Calcineurin signaling regulates human islet {beta}-cell survival

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic β-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant β-cell failure. We sought to determine the effect of calcineurin inhibition on β-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue β-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human β-cell apoptosis was significantly increased. Although we detected no human β-cell replication, tacrolimus significantly decreased rodent β-cell replication. Ex-4 nearly normalized both human β-cell survival and rodent β-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in β-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human β-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.     

β-Cell Regeneration Mediated by Human Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.     

Altered insulin receptor signalling and β-cell cycle dynamics in type 2 diabetes mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells--which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM.     

Emerging roles for β-arrestin-1 in the control of the pancreatic β-cell function and mass: New therapeutic strategies and consequences for drug screening

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase in β-cell mass, decreased β-cell survival and impaired glucose-dependent insulin release. Pancreatic β-cell proliferation, survival and secretion are thought to be regulated by signalling pathways linked to G-protein coupled receptors (GPCRs), such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors. β-arrestin-1 serves as a multifunctional adaptor protein that mediates receptor desensitization, receptor internalization, and links GPCRs to downstream pathways such as tyrosine kinase Src, ERK1/2 or Akt/PKB. Importantly, recent studies found that β-arrestin-1 mediates GLP-1 signalling to insulin secretion, GLP-1 antiapoptotic effect by phosphorylating the proapoptotic protein Bad through ERK1/2 activation, and PACAP potentiation of glucose-induced long-lasting ERK1/2 activation controlling IRS-2 expression. Together, these novel findings reveal an important functional role for β-arrestin-1 in the regulation of insulin secretion and β-cell survival by GPCRs.     

Puerarin ameliorates hyperglycemia in HFD diabetic mice by promoting β-cell neogenesis via GLP-1R signaling activation

BackgroundDiabetes is characterized by β-cell loss and dysfunction. A strategy for diabetes treatment is to promote new β-cell formation. Puerarin is an isoflavone from the root of Pueraria lobata (Willd.) Ohwi. Our previous study demonstrated puerarin could ameliorate hyperglycemia in diabetic mice. However, related mechanisms and potential roles of puerarin in β-cell neogenesis have not been elucidated.PurposeThe present study aims to investigate whether anti-diabetic effect of puerarin is dependent on promoting β-cell neogenesis via GLP-1R signaling activation.MethodsA high-fat diet (HFD) induced diabetic mouse model was applied to investigate effects of puerarin in vivo, exendin-4 (GLP-1R agonist) and metformin were used as positive controls. Moreover, related mechanisms and GLP-1R downstream signal transduction were explored in isolated cultured mouse pancreatic ductal cells.ResultsPuerarin improved glucose homeostasis in HFD diabetic mice significantly. Markers of new β-cell formation (insulin, PDX1 and Ngn3) were observed in pancreatic ducts of HFD mice treated by puerarin. Of note, efficacy of puerarin in vivo was suppressed by GLP-1R antagonist exendin9-39, but enhanced by exendin-4 respectively. In cultured mouse pancreatic ductal cells, puerarin induced expressions of insulin and PDX1, upregulated GLP-1R expression and activated β-catenin and STAT3 subsequently. Expressions of insulin and PDX1 in ductal cells could be blocked by exendin9-39, or β-catenin inhibitor ICG001, or JAK2 inhibitor AG490.ConclusionThese data clarified puerarin ameliorated hyperglycemia of HFD mice via a novel mechanism involved promoting β-cell neogenesis. Our finding highlights the potential value of puerarin developing as an anti-diabetic agent.     

Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization

Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature β-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in β-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 β-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51 ± 0.2% decrease in Ser-269 phosphorylation in MIN6 β-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.     

Voluntary running exercise prevents β-cell failure in susceptible islets of the Zucker diabetic fatty rat

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving β-cell function is uncertain. We evaluated the role of physical activity on β-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with β-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key β-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining β-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and β-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered β-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.     

Pleiotropic and age-dependent effects of decreased protein modification by O-linked N-acetylglucosamine on pancreatic β-cell function and vascularization

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of β-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal β-cell function, however, is not well understood. We studied transgenic mice that over express β-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic β-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in β-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.     

FTY720 normalizes hyperglycemia by stimulating β-cell in vivo regeneration in db/db mice through regulation of cyclin D3 and p57(KIP2)

Loss of insulin-producing β-cell mass is a hallmark of type 2 diabetes in humans and diabetic db/db mice. Pancreatic β-cells can modulate their mass in response to a variety of physiological and pathophysiological cues. There are currently few effective therapeutic approaches targeting β-cell regeneration although some anti-diabetic drugs may positively affect β-cell mass. Here we show that oral administration of FTY720, a sphingosine 1-phosphate (S1P) receptor modulator, to db/db mice normalizes fasting blood glucose by increasing β-cell mass and blood insulin levels without affecting insulin sensitivity. Fasting blood glucose remained normal in the mice even after the drug was withdrawn after 23 weeks of treatment. The islet area in the pancreases of the FTY720-treated db/db mice was more than 2-fold larger than that of the untreated mice after 6 weeks of treatment. Furthermore, BrdU incorporation assays and Ki67 staining demonstrated cell proliferation in the islets and pancreatic duct areas. Finally, islets from the treated mice exhibited a significant decrease in the level of cyclin-dependent kinase inhibitor p57(KIP2) and an increase in the level of cyclin D3 as compared with those of untreated mice, which could be reversed by the inhibition of phosphatidylinositol 3-kinase (PI3K). Our findings reveal a novel network that controls β-cell regeneration in the obesity-diabetes setting by regulating cyclin D3 and p57(KIP2) expression through the S1P signaling pathway. Therapeutic strategies targeting this network may promote in vivo regeneration of β-cells in patients and prevent and/or cure type 2 diabetes.     

Obesity, insulin resistance and diabetes in the sand rat exposed to a hypercaloric diet; possible protective effect for IL1-β

It is well established that, upon changing their natural desert low caloric (succulent halophilic plants) to a regular laboratory high caloric diet, sand rats undergo various phenotypic changes depending on their genetic background and including obesity and various degrees of insulin resistance. Our aim was to investigate the acute effects of Interleukin-1β (IL-1β) and Interferon-γ (IFN-γ) on glucose-induced insulin secretion in normal lean sand rats maintained on their natural diet and in obese insulin resistant normoglycemic or type 2 diabetic animals after a 9-month high caloric diet. Animals were fed either a low or a high caloric diet; after 9 months, pancreatic islets were isolated and incubated in the presence of increasing cytokine concentrations. At the end of the high-energy diet, animals were all over-weight, and probably due to a different genetic background, they displayed either insulin resistance, hyperinsulinemia and normoglycemia or a marked type-2 diabetic state. Pancreatic islets from obese insulin resistant normoglycemic animals were much more sensitive and responsive to IL-1β when compared to lean controls. The cytokine was inefficient in diabetic islets. In conclusion, the markedly increased insulinotropic effect of IL-1β in obese diabetes-resistant sand rat could participate and be involved in pancreatic β-cell hyperactivity that compensates for insulin resistance and thereby prevent the development of type 2 diabetes in these animals.     

Betacellulin and nicotinamide sustain PDX1 expression and induce pancreatic beta-cell differentiation in human embryonic stem cells

The major obstacle in cell therapy of diabetes mellitus is the limited source of insulin-producing β cells. Very recently, it was shown that a five-stage protocol recapitulating in vivo pancreatic organogenesis induced pancreatic β cells in vitro; however, this protocol is specific to certain cell lines and shows much line-to-line variation in differentiation efficacy. Here, we modified the five-stage protocol for the human embryonic stem cell line SNUhES3 by the addition of betacellulin and nicotinamide. We reproduced in vivo pancreatic islet differentiation by directing the cells through stages that resembled in vivo pancreatic organogenesis. The addition of betacellulin and nicotinamide sustained PDX1 expression and induced β-cell differentiation. C-peptide—a genuine marker of de novo insulin production—was identified in the differentiated cells, although the insulin mRNA content was very low. Further studies are necessary to develop more efficient and universal protocols for β-cell differentiation.     

Effect of hTIMP-1 overexpression in human umbilical cord mesenchymal stem cells on the repair of pancreatic islets in type-1 diabetic mice

Mesenchymal stem cells (MSCs) have been suggested for pancreatic islet repair in Type 1 diabetes mellitus (T1DM). This study aimed to investigate the effect of human umbilical cord MSCs (hUC-MSCs) transfected with tissue inhibitors of matrix metalloproteinase (TIMP)-1 on the regeneration of β-cell islets in vitro and in vivo. hUC-MSCs were isolated, cultured, and transfected with lentiviruses for the overexpression of hTIMP-1. An in vitro coculture system of hUC-MSCs and streptozotocin-induced islets was established to examine the morphology, apoptosis, and insulin secretion of the cocultured islets. Diabetic mouse models were injected with lenti-TIMP-1-enhanced green fluorescent protein (EGFP)-hUC-MSCs to test the effect of hTIMP-1 on insulin levels and glucose tolerance in vivo. The expression of insulin and glucagon was evaluated by immunofluorescence staining. The results showed that coculture with hUC-MSCs or Lenti-TIMP-1-EGFP-hUC-MSCs improved islet viability rates. Lenti-TIMP-1-EGFP-hUC-MSC coculture increased the insulin and C-peptide secretion function of the cultured islets and increased the secretion of tumor necrosis factor-β1, interleukin-6, IL-10, and hTIMP-1. hUC-MSCs, especially those transfected with Lenti-hTIMP-1-EGFP, showed a strong protective effect in diabetic mice by alleviating weight loss and improving glucose and insulin metabolism. In addition, transplantation rescued islet histology and function in vivo. The overexpression of TIMP-1 by hUC-MSCs seems to exert beneficial effects on pancreatic islet cells. In conclusion, this study may provide a new perspective on the development of hUC-MSC-based cell transplantation therapy for T1DM.