 6,867) from the National Health and Nutrition Examination Survey (NHANES), 1999–2006. Type 2 diabetes was defined as clinical diagnosis after age 30 without initiation of insulin therapy within 1 year. Those with diabetes were classified according to their current metformin use. Biochemical B₁₂ deficiency was defined as serum B₁₂concentrations ≤148 pmol/L and borderline deficiency was defined as >148 to ≤221 pmol/L.

RESULTS Biochemical B₁₂ deficiency was present in 5.8% of those with diabetes using metformin compared with 2.4% of those not using metformin (P = 0.0026) and 3.3% of those without diabetes (P = 0.0002). Among those with diabetes, metformin use was associated with biochemical B₁₂ deficiency (adjusted odds ratio 2.92; 95% CI 1.26–6.78). Consumption of any supplement containing B₁₂ was not associated with a reduction in the prevalence of biochemical B₁₂deficiency among those with diabetes, whereas consumption of any supplement containing B₁₂ was associated with a two-thirds reduction among those without diabetes.

CONCLUSIONS Metformin therapy is associated with a higher prevalence of biochemical B₁₂ deficiency. The amount of B₁₂recommended by the Institute of Medicine (IOM) (2.4 μg/day) and the amount available in general multivitamins (6 μg) may not be enough to correct this deficiency among those with diabetes.

It is well known that the risks of both type 2 diabetes and B₁₂deficiency increase with age (1,2). Recent national data estimate a 21.2% prevalence of diagnosed diabetes among adults ≥65 years of age and a 6 and 20% prevalence of biochemical B₁₂ deficiency (serum B₁₂<148 pmol/L) and borderline deficiency (serum B₁₂ ≥148–221 pmol/L) among adults ≥60 years of age (3,4).

The diabetes drug metformin has been reported to cause a decrease in serum B₁₂ concentrations. In the first efficacy trial, DeFronzo and Goodman (5) demonstrated that although metformin offers superior control of glycosylated hemoglobin levels and fasting plasma glucose levels compared with glyburide, serum B₁₂ concentrations were lowered by 22% compared with placebo, and 29% compared with glyburide therapy after 29 weeks of treatment. A recent, randomized control trial designed to examine the temporal relationship between metformin and serum B₁₂ found a 19% reduction in serum B₁₂ levels compared with placebo after 4 years (6). Several other randomized control trials and cross-sectional surveys reported reductions in B₁₂ranging from 9 to 52% (7–16). Although classical B₁₂ deficiency presents with clinical symptoms such as anemia, peripheral neuropathy, depression, and cognitive impairment, these symptoms are usually absent in those with biochemical B₁₂ deficiency (17).

Several researchers have made recommendations to screen those with type 2 diabetes on metformin for serum B₁₂ levels (6,7,14–16,18–21). However, no formal recommendations have been provided by the medical community or the U.S. Prevention Services Task Force. High-dose B₁₂ injection therapy has been successfully used to correct the metformin-induced decline in serum B₁₂ (15,21,22). The use of B₁₂supplements among those with type 2 diabetes on metformin in a nationally representative sample and their potentially protective effect against biochemical B₁₂ deficiency has not been reported. It is therefore the aim of the current study to use the nationally representative National Health and Nutrition Examination Survey (NHANES) population to determine the prevalence of biochemical B₁₂deficiency among those with type 2 diabetes ≥50 years of age taking metformin compared with those with type 2 diabetes not taking metformin and those without diabetes, and to explore how these relationships are modified by B₁₂ supplement consumption.

Design overview

NHANES is a nationally representative sample of the noninstitutionalized U.S. population with targeted oversampling of U.S. adults ≥60 years of age, African Americans, and Hispanics. Details of these surveys have been described elsewhere (23). All participants gave written informed consent, and the survey protocol was approved by a human subjects review board.

Setting and participants

Our study included adults ≥50 years of age from NHANES 1999–2006. Participants with positive HIV antibody test results, high creatinine levels (>1.7 mg/dL for men and >1.5 mg/dL for women), and prescription B₁₂ injections were excluded from the analysis. Participants who reported having prediabetes or borderline diabetes (n = 226) were removed because they could not be definitively grouped as having or not having type 2 diabetes. We also excluded pregnant women, those with type 1 diabetes, and those without diabetes taking metformin. Based on clinical aspects described by the American Diabetes Association and previous work in NHANES, those who were diagnosed before the age of 30 and began insulin therapy within 1 year of diagnosis were classified as having type 1 diabetes (24,25). Type 2 diabetes status in adults was dichotomized as yes/no. Participants who reported receiving a physician’s diagnosis after age 30 (excluding gestational diabetes) and did not initiate insulin therapy within 1 year of diagnosis were classified as having type 2 diabetes.

Outcomes and follow-up

The primary outcome was biochemical B₁₂ deficiency determined by serum B₁₂ concentrations. Serum B₁₂ levels were quantified using the Quantaphase II folate/vitamin B₁₂ radioassay kit from Bio-Rad Laboratories (Hercules, CA). We defined biochemical B₁₂ deficiency as serum levels ≤148 pmol/L, borderline deficiency as serum B₁₂ >148 to ≤221 pmol/L, and normal as >221 pmol/L (26).

The main exposure of interest was metformin use. Using data collected in the prescription medicine questionnaire, those with type 2 diabetes were classified as currently using metformin therapy (alone or in combination therapy) versus those not currently using metformin. Length of metformin therapy was used to assess the relationship between duration of metformin therapy and biochemical B₁₂ deficiency. In the final analysis, two control groups were used to allow the comparison of those with type 2 diabetes taking metformin with those with type 2 diabetes not taking metformin and those without diabetes.

To determine whether the association between metformin and biochemical B₁₂ deficiency is modified by supplemental B₁₂ intake, data from the dietary supplement questionnaire were used. Information regarding the dose and frequency was used to calculate average daily supplemental B₁₂ intake. We categorized supplemental B₁₂ intake as 0 μg (no B₁₂ containing supplement), >0–6 μg, >6–25 μg, and >25 μg. The lower intake group, >0–6 μg, includes 6 μg, the amount of vitamin B₁₂ typically found in over-the-counter multivitamins, and 2.4 μg, the daily amount the IOM recommends for all adults ≥50 years of age to consume through supplements or fortified food (1). The next group, >6–25 μg, includes 25 μg, the amount available in many multivitamins marketed toward senior adults. The highest group contains the amount found in high-dose B-vitamin supplements.

 

In the final analysis, there were 575 U.S. adults ≥50 years of age with type 2 diabetes using metformin, 1,046 with type 2 diabetes not using metformin, and 6,867 without diabetes. The demographic and biological characteristics of the groups are shown in Table 1. Among metformin users, mean age was 63.4 ± 0.5 years, 50.3% were male, 66.7% were non-Hispanic white, and 40.7% used a supplement containing B₁₂. The median duration of metformin use was 5 years. Compared with those with type 2 diabetes not taking metformin, metformin users were younger (P < 0.0001), reported a lower prevalence of insulin use (P < 0.001), and had a shorter duration of diabetes (P = 0.0207). Compared with those without diabetes, metformin users had a higher proportion of nonwhite racial groups (P< 0.0001), a higher proportion of obesity (P < 0.0001), a lower prevalence of macrocytosis (P = 0.0017), a lower prevalence of supplemental folic acid use (P = 0.0069), a lower prevalence of supplemental vitamin B₁₂ use (P = 0.0180), and a lower prevalence of calcium supplement use (P = 0.0002). There was a twofold difference in the prevalence of anemia among those with type 2 diabetes versus those without, and no difference between the groups with diabetes.    

Association of Biochemical B₁₂Deficiency With Metformin Therapy and Vitamin B₁₂Supplements

Demographic and biological characteristics of U.S. adults ≥50 years of age: NHANES 1999–2006

The geometric mean serum B₁₂ concentration among those with type 2 diabetes taking metformin was 317.5 pmol/L. This was significantly lower than the geometric mean concentration in those with type 2 diabetes not taking metformin (386.7 pmol/L; P = 0.0116) and those without diabetes (350.8 pmol/L; P = 0.0011). As seen in Fig. 1, the weighted prevalence of biochemical B₁₂ deficiency adjusted for age, race, and sex was 5.8% for those with type 2 diabetes taking metformin, 2.2% for those with type 2 diabetes not taking metformin (P = 0.0002), and 3.3% for those without diabetes (P = 0.0026). Among the three aforementioned groups, borderline deficiency was present in 16.2, 5.5, and 8.8%, respectively (P < 0.0001). Applying the Fleiss formula for calculating attributable risk from cross-sectional data (27), among all of the cases of biochemical B₁₂ deficiency, 3.5% of the cases were attributable to metformin use; and among those with diabetes, 41% of the deficient cases were attributable to metformin use. When the prevalence of biochemical B₁₂ deficiency among those with diabetes taking metformin was analyzed by duration of metformin therapy, there was no notable increase in the prevalence of biochemical B₁₂ deficiency as the duration of metformin use increased. The prevalence of biochemical B₁₂ deficiency was 4.1% among those taking metformin <1 year, 6.3% among those taking metformin ≥1–3 years, 4.1% among those taking metformin >3–10 years, and 8.1% among those taking metformin >10 years (P = 0.3219 for <1 year vs. >10 years). Similarly, there was no clear increase in the prevalence of borderline deficiency as the duration of metformin use increased (15.9% among those taking metformin >10 years vs. 11.4% among those taking metformin <1 year; P = 0.4365).

Weighted prevalence of biochemical B₁₂ deficiency and borderline deficiency adjusted for age, race, and sex in U.S. adults ≥50 years of age: NHANES 1999–2006. Black bars are those with type 2 diabetes on metformin, gray bars are those with type 2 diabetes not on metformin, and the white bars are those without diabetes. *P = 0.0002 vs. type 2 diabetes on metformin. †P < 0.0001 vs. type 2 diabetes on metformin. ‡P = 0.0026 vs. type 2 diabetes on metformin.

Table 2 presents a stratified analysis of the weighted prevalence of biochemical B₁₂ deficiency and borderline deficiency by B₁₂supplement use. For those without diabetes, B₁₂ supplement use was associated with an ∼66.7% lower prevalence of both biochemical B₁₂deficiency (4.8 vs. 1.6%; P < 0.0001) and borderline deficiency (16.6 vs. 5.5%; P < 0.0001). A decrease in the prevalence of biochemical B₁₂deficiency was seen at all levels of supplemental B₁₂ intake compared with nonusers of supplements. Among those with type 2 diabetes taking metformin, supplement use was not associated with a decrease in the prevalence of either biochemical B₁₂ deficiency (5.6 vs. 5.3%; P= 0.9137) or borderline deficiency (15.5 vs. 8.8%; P = 0.0826). Among the metformin users who also used supplements, those who consumed >0–6 μg of B₁₂ had a prevalence of biochemical B₁₂ deficiency of 14.1%. However, consumption of a supplement containing >6 μg of B₁₂ was associated with a prevalence of biochemical B₁₂ deficiency of 1.8% (P = 0.0273 for linear trend). Similar trends were seen in the association of supplemental B₁₂ intake and the prevalence of borderline deficiency. For those with type 2 diabetes not taking metformin, supplement use was also not associated with a decrease in the prevalence of biochemical B₁₂ deficiency (2.1 vs. 2.0%; P = 0.9568) but was associated with a 54% reduction in the prevalence of borderline deficiency (7.8 vs. 3.4%; P = 0.0057 for linear trend).

Comparison of average daily B₁₂ supplement intake by weighted prevalence of biochemical B₁₂ deficiency (serum B₁₂ ≤148 pmol/L) and borderline deficiency (serum B₁₂ >148 to ≤221 pmol/L) among U.S. adults ≥50 years of age: NHANES 1999–2006.

Table 3 demonstrates the association of various risk factors with biochemical B₁₂ deficiency. Metformin therapy was associated with biochemical B₁₂ deficiency (odds ratio [OR] 2.89; 95% CI 1.33–6.28) and borderline deficiency (OR 2.32; 95% CI 1.31–4.12) in a crude model (results not shown). After adjusting for age, BMI, and insulin and supplement use, metformin maintained a significant association with biochemical B₁₂ deficiency (OR 2.92; 95% CI 1.28–6.66) and borderline deficiency (OR 2.16; 95% CI 1.22–3.85). Similar to Table 2, B₁₂ supplements were protective against borderline (OR 0.43; 95% CI 0.23–0.81), but not biochemical, B₁₂ deficiency (OR 0.76; 95% CI 0.34–1.70) among those with type 2 diabetes. Among those without diabetes, B₁₂ supplement use was ∼70% protective against biochemical B₁₂ deficiency (OR 0.26; 95% CI 0.17–0.38) and borderline deficiency (OR 0.27; 95% CI 0.21–0.35).

Polytomous logistic regression for potential risk factors of biochemical B₁₂ deficiency and borderline deficiency among U.S. adults ≥50 years of age: NHANES 1999–2006, OR (95% CI)

The IOM has highlighted the detection and diagnosis of B₁₂ deficiency as a high-priority topic for research (1). Our results suggest several findings that add to the complexity and importance of B₁₂ research and its relation to diabetes, and offer new insight into the benefits of B₁₂ supplements. Our data confirm the relationship between metformin and reduced serum B₁₂ levels beyond the background prevalence of biochemical B₁₂ deficiency. Our data demonstrate that an intake of >0–6 μg of B₁₂, which includes the dose most commonly found in over-the-counter multivitamins, was associated with a two-thirds reduction of biochemical B₁₂ deficiency and borderline deficiency among adults without diabetes. This relationship has been previously reported with NHANES and Framingham population data (4,29). In contrast, we did not find that >0–6 μg of B₁₂ was associated with a decrease in the prevalence of biochemical B₁₂ deficiency or borderline deficiency among adults with type 2 diabetes taking metformin. This observation suggests that metformin reduces serum B₁₂ by a mechanism that is additive to or different from the mechanism in older adults. It is also possible that metformin may exacerbate the deficiency among older adults with low serum B₁₂. Our sample size was too small to determine which amount >6 μg was associated with maximum protection, but we did find a dose-response trend.

We were surprised to find that those with type 2 diabetes not using metformin had the lowest prevalence of biochemical B₁₂ deficiency. It is possible that these individuals may seek medical care more frequently than the general population and therefore are being treated for their biochemical B₁₂ deficiency. Or perhaps, because this population had a longer duration of diabetes and a higher proportion of insulin users compared with metformin users, they have been switched from metformin to other diabetic treatments due to low serum B₁₂ concentrations or uncontrolled glucose levels and these new treatments may increase serum B₁₂ concentrations. Despite the observed effects of metformin on serum B₁₂ levels, it remains unclear whether or not this reduction is a public health concern. With lifetime risks of diabetes estimated to be one in three and with metformin being a first-line intervention, it is important to increase our understanding of the effects of oral vitamin B₁₂ on metformin-associated biochemical deficiency (20,21).

The strengths of this study include its nationally representative, population-based sample, its detailed information on supplement usage, and its relevant biochemical markers. This is the first study to use a nationally representative sample to examine the association between serum B₁₂ concentration, diabetes status, and metformin use as well as examine how this relationship may be modified by vitamin B₁₂ supplementation. The data available regarding supplement usage provided specific information regarding dose and frequency. This aspect of NHANES allowed us to observe the dose-response relationship in Table 2 and to compare it within our three study groups.

This study is also subject to limitations. First, NHANES is a cross-sectional survey and it cannot assess time as a factor, and therefore the results are associations and not causal relationships. A second limitation arises in our definition of biochemical B₁₂ deficiency. There is no general consensus on how to define normal versus low serum B₁₂levels. Some researchers include the functional biomarker methylmalonic acid (MMA) in the definition, but this has yet to be agreed upon (30–34). Recently, an NHANES roundtable discussion suggested that definitions of biochemical B₁₂ deficiency should incorporate one biomarker (serum B₁₂ or holotranscobalamin) and one functional biomarker (MMA or total homocysteine) to address problems with sensitivity and specificity of the individual biomarkers. However, they also cited a need for more research on how the biomarkers are related in the general population to prevent misclassification (34). MMA was only measured for six of our survey years; one-third of participants in our final analysis were missing serum MMA levels. Moreover, it has recently been reported that MMA values are significantly greater among the elderly with diabetes as compared with the elderly without diabetes even when controlling for serum B₁₂ concentrations and age, suggesting that having diabetes may independently increase the levels of MMA (35). This unique property of MMA in elderly adults with diabetes makes it unsuitable as part of a definition of biochemical B₁₂ deficiency in our specific population groups. Our study may also be subject to misclassification bias. NHANES does not differentiate between diabetes types 1 and 2 in the surveys; our definition may not capture adults with type 2 diabetes exclusively. Additionally, we used responses to the question “Have you received a physician’s diagnosis of diabetes” to categorize participants as having or not having diabetes. Therefore, we failed to capture undiagnosed diabetes. Finally, we could only assess current metformin use. We cannot determine if nonmetformin users have ever used metformin or if they were not using it at the time of the survey.

Our data demonstrate several important conclusions. First, there is a clear association between metformin and biochemical B₁₂ deficiency among adults with type 2 diabetes. This analysis shows that 6 μg of B₁₂ offered in most multivitamins is associated with two-thirds reduction in biochemical B₁₂ deficiency in the general population, and that this same dose is not associated with protection against biochemical B₁₂ deficiency among those with type 2 diabetes taking metformin. Our results have public health and clinical implications by suggesting that neither 2.4 μg, the current IOM recommendation for daily B₁₂ intake, nor 6 μg, the amount found in most multivitamins, is sufficient for those with type 2 diabetes taking metformin.

This analysis suggests a need for further research. One research design would be to identify those with biochemical B₁₂ deficiency and randomize them to receive various doses of supplemental B₁₂chronically and then evaluate any improvement in serum B₁₂concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B₁₂ deficiency and how they are affected by B₁₂supplements at various doses. Given that a significant proportion of the population ≥50 years of age have biochemical B₁₂ deficiency and that those with diabetes taking metformin have an even higher proportion of biochemical B₁₂ deficiency, we suggest that support for further research is a reasonable priority.

 

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Discussion:

One research design would be to identify those with biochemical B₁₂ deficiency and randomize them to receive various doses of supplemental B₁₂chronically and then evaluate any improvement in serum B₁₂concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B₁₂ deficiency and how they are affected by B₁₂supplements at various doses.

This is of considerable interest.  As far as I can see, there is insufficient data presented to discern all of the variables entangled.  In a study of 8000 hemograms several years ago, it was of some interest that there were a large percentage of patients who were over age 75 years having a MCV of 94 – 100, not considered indicative of macrocytic anemia.  It would have been interesting to explore that set of the data further.

UPDATED 3/17/2020

Nutrients. 2019 May 7;11(5). pii: E1020. doi: 10.3390/nu11051020.

Monitoring Vitamin B₁₂ in Women Treated with Metformin for Primary Prevention of Breast Cancer and Age-Related Chronic Diseases.

Mastroianni A¹, Ciniselli CM², Panella R³, Macciotta A⁴, Cavalleri A⁵, Venturelli E⁶, Taverna F⁷, Mazzocchi A⁸, Bruno E⁹, Muti P¹⁰, Berrino F¹¹, Verderio P¹², Morelli D¹³, Pasanisi P¹⁴.

Author information

Abstract

Metformin (MET) is currently being used in several trials for cancer prevention or treatment in non-diabetics. However, long-term MET use in diabetics is associated with lower serum levels of total vitamin B₁₂. In a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET, whose participants were characterized by different components of metabolic syndrome, we tested the effect of MET on serum levels of B₁₂, holo transcobalamin II (holo-TC-II), and methylmalonic acid (MMA). The study was conducted on 165 women receiving MET or placebo for three years. Results of the study indicate a significant overall reduction in both serum total B₁₂ and holo-TC-II levels according with MET-treatment. In particular, in the MET group 26 of 81 patients and 10 of the 84 placebo-treated subjects had B₁₂ below the normal threshold (<221 pmol/L) at the end of the study. Considering jointly all B₁₂, Holo-TC-II, and MMA, 13 of the 165 subjects (10 MET and 3 placebo-treated) had at least two deficits in the biochemical parameters at the end of the study, without reporting clinical signs. Although our results do not affect whether women remain in the trial, B₁₂ monitoring for MET-treated individuals should be implemented.

ntroduction

Metformin (MET) is the first-line treatment for type-2 diabetes and has been used for decades to treat this chronic condition [1]. Given its favorable effects on glycemic control, weight patterns, insulin requirements, and cardiovascular outcomes, MET has been recently proposed in addition to lifestyle interventions to reduce metabolic syndrome (MS) and age-related chronic diseases [2]. Observational studies have also suggested that diabetic patients treated with MET had a significantly lower risk of developing cancer or lower cancer mortality than those untreated or treated with other drugs [3,4]. For this reason, a number of clinical trials are in progress in different solid cancers.

One of the limitations in implementing long-term use of MET to prevent chronic conditions in healthy subjects relates to its potential lowering effect on vitamin B₁₂ (B₁₂). The aim of the present study was to assess the effect of three years of MET treatment in a randomized, controlled trial considering both B₁₂ levels and biomarkers of its metabolism and biological effectiveness.

Cobalamin, also known as B₁₂, is a water-soluble, cobalt-containing vitamin. All forms of B₁₂ are converted intracellularly into adenosyl-Cbl and methylcobalamin—the biologically active forms at the cellular level [5]. Vitamin B₁₂ is a vital cofactor of two enzymes: methionine synthase and L-methyl-malonyl-coenzyme. A mutase in intracellular enzymatic reactions related to DNA synthesis, as well as in amino and fatty acid metabolism. Vitamin B₁₂, under the catalysis of the enzyme l-methyl-malonyl-CoA mutase, synthesizes succinyl-CoA from methylmalonyl-CoA in the mitochondria. Deficiency of B₁₂, thus results in elevated methylmalonic acid (MMA) levels.

Dietary B₁₂ is normally bound to proteins. Food-bound B₁₂ is released in the stomach under the effect of gastric acid and pepsin. The free vitamin is then bound to an R-binder, a glycoprotein in gastric fluid and saliva that protects B₁₂ from the highly acidic stomach environment. Pancreatic proteases degrade R-binder in the duodenum and liberate B₁₂; finally, the free vitamin is then bound by the intrinsic factor (IF)—a glycosylated protein secreted by gastric parietal cells—forming an IF-B₁₂ complex [6]. The IF resists proteolysis and serves as a carrier for B₁₂ to the terminal ileum where the IF-B₁₂ complex undergoes receptor (cubilin)-mediated endocytosis [7]. The vitamin then appears in circulation bound to holo-transcobalamin-I (holo-TC-I), holo-transcobalamin-II (holo-TC-II), and holo-transcobalamin-III (holo-TC-III). It is estimated that 20–30% of the total circulating B₁₂ is bound to holo-TC-II and only this form is available to the cells [7]. Holo-TC-I binds 70–80% of circulating B₁₂, preventing the loss of the free unneeded portion [6]. Vitamin B₁₂ is stored mainly in the liver and kidneys.

Many mechanisms have been proposed to explain how MET interferes with the absorption of B₁₂: diminished absorption due to changes in bacterial flora, interference with intestinal absorption of the IF–B₁₂ complex (and)/or alterations in IF levels. The most widely accepted current mechanism suggests that MET antagonizes the calcium cation and interferes with the calcium-dependent IF–B₁₂ complex binding to the ileal cubilin receptor [8,9]. The recognition and treatment of B₁₂ deficiency is important because it is a cause of bone marrow failure, macrocytic anemia, and irreversible neuropathy [10].

In general, previous studies on diabetics have observed a reduction in serum levels of B₁₂ after both short- and long-term MET treatment [1]. A recent review on observational studies showed significantly lower levels of B₁₂ and an increased risk of borderline or frank B₁₂ deficiency in patients on MET than not on MET [1]. The meta-analysis of four trials (only one double-blind) found a significant overall mean B₁₂ reducing effect of MET after six weeks to three months of use [1]. A secondary analysis (13 years after randomization) of the Diabetes Prevention Program Outcomes Study, which randomized over 3000 persons at high risk for type 2 diabetes to MET or placebo, showed a 13% increase in the risk of B₁₂ deficiency per year of total MET use [3]. In this study, B₁₂ levels were measured from samples obtained in years 1 and 9. Stored serum samples from other time points, including baseline, were not available, and potentially informative red blood cell indices that might have demonstrated the macrocytic anemia, typical of B₁₂ deficiency, were not recorded [3]. The HOME (Hyperinsulinaemia: the Outcome of its Metabolic Effects) study, a large randomized controlled trial investigating the long-term effects of MET versus placebo in patients with type 2 diabetes treated with insulin, showed that the addition of MET improved glycemic control, reduced insulin requirements, prevented weight gain but lowered serum B₁₂ over time, and raised serum homocysteine, suggesting tissue B₁₂ deficiency [4]. A recent analysis of 277 diabetics from the same trial showed that serum levels of MMA, the specific biomarker for tissue B₁₂ deficiency [5], were significantly higher in people treated with MET than those receiving placebo after four years (on average) [4].

The risk of MET-associated B₁₂ deficiency may be higher in older individuals and those with poor dietary habits. Prospective studies have found negative associations between obesity and B₁₂ in numerous ethnicities [11,12]. An energy-dense but micronutrient-insufficient diet consumed by individuals who are overweight or obese might explain this [12]. Furthermore, obesity is associated with low-grade inflammation and these physiological changes have been shown to be associated, in several studies, with elevated C-reactive protein and homocysteine and with low concentrations of B₁₂ and other vitamins [13,14].

As part of a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET for primary prevention of breast cancer and other chronic age-related diseases in healthy women with tracts of MS [15] we tested the effect of MET on serum levels of B₁₂, holo-TC-II, and MMA.

Other articles of note on the Mediterranean Diet in this Online Open Access Scientific Journal Include

Micronutrients, Macronutrients and Dietary Patterns: Nutrition and Fertility

Live 2:30-4:30 PM  Mediterranean Diet and Lifestyle: A Symposium on Diet and Human Health:  October 19, 2018

Live 12:00 – 1:00 P.M  Mediterranean Diet and Lifestyle: A Symposium on Diet and Human Health : October 19, 2018

Announcement 11AM- 5PM: Live Conference Coverage  from Mediterranean Diet and Lifestyle: A Symposium on Diet and Human Health @S.H.R.O. and Temple University October 19, 2018

 

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Posted in Clinical & Translational, Clinical Diagnostics, Curation, Diabetes Mellitus, Endocrine Diseases, Folate and B12, Hematology, Hematopoiesis | Tagged Drug therapy, metformin, Nutrition Disorders, Outcomes research, type 2 diabetes | Leave a Comment »

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