Apoptosis is probably the main form of beta-cell death in both type 1 diabetes mellitus (T1DM) and T2DM. In T1DM, cytokines contribute to beta-cell destruction through nuclear factor-kappaB (NF-kappaB) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are beta-cell toxic also via NF-kappaB activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced beta-cell apoptosis and determine whether TNFalpha, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-kappaB activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified beta-cells by oleate, palmitate, and/or cytokines (IL-1beta, interferon-gamma, TNFalpha). Palmitate and IL-1beta induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNFalpha did not potentiate FFA toxicity in primary beta-cells. The NF-kappaB-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1beta but not by FFAs. Cytokines activated NF-kappaB in INS-1E and beta-cells, but FFAs did not. Moreover, FFAs did not enhance NF-kappaB activation by TNFalpha. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA- and cytokine-induced beta-cell death but the mechanisms involved are different. Whereas cytokines induce NF-kappaB activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-kappaB- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of beta-cell death in T1DM and T2DM.

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Different degrees of ␤-cell failure and apoptosis are present in type 1 and type 2 diabetes. It has been recently suggested that high glucose-induced ␤-cell apoptosis in type 2 diabetes shares a final common pathway with type 1 diabetes, involving interleukin-1␤ (IL-1␤) production by ␤-cells, nuclear factor-B (NF-B) activation, and death via Fas-FasL. The aim of this study was to test whether human islet exposure to high glucose in vitro, or to the type 2 diabetes environment in vivo, induces IL-1␤ expression and consequent activation of NF-B-dependent genes. Human islets were isolated from five normoglycemic organ donors. The islets were cultured for 48 h to 7 days at 5.6, 11, or 28 mmol/l glucose. For comparative purposes, islets were also exposed to IL-1␤. Gene mRNA expression levels were assessed by real-time RT-PCR in a blinded fashion. Culture of the human islets at 11 and 28 mmol/l glucose induced a four-to fivefold increase in medium insulin as compared with 5.6 mmol/l glucose, but neither IL-1␤ nor IL-1 receptor antagonist (IL-1ra) expression changed. IL-1␤ and IL-1ra protein release to the medium was also unchanged. Stimulated human monocytes, studied in parallel, released >50-fold more IL-1␤ than the islets. There was also no glucose-induced islet Fas expression. Expression of the NF-B-dependent genes IB-␣ and monocyte chemoattractant protein (MCP)-1 was induced in human islets by IL-1␤ but not by high glucose. In a second set of experiments, human islets were isolated from seven type 2 diabetic patients and eight control subjects. The findings on mRNA levels were essentially the same as in the in vitro experiments, namely the in vivo diabetic state did not induce IL-1␤, Fas, or MCP-1 expression in human islets, and also did not modify IL-1ra expression. The present findings suggest that high glucose in vitro, or the diabetic milieu in vivo, does not induce IL-1␤ production or NF-B activation in human islets. This makes it unlikely that locally produced IL-1␤ is an important mediator of glucotoxicity to human islets and argues against the IL-1␤-NF-B-Fas pathway as a common mediator for ␤-cell death in type 1 and type 2 diabetes. Diabetes 54:3238 -3244, 2005

COUP-TFII has an important role in regulating metabolism in vivo. We showed this previously by deleting COUP-TFII from pancreatic beta cells in heterozygous mutant mice, which led to abnormal insulin secretion. Here, we report that COUP-TFII expression is reduced in the pancreas and liver of mice refed with a carbohydrate-rich diet and in the pancreas and liver of hyperinsulinemic and hyperglycemic mice. In pancreatic beta cells, COUP-TFII gene expression is repressed by secreted insulin in response to glucose through Foxo1 signaling. Ex vivo COUP-TFII reduces insulin production and secretion. Our results suggest that beta cell insulin secretion is under the control of an autocrine positive feedback loop by alleviating COUP-TFII repression. In hepatocytes, both insulin, through Foxo1, and high glucose concentrations repress COUP-TFII expression. We demonstrate that this negative glucose effect involves ChREBP expression. We propose that COUP-TFII acts in a coordinate fashion to control insulin secretion and glucose metabolism.Homeostatic mechanisms maintain blood glucose levels within a narrow range, protecting the body against hypoglycemia during periods of fasting and against excess glucose following the ingestion of a high-carbohydrate (HCHO) meal. Many genes are tightly controlled at the transcriptional level by key hormones, particularly insulin, and by glucose, which act through distinct, but synergistic, signaling pathways in the liver and pancreas. Elevated levels of glucose and insulin resistance in type 2 diabetes are the consequence of progressive defects in liver function and the insulin secretory capacity and/or beta cell mass. Only a few transcription factors are known to be controlled by insulin or glucose and to have an essential role in the pathogenesis of this disease. Among them, Foxo1, a forkhead family member, has been shown to regulate pancreatic beta cell mass and function and to control multiple metabolic pathways in liver. It can function as a transcription factor or as a coregulator, depending on the gene being regulated (9, 16). Both insulin and oxidative stress (chronic exposure to high glucose concentrations) regulate its activity through dynamic relocalization in the cell after posttranslational modification (9, 16). The carbohydrate response element binding protein ChREBP is a glucose-sensitive transcription factor regulated at the transcriptional and posttranslational levels that promotes the hepatic conversion of excess carbohydrate to fatty acids (6,8,14,15). The identification of the transcription factors that mediate glucose and insulin effects on metabolic pathways is of great interest, as any modulation in these pathways will have consequences on glucose homeostasis.Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) (also called NR2F2) is an orphan member of the steroid/thyroid hormone receptor superfamily that binds DNA by a Zn finger DNA binding domain on a variety of hormone response elements that contain direct or inverted imperfect AGGTCA repeats wi...

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Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4␣ (HNF4␣) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4␣ activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4␣ with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4␣ transactivation of COUP-TFII. The dominant negative suppression of HNF4␣ function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4␣ mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4␣ by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4␣ mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4␣/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, also called NR2F2) is an orphan member of the steroid/thyroid hormone receptor superfamily classed in the same subfamily as hepatocyte nuclear factor 4␣ (HNF4␣)/ maturity-onset diabetes of the young 1 (MODY1) and retinoid X receptor (RXR) (4, 11). Several molecular mechanisms by which COUP-TFII controls gene expression in pancreatic islet beta cell differentiation and function have been shown previously. COUP-TFII binds DNA by a Zn finger DNA binding domain in a variety of hormone response elements (HRE) that contain imperfect AGGTCA direct or inverted repeats with various spacing patterns (3, 14). It can form heterodimeric complexes with RXR, the universal partner of many nuclear receptors, and as such acts as a repressor (15). We previously showed that COUP-TFII acts as an inhibitor of the glucose activation of the liver pyruvate kinase gene by binding to the glucose-responsive element (9). On most promoters, HNF4␣ response elements are also bound by COUP-TFII, which often behaves as a transcriptional repressor antagonizing the enhancement of transcription by HNF4␣ (8,12,24). In a functional study, the impaired synergy between COUP-TFII and the E276Q mutant form of...

Cytokines activate the PPAR-delta gene network in beta cells. This network does not, however, regulate the pro-inflammatory response to cytokines because beta cells lack constitutive BCL-6 expression. This may render beta cells particularly susceptible to propagating inflammation in type 1 diabetes.

Human bone marrow-derived mesenchymal stem cells (hBMSC) are able to differentiate into cells of connective tissue lineages, including bone and cartilage. They are therefore considered as a promising tool for the treatment of bone degenerative diseases. One of the major issues in regenerative cell therapy is the biosafety of fetal bovine serum used for cell culture. Therefore, the development of a culture medium devoid of serum but preserving hBMSC viability will be of clinical value. The glucose-dependent insulinotropic peptide (GIP) has an anti-apoptotic action in insulin-producing cells. Interestingly, GIP also exerts beneficial effects on bone turnover by acting on osteoblasts and osteoclasts. We therefore evaluated the ability of GIP to prevent cell death in osteoblastic cells cultured in serum-free conditions. In hBMSC and SaOS-2 cells, activation of the GIP receptor increased intracellular cAMP levels. Serum deprivation induced apoptosis in SaOS-2 and hBMSC that was reduced by 30 and 50 %, respectively, in the presence of GIP. The protective effect of GIP involves activation of the adenylate cyclase pathway and inhibition of caspases 3/7 activation. These findings demonstrate that GIP exerts a protective action against apoptosis in hBMSC and suggest a novel approach to preserve viability of hBMSC cultured in the absence of serum.