GDF15 Protects Insulin-Producing Beta Cells against Pro-Inflammatory Cytokines and Metabolic Stress via Increased Deamination of Intracellular Adenosine.

It has been proposed that antidiabetic drugs, such as metformin and imatinib, at least in part, promote improved glucose tolerance in type 2 diabetic patients via increased production of the inflammatory cytokine GDF15. This is supported by studies, performed in rodent cell lines and mouse models, in which the addition or production of GDF15 improved beta-cell function and survival. The aim of the present study was to determine whether human beta cells produce GDF15 in response to antidiabetic drugs and, if so, to further elucidate the mechanisms by which GDF15 modulates the function and survival of such cells. The effects and expression of GDF15 were analyzed in human insulin-producing EndoC-betaH1 cells and human islets. We observed that alpha and beta cells exhibit considerable heterogeneity in GDF15 immuno-positivity. The predominant form of GDF15 present in islet and EndoC-betaH1 cells was pro-GDF15. Imatinib, but not metformin, increased pro-GDF15 levels in EndoC-betaH1 cells. Under basal conditions, exogenous GDF15 increased human islet oxygen consumption rates. In EndoC-betaH1 cells and human islets, exogenous GDF15 partially ameliorated cytokine- or palmitate + high-glucose-induced loss of function and viability. GDF15-induced cell survival was paralleled by increased inosine levels, suggesting a more efficient disposal of intracellular adenosine. Knockdown of adenosine deaminase, the enzyme that converts adenosine to inosine, resulted in lowered inosine levels and loss of protection against cytokine- or palmitate + high-glucose-induced cell death. It is concluded that imatinib-induced GDF15 production may protect human beta cells partially against inflammatory and metabolic stress. Furthermore, it is possible that the GDF15-mediated activation of adenosine deaminase and the increased disposal of intracellular adenosine participate in protection against beta-cell death.

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine.

Introduction: High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions. Methods: Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied. Results: We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents. Discussion: It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

Imatinib protects against human beta-cell death via inhibition of mitochondrial respiration and activation of AMPK.

The protein tyrosine kinase inhibitor imatinib is used in the treatment of various malignancies but may also promote beneficial effects in the treatment of diabetes. The aim of the present investigation was to characterize the mechanisms by which imatinib protects insulin producing cells. Treatment of non-obese diabetic (NOD) mice with imatinib resulted in increased beta-cell AMP-activated kinase (AMPK) phosphorylation. Imatinib activated AMPK also in vitro, resulting in decreased ribosomal protein S6 phosphorylation and protection against islet amyloid polypeptide (IAPP)-aggregation, thioredoxin interacting protein (TXNIP) up-regulation and beta-cell death. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) mimicked and compound C counteracted the effect of imatinib on beta-cell survival. Imatinib-induced AMPK activation was preceded by reduced glucose/pyruvate-dependent respiration, increased glycolysis rates, and a lowered ATP/AMP ratio. Imatinib augmented the fractional oxidation of fatty acids/malate, possibly via a direct interaction with the beta-oxidation enzyme enoyl coenzyme A hydratase, short chain, 1, mitochondrial (ECHS1). In non-beta cells, imatinib reduced respiratory chain complex I and II-mediated respiration and acyl-CoA carboxylase (ACC) phosphorylation, suggesting that mitochondrial effects of imatinib are not beta-cell specific. In conclusion, tyrosine kinase inhibitors modestly inhibit mitochondrial respiration, leading to AMPK activation and TXNIP down-regulation, which in turn protects against beta-cell death.

Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro.

PURPOSE: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing ß-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [ 1, 2 ]. Recently, we have shown that beneficial effects on ß-cells require cadherin contacts between bNCSCs and ß-cells [ 3, 4 ]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on ß-cells. MATERIALS AND METHODS: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of ß-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. RESULTS: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced ß-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and ß-cells. Even in direct contact between ß-cells and HF-NCSCs, no cadherin expression was detected. CONCLUSIONS: These observations indicate that HF-NCSCs do not confer the same positive effect on ß-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support ß-cells in coculture and after transplantation.

Co-culture of neural crest stem cells (NCSC) and insulin producing beta-TC6 cells results in cadherin junctions and protection against cytokine-induced beta-cell death.

PURPOSE: Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs) together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death. PROCEDURES: Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1ß and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry), nitrite production (Griess reagent), protein localization (immunofluorescence) and protein phosphorylation (flow cytometry). RESULTS: We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i) augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii) NCSC-derived laminin production; (iii) decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv) decreased beta-TC6 cell phosphorylation of ERK(T202/Y204), FAK(Y397) and FAK(Y576). Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs. CONCLUSION: In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta-cell survival even though ERK and FAK signaling are suppressed. It may be that future strategies to improve islet transplantation outcome may benefit from attempts to increase beta-cell cadherin junctions to neighboring cells.

Forced Runx1 expression in human neural stem/progenitor cells transplanted to the rat dorsal root ganglion cavity results in extensive axonal growth specifically from spinal cord-derived neurospheres.

Cell replacement therapy holds great promise for treating a wide range of human disorders. However, ensuring the predictable differentiation of transplanted stem cells, eliminating their risk of tumor formation, and generating fully functional cells after transplantation remain major challenges in regenerative medicine. Here, we explore the potential of human neural stem/progenitor cells isolated from the embryonic forebrain (hfNSPCs) or the spinal cord (hscNSPCs) to differentiate to projection neurons when transplanted into the dorsal root ganglion cavity of adult recipient rats. To stimulate axonal growth, we transfected hfNSPC- and hscNSPC-derived neurospheres, prior to their transplantation, with a Tet-Off Runx1-overexpressing plasmid to maintain Runx1 expression in vivo after transplantation. Although pronounced cell differentiation was found in the Runx1-expressing transplants from both cell sources, we observed extensive, long-distance growth of axons exclusively from hscNSPC-derived transplants. These axons ultimately reached the dorsal root transitional zone, the boundary separating peripheral and central nervous systems. Our data show that hscNSPCs have the potential to differentiate to projection neurons with long-distance axonal outgrowth and that Runx1 overexpression is a useful approach to induce such outgrowth in specific sources of NSPCs.