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Endoplasmic reticulum (ER) stress is a key pathogenic factor in type 1 and 2 diabetes. Glycogen synthase kinase 3 (Gsk-3) contributes to β-cell loss in mice. However, the mechanism by which Gsk-3 leads β-cell death remains unclear. ER stress was pharmacologically induced in mouse primary islets and insulinoma cells. We used insulinoma cells derived from Akita mice as a model of genetic ER stress. Gsk-3 activity was blocked by treating with Gsk-3 inhibitors or by introducing catalytically inactive Gsk-3β. Gsk-3 inhibition prevented proteasomal degradation of activating transcriptional factor 4 (ATF4) and alleviated apoptosis. We found that ATF4-S214 was phosphorylated by Gsk-3, and that this was required for a binding of ATF4 with βTrCP, which mediates polyubiquitination. The anti-apoptotic effect of Gsk-3 inhibition was attenuated by introducing DN-ATF4 or by knockdown of ATF4. Mechanistically, Gsk-3 inhibition modulated transcription targets of ATF4 and in turn facilitated dephosphorylation of eIF2α, altering the protein translational dynamism under ER stress. These observations were reproduced in the Akita mouse-derived cells. Thus, these results reveal the role of Gsk-3 in the regulation of the integrated stress response, and provide a rationale for inhibiting this enzyme to prevent β-cell death under ER stress conditions.

Wolfram syndrome (WS) is mainly caused by mutations in the WFS1 gene and characterized by diabetes mellitus, optic atrophy, hearing loss, and central diabetes insipidus (CDI). WFS1 is an endoplasmic reticulum (ER)-resident transmembrane protein, and Wfs1 knockout (Wfs1−/−) mice, which have been used as a mouse model for WS, reportedly manifested impairment of glucose tolerance due to pancreatic β-cell loss. In the present study, we examined water balance, arginine vasopressin (AVP) secretion, and ER stress in AVP neurons of the hypothalamus in Wfs1−/− mice. There were no differences in urine volumes between Wfs1−/− and wild-type mice with free access to water. Conversely, when mice were subjected to intermittent water deprivation (WD) for 20 weeks, during which water was unavailable for 2 days a week, urine volumes were larger in Wfs1−/− mice, accompanied by lower urine AVP concentrations and urine osmolality, compared to wild-type mice. The mRNA expression of immunoglobulin heavy chain binding protein, a marker of ER stress, was significantly increased in the supraoptic nucleus and paraventricular nuclei in Wfs1−/− mice compared to wild-type mice after WD. Our results thus showed that Wfs1 knockout leads to a decrease in AVP secretion during dehydration, which could explain in part the mechanisms by which Wfs1 mutations cause CDI in humans.

Dedifferentiation has been implicated in β cell dysfunction and loss in rodent diabetes. However, the pathophysiological significance in humans remains unclear. To elucidate this, we analyzed surgically resected pancreatic tissues of 26 Japanese subjects with diabetes and 11 nondiabetic subjects, who had been overweight during adulthood but had no family history of diabetes. The diabetic subjects were subclassified into 3 disease stage categories, early, advanced, and intermediate. Despite no numerical changes in endocrine cells immunoreactive for chromogranin A (ChgA), diabetic islets showed profound β cell loss, with an increase in α cells without an increase in insulin and glucagon double-positive cells. The proportion of dedifferentiated cells that retain ChgA immunoreactivity without 4 major islet hormones was strikingly increased in diabetic islets and rose substantially during disease progression. The increased dedifferentiated cell ratio was inversely correlated with declining C-peptide index. Moreover, a subset of islet cells converted into exocrine-like cells during disease progression. These results indicate that islet remodeling with dedifferentiation is the underlying cause of β cell failure during the course of diabetes progression in humans., Islet remodeling with dedifferentiation is a pathologic mechanism of β celldysfunction and lost during the course of progression in human type 2 diabetes.

To elucidate the pathogenesis of post-pancreatectomy diabetes (PPDM), we carried out a prospective observational study in patients without diabetes undergoing partial pancreatectomy, investigating the effects of gut microbiota, incretins, and short-chain fatty acids (SCFAs) on postsurgical glucose metabolism. In addition, we analyzed histologically the resected pancreas to investigate whether increased cellular-plasticity and expansion of the islets could predict the development of PPDM. Forty-eight patients without diabetes, who underwent either pancreatoduodenectomy (PD) (n=20) or distal pancreatectomy (DP) (n=28), were included. To assess the post-surgical glucose metabolism, 75-g oral glucose tolerance test (OGTT) was performed every 6 months. Microbiome composition and SCFAs in feces were examined before and 6 months after the surgery. We also underwent immunostainings of insulin, glucagon, and ALDH1A3 (a progenitor cell marker) in the resected pancreatic samples obtained from patients following DP. During the follow-up (median 2.50 years), 2 out of 18 patients with PD and 16 out of 28 patients with DP developed PPDM. Microbiota shifted to a higher relative abundance of Proteobacteria after PD. After PD, fecal butyrate and plasma GLP-1 concentrations were significantly increased, which were not increased after DP. In all patients, postsurgical change in butyrate was strongly correlated with increased GLP-1 secretion. In DP patients, expansion of both α- and β-cell areas in the resected pancreas were significantly associated with the development of PPDM, and HR (95% CI) of ALDH1A3 positive cells (%) for PPDM was 1.127 (1.000-1.270). This study demonstrates that incidence of PPDM is much lower after PD than that after DP, which may be explained by marked changes in intestinal environment after PD. On the other hand, high cellular-plasticity and expansion of the islets in the resected pancreas can predict the development of PPDM after DP. Disclosure R. Bouchi: None. T. Fukuda: None. T. Takeuchi: None. K. Amo-Shiinoki: None. K. Tanabe: None. Y. Tanizawa: None. T. Yamada: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Novartis Pharma K.K., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Y. Ogawa: Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker’s Bureau; Self; Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation. Funding Ministry of Education, Culture, Sports, Science and Technology of Japan (15K19507); Japan Foundation for Applied Enzymology; Takeda Science Foundation; AstraZeneca

T2D is associated with deficits of β-cell function and number. But it remains unclear how these processes interact during disease progression. It has been described that islet cell plasticity, i.e., β-cell dedifferentiation and conversion into other islet cells, is implicated to β-cell failure in murine diabetes of insulin resistance. We aimed to examine it in human T2D and to elucidate its involvement in disease progression. Methods: We evaluated pancreata obtained during pancreatectomy from Japanese subjects who had been over-weighed during adulthood and had no diabetic family history and analyzed patient’s clinical information. The diabetics in the early- and the advanced-stage that were defined by combining clinical information on disease duration, microvascular complications and insulin replacement were evaluated. The islet area was determined by measuring ChgA- positive area. We detected islet major 4 hormones-negative/ChgA-positive cells as dedifferentiated cells. Results: There was a diversity in relative β-, α- and islet area to pancreatic area in the T2D subjects, suggesting metabolic heterogeneity associated with various capabilities of islet compensation in response to insulin resistance. We found that a decreased relative β-cell number and an increased relative α-cell number to entire ChgA-positive cell number in individual T2D islets. But there was no further increase in α-cells along disease progression despite progressive β-cell loss, and only little INS/GCG double positive cells. Instead, a strikingly elevated number of the dedifferentiated cells in T2D islets compared to control subjects and its significant increase with disease progression was observed. Importantly, there was a significant inverse correlation between increasing dedifferentiated cells and declining C-peptide index among the groups. These finding indicates that islet cells exert plasticity during diabetes progression and that β-cell dedifferentiation is a pathologic process leading β-cell failure. Disclosure K. Amo-Shiinoki: None. K. Tanabe: None. R. Bouchi: None. W. Nishimura: None. Y. Ogawa: Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker’s Bureau; Self; Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation. Y. Tanizawa: None. Funding Japan Society for the Promotion of Science (20390093, 25461357, 16K09752, 15K21198); Banyu International Life Science Foundation; Novo Nordisk; Japan Association for Diabetes Education and Care; Merck Sharp & Dohme Corp; Public Interest Incorporated Foundation

OBJECTIVE To elucidate the pathogenesis of postpancreatectomy diabetes mellitus (PPDM). RESEARCH DESIGN AND METHODS Forty-eight patients without diabetes undergoing either pancreatoduodenectomy (PD) (n = 20) or distal pancreatectomy (DP) (n = 28) were included. A 75-g oral glucose tolerance test was performed every 6 months. Microbiome composition and short-chain fatty acids (SCFAs) in feces were examined before and 6 months after surgery. The association of histological characteristics of the resected pancreas with PPDM was examined. RESULTS During follow-up (median 3.19 years), 2 of 20 PD patients and 16 of 28 DP patients developed PPDM. Proteobacteria relative abundance, plasma glucagon-like peptide 1 (GLP-1), and fecal butyrate levels increased only after PD. Postsurgical butyrate levels were correlated with postsurgical GLP-1 levels. With no significant difference in the volume of the resected pancreas between the surgical procedures, both β-cell and α-cell areas in the resected pancreas were significantly higher in DP patients than in PD patients. In DP patients, the progressors to diabetes showed preexisting insulin resistance compared with nonprogressors, and both increased α- and β-cell areas were predictors of PPDM. Furthermore, in DP patients, α-cell and β-cell areas were associated with ALDH1A3 expression in islets. CONCLUSIONS We postulate that a greater removal of β-cells contributes to the development of PPDM after DP. Islet expansion along with preexisting insulin resistance is associated with high cellular plasticity, which may predict the development of PPDM after DP. In contrast, PD is associated with alterations of gut microbiome and increases in SCFA production and GLP-1 secretion, possibly protecting against PPDM development.

Type 2 diabetes mellitus (T2DM) results from pancreatic β-cell failure in the setting of insulin resistance. In the early stages of this disease, pancreatic β-cells meet increased insulin demand by both enhancing insulin-secretory capacity and increasing β-cell mass. As the disease progresses, β-cells fail to maintain these compensatory responses. This involves both extrinsic signals and mediators intrinsic to β-cells, which adversely affect β-cells by impairing insulin secretion, decreasing proliferative capacities, and ultimately causing apoptosis. In recent years, it has increasingly been recognized that changes in circulating levels of various factors from other organs play roles in β-cell dysfunction and cellular loss. In this review, we discuss current knowledge of interorgan communications underlying β-cell failure during the progression of T2DM.

Loss of functional β cells results in a gradual progression of insulin insufficiency in Wolfram syndrome caused by recessive WFS1 mutations. Currently, there are no specific treatment recommendations for the disease. We have so far evidenced that, in the Wfs1-/- mice, β cells become dedifferentiated and revert to endocrine progenitor-like cells expressing Ngn3, and a subset of β cells takes α cell fate. Such β cell plasticity appears after nursing, independently of hyperglycaemia, and becomes more apparent along with diabetes progression accompanied with no significant increase in apoptosis. In this study, we examined the potential effects of a GLP-1 receptor agonist on β cell plasticity and glucose homeostasis in young Wfs1−/− mice without noticeable hyperglycaemia. Results: Four weeks administration of Ex-4 resulted in an off-drug amelioration of glucose excursions after glucose loading in Wfs1−/− mice, with insulin secretory dynamics that were indistinguishable from those in WT mice, despite no alteration in beta cell mass. In association with the functional improvements, Ex-4 alleviated β cell dedifferentiation evidenced by a decrease of MafA and a Ngn3 emergence. At the same time, Ex-4 reversed a robust increase in thioredoxin interacting protein (TXNIP) in Wfs1-/- β cells. Mechanistically, Ex-4 suppressed it at both transcriptional and post-translational levels. Furthermore, we have found that genetic ablation of Txnip in Wfs1-/- mice inhibited β cell dedifferentiation, resulting in prevention of diabetes progression with preservation of β cell mass. Our finding implies that activation of the GLP-1 receptor signaling may prevent β cells from becoming dedifferentiated by modulating Txnip and may slow diabetes progression in Wolfram syndrome. Disclosure K. Tanabe: None. K. Amo-Shiinoki: None. M. Hatanaka: None. Y. Tanizawa: None. Funding Japan Society for the Promotion of Science (16K09752, 15K21198, 15K09390); Japan Diabetes Society; Japan Association for Diabetes Education and Care

A 39-year-old man with type 1 diabetes, who had a 4-year history of Graves' disease being treated with propylthiouracil (PTU), had developed sudden hearing loss. However, he showed no other clinical manifestations. Intratympanic administration with dexamethasone had failed, and his hearing had deteriorated. Magnetic resonance imaging showed the contrast effect on T1-weighted image in both cochleae, and the serum immunological analysis showed the high titers for anti-neutrophil cytoplasmic antibodies (ANCA). Therefore, his sudden hearing loss was presumed to be initial presentation of ANCA-associated vasculitis owing to PTU. His hearing was rapidly restored by a PTU withdrawal while no use of immunosuppressive agents, and he confirmed his hearing improvement in ordinary conversation. The patient's clinical course suggests that bilateral sensorineural hearing loss that occurs during treating hyperthyroidism could be initial presentation of ANCA-associated vasculitis, and discontinuing anti-thyroid drugs should be considered before treating with glucocorticoids., Sudden hearing loss; Endocrinology; Internal medicine; Clinical research; Hyperthyroidism; Anti-thyroid drugs.

Pancreatic beta cell loss is associated with augmented ER and oxidative stresses in Wolfram syndrome caused by mutations in the WFS1. In the Wfs1-/- mice, beta cells become dedifferentiated and revert to endocrine progenitor-like cells, and a subset of them takes alpha cell fate. Such beta cell plasticity appears after nursing, independently of hyperglycemia, and becomes more apparent along with diabetes progression accompanied with no significant increase in apoptosis. We have found that genetic inhibition of Txnip, which is a stress response molecule involving in various cellular processes, preserved beta cell mass and maintained glucose homeostasis in the Wfs1-/- mice. This suggests its roles in the regulation of beta cell plasticity in the setting of Wfs1 deficiency. One clue to the mechanisms underlying beta cell dedifferentiation was the paradoxical reduction of acetyl-CoA, citrate and ATP content in the Wfs1-/- islets in spite of the enhanced glycolysis with an increase in pyruvate. This metabolic dissociation was correlated with an increase in phosphorylated pyruvate dehydrogenase (PDH). Importantly, Txnip directly interacts with both PDH kinase and PDH, indicating its involvement in the regulation of PDH activity. Indeed, islets of Wfs1-/- mice lacking Txnip demonstrated a robust reduction of phosphorylated PDH and a restoration of capabilities of ATP production in response to glucose. Thus, these finding illustrate impaired energy metabolism in beta cells under the chronic stress conditions and suggest that beta cells may possibly become dedifferentiated to adapt to metabolic insufficiency caused by unresolvable stresses. This provides new insights into molecular mechanisms underlying beta cell loss in diabetes related to cellular stresses, such as Wolfram syndrome. Disclosure K. Amo-Shiinoki: None. K. Tanabe: None. M. Hatanaka: None. Y. Tanizawa: None.

Loss of functional beta cells results in a gradual progression of insulin insufficiency in Wolfram syndrome caused by recessive WFS1 mutations. However, beta cell dysfunction in Wolfram syndrome has yet to be fully characterised, and there are also no specific treatment recommendations. In this study, we aimed to characterise beta cell secretory defects and to examine the potential effects of a glucagon-like peptide-1 (GLP-1) receptor agonist on diabetes in Wolfram syndrome. Insulin secretory function was assessed by the pancreatic perfusion method in mice used as a model of Wolfram syndrome. In addition, granule dynamics in living beta cells were examined using total internal reflection fluorescence microscopy. Acute and chronic effects of exendin-4 (Ex-4) on glucose tolerance and insulin secretion were examined in young Wfs1−/− mice without hyperglycaemia. Molecular events associated with Ex-4 treatment were investigated using pancreatic sections and isolated islets. In addition, we retrospectively observed a woman with Wolfram syndrome who had been treated with liraglutide for 24 weeks. Treatment with liraglutide ameliorated our patient’s glycaemic control and resulted in a 20% reduction of daily insulin dose along with an off-drug elevation of fasting C-peptide immunoreactivity. Glucose-stimulated first-phase insulin secretion and potassium-stimulated insulin secretion decreased by 53% and 59%, respectively, in perfused pancreases of 10-week-old Wfs1−/− mice compared with wild-type (WT) mice. The number of insulin granule fusion events in the first phase decreased by 41% in Wfs1−/− beta cells compared with WT beta cells. Perfusion with Ex-4 increased insulin release in the first and second phases by 3.9-fold and 5.6-fold, respectively, in Wfs1−/− mice compared with perfusion with saline as a control. The physiological relevance of the effects of Ex-4 was shown by the fact that a single administration potentiated glucose-stimulated insulin secretion and improved glucose tolerance in Wfs1−/− mice. Four weeks of administration of Ex-4 resulted in an off-drug amelioration of glucose excursions after glucose loading in Wfs1−/− mice, with insulin secretory dynamics that were indistinguishable from those in WT mice, despite the fact that there was no alteration in beta cell mass. In association with the functional improvements, Ex-4 treatment reversed the increases in phosphorylated eukaryotic initiation factor (EIF2α) and thioredoxin interacting protein (TXNIP), and the decrease in phosphorylated AMP-activated kinase (AMPK), in the beta cells of the Wfs1−/− mice. Furthermore, Ex-4 treatment modulated the transcription of oxidative and endoplasmic reticulum stress-related markers in isolated islets, implying that it was able to mitigate the cellular stresses resulting from Wfs1 deficiency. Our study provides deeper insights into the pathophysiology of beta cell dysfunction caused by WFS1 deficiency and implies that activation of the GLP-1 receptor signal may alleviate insulin insufficiency and aid glycaemic control in Wolfram syndrome.

We report on two Japanese siblings with Wolfram syndrome (WS) who were identified as carrying a novel mutation in Wolfram syndrome 1 (WFS1). The homozygous mutation, Y652X in exon 8, has not been reported in WS or low-frequency sensorineural hearing loss previously. The substitution is located at the beginning of the C-terminus of the protein, which is typically localized to the lumen of the endoplasmic reticulum (ER). This mutation predicts a truncated protein of 651 aa, with deletion of the entire C-terminal peptide, and was suggested to cause a severe phenotype. They developed a typical presentation, including symptoms such as deafness, diabetes insipidus, and visual disturbances at younger ages compared to those reported in the review. Moreover, they were also diagnosed with attention deficit hyperactivity disorder (ADHD), a neuropsychiatric disorder that manifests in early childhood as a deviation from normal development at the ages of 3 and 5 years. WS is known to be associated with neurological and psychiatric disorders, such as severe depression. However, co-occurrence of ADHD and WS has not been found in the literature; these cases were also interesting in this aspect.

Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1(-/-) A(y)/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

Glycogen synthase kinase 3β (GSK-3β) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3β in mice would result in enhanced beta cell mass and function.Mice with beta cell deficiency of GSK-3β (β-Gsk-3β [also known as Gsk3b](-/-)) were generated by breeding Gsk-3β (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting.On a normal diet β-Gsk-3β ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet β-Gsk-3β (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation.These results are consistent with a mechanism whereby endogenous GSK-3β activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.

OBJECTIVE—β-Cells are particularly susceptible to fatty acid–induced apoptosis associated with decreased insulin receptor/phosphatidylinositol-3 kinase/Akt signaling and the activation of stress kinases. We examined the mechanism of fatty acid–induced apoptosis of mouse β-cells especially as related to the role played by endoplasmic reticulum (ER) stress–induced Foxo1 activation and whether decreasing Foxo1 activity could enhance cell survival.RESEARCH DESIGN AND METHODS—Mouse insulinoma (MIN6) cells were administered with fatty acids, and the role of Foxo1 in mediating effects on signaling pathways and apoptosis was examined by measuring Foxo1 activity and using dominant-negative Foxo1.RESULTS—Increasing fatty acid concentrations (100–400 μmol/l palmitate or oleate) led to early Jun NH2-terminal kinase (JNK) activation that preceded induction of ER stress markers and apoptosis. Foxo1 activity was increased with fatty acid administration and by pharmacological inducers of ER stress, and this increase was prevented by JNK inhibition. Fatty acids induced nuclear localization of Foxo1 at 4 h when Akt activity was increased, indicating that FoxO1 activation was not mediated by JNK inhibition of Akt. In contrast, fatty acid administration for 24 h was associated with decreased insulin signaling. A dominant-negative Foxo1 adenovirus (Adv-DNFoxo) conferred cells with protection from ER stress and fatty acid–mediated apoptosis. Microarray analysis revealed that fatty acid induction of gene expression was in most cases reversed by Adv-DNFoxo, including the proapoptotic transcription factor CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein).CONCLUSIONS—Early induction of JNK and Foxo1 activation plays an important role in fatty acid–induced apoptosis. Expressing a dominant-negative allele of Foxo1 reduces expression of apoptotic and ER stress markers and promotes β-cell survival from fatty acid and ER stress, identifying a potential therapeutic target for preserving β-cells in type 2 diabetes.

Glycogen synthase kinase-3 (GSK3) has been implicated in the pathophysiology of several prevalent diseases, including diabetes. However, despite recent progress in our understanding of the role of GSK3 in the regulation of glucose metabolism in peripheral tissues, the involvement of GSK3 in islet beta cell growth and function in vivo is unknown. We therefore sought to determine whether over-activation of GSK3beta would lead to alterations in islet beta cell mass and/or function.Transgenic mice overexpressing a constitutively active form of human GSK3beta (S9A) under the control of the rat insulin promoter (RIP-GSK3betaCA) were created. Studies using mouse insulinoma cells (MIN6) were conducted to investigate the regulation of GSK3beta activity and its impact on pancreas/duodenum homeobox protein-1 (PDX-1) levels.We demonstrated that phosphorylation of GSK3beta was decreased, indicating increased GSK3beta activity in two animal models of diabetes, Lepr(-/- ) mice and Ins2 (Akita/+) mice. In MIN6 cells, the activity of GSK3beta was regulated by glucose, in a fashion largely dependent on phosphatidylinositol 3-kinase. RIP-GSK3betaCA transgenic mice showed impaired glucose tolerance after 5 months of age. Histological studies revealed that transgenic mice had decreased beta cell mass and decreased beta cell proliferation, with a 50% decrease (p0.05) in the level of PDX-1.We showed direct evidence that GSK3beta activity is associated with beta cell failure in diabetic mouse models and that its overactivation resulted in decreased pancreatic beta cell proliferation and mass. GSK3 modulates PDX-1 stability in both cultured insulinoma cells and islets in vivo. These results may ultimately facilitate the development of potential therapeutic interventions targeting type 2 diabetes and/or islet transplantation.

Wolfram syndrome(WFS: OMIM 222300) is a rare recessive neuro-endocrine degenerative disorder, known as DIDMOAD(Diabetes Insipidus, early-onset Diabetes Mellitus, Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene(WFS1). The WFS1 protein is an endoplasmic reticulum(ER) embedded protein, which functions in ER calcium homeostasis and unfolded protein responses. Dysregulation of these cellular processes results in the development of ER stress, leading to apoptosis. In addition, abundantly present WFS1 protein in insulin secretory granules plays a role in the intra-granular acidification. However, the phenotypic pleiomorphism and molecular complexity of this disease limit the understanding of WFS. Here we review clinical features, molecular mechanisms and mutations of WFS1 gene that relate to this syndrome.

Background Wolfram syndrome (WFS) is a recessive neurologic and endocrinologic degenerative disorder, and is also known as DIDMOAD (Diabetes Insipidus, early-onset Diabetes Mellitus, progressive Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene (WFS1). However, the phenotypic pleiomorphism, rarity and molecular complexity of this disease complicate our efforts to understand WFS. To address this limitation, we aimed to describe complications and to elucidate the contributions of WFS1 mutations to clinical manifestations in Japanese patients with WFS. Methodology The minimal ascertainment criterion for diagnosing WFS was having both early onset diabetes mellitus and bilateral optic atrophy. Genetic analysis for WFS1 was performed by direct sequencing. Principal Findings Sixty-seven patients were identified nationally for a prevalence of one per 710,000, with 33 patients (49%) having all 4 components of DIDMOAD. In 40 subjects who agreed to participate in this investigation from 30 unrelated families, the earliest manifestation was DM at a median age of 8.7 years, followed by OA at a median age of 15.8 years. However, either OA or DI was the first diagnosed feature in 6 subjects. In 10, features other than DM predated OA. Twenty-seven patients (67.5%) had a broad spectrum of recessive mutations in WFS1. Two patients had mutations in only one allele. Eleven patients (27.5%) had intact WFS1 alleles. Ages at onset of both DM and OA in patients with recessive WFS1 mutations were indistinguishable from those in patients without WFS1 mutations. In the patients with predicted complete loss-of-function mutations, ages at the onsets of both DM and OA were significantly earlier than those in patients with predicted partial-loss-of function mutations. Conclusion/Significance This study emphasizes the clinical and genetic heterogeneity in patients with WFS. Genotype-phenotype correlations may exist in patients with WFS1 mutations, as demonstrated by the disease onset.

Leptin at 1-5 nM, the concentrations observed in obese subjects, caused an increase in the active form of mitogen-activated protein kinase (MAPK) that was accompanied by increased tyrosine phosphorylation of STAT-1 and STAT-3 in a mouse pancreatic beta cell line, MIN6. Leptin also increased DNA synthesis and cell viability in MIN6 cells based on the results of [3H]-thymidine incorporation and colorimetric MTT assay, respectively. The specific MAPK-inhibitor PD98059 blocked not only the MAPK activation but also the increment in DNA synthesis and cell viability caused by leptin. Thus, leptin stimulates both the MAPK and the Janus kinase (JAK)-STAT cascade as well as inducing proliferation through the MAPK cascade in MIN6 cells. This mechanism might account, at least in part, for obesity-induced pancreatic islet hypertrophy.

Background The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined. Principal Findings Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β. Conclusions/Significance Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.

Insulin stimulates glucose uptake in fat and muscle primarily by stimulating the translocation of vesicles containing facilitative glucose transporters, GLUT4, from intracellular compartments to the plasma membrane. Although cell surface externalization of GLUT4 is critical for glucose transport, the mechanism regulating cell surface GLUT4 remains unknown. Using a yeast two-hybrid screening system, we have screened GLUT4-binding proteins, and identified a novel glycosyl phosphatidyl inositol (GPI)-linked proteoglycan, Glypican3 (GPC3). We confirmed their interaction using immunoprecipitation and a GST pull-down assay. We also revealed that GPC3 and GLUT4 to co-localized at the plasma membrane, using immunofluorescent microscopy. Furthermore, we observed that glucose uptake in GPC3-overexpressing adipocytes was increased by 30% as compared to control cells. These findings suggest that GPC3 may play roles in glucose transport through GLUT4.

Despite treatment with agents that enhance β-cell function and insulin action, reduction in β-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3β (Gsk-3β). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3β to regulation of β-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/−) exhibit insulin resistance and a doubling of β-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3β (Gsk-3β+/−) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced β-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2−/−), like the Ir+/− mice, are insulin resistant, but develop profound β-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3β activity associated with a marked reduction of β-cell proliferation and increased apoptosis. Irs2−/− mice crossed with Gsk-3β+/− mice preserved β-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2−/− mice had increased cyclin-dependent kinase inhibitor p27kip1 that was limiting for β-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of β-cell mass in Gsk-3β+/−Irs2−/− mice was accompanied by suppressed p27kip1 levels and increased Pdx1 levels. To separate peripheral versus β-cell–specific effects of reduction of Gsk3β activity on preservation of β-cell mass, mice homozygous for a floxed Gsk-3β allele (Gsk-3F/F) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce β-cell–specific knockout of Gsk-3β (βGsk-3β−/−). Like Gsk-3β+/− mice, βGsk-3β−/− mice also prevented the diabetes of the Irs2−/− mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within β-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of β-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve β-cells and prevent diabetes onset., Author Summary Diabetes is often characterized by a failure of insulin production by pancreatic β-cells to properly regulate glucose homeostasis. Insulin resistance can lead to β-cell failure, and our studies have focused on elucidating the mechanisms involved in this postnatal failure. In this study, we evaluated a new, negatively regulated enzyme of the insulin signaling pathway, glycogen synthase kinase 3 (Gsk-3), specifically within insulin-producing pancreatic β-cells. When this enzyme is elevated, it can impair replication and increase cell death, resulting in loss of insulin-producing cells and diabetes. Gsk-3 is also known to regulate cell death and proliferation in neurons. We assessed the role of Gsk-3 on glucose homeostasis in two different mouse models of insulin resistance. We demonstrated that genetically reducing the levels of Gsk-3β in the insulin-resistant mouse improved glucose homeostasis. In another model in which severe insulin resistance is associated with destruction of β-cells, reducing Gsk-3β not only preserved β-cells by increasing proliferation and reducing cell death, but it also corrected diabetes. Controlling activity of Gsk-3 could lead to new hopes for maintaining or improving β-cell number and prevention of diabetes., The insulin signaling pathway negatively regulates glycogen synthase kinase 3. The current studies demonstrate that this enzyme is rate limiting for pancreatic β-cell replication and survival.

Objective: TheWFS1gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. HomozygousWFS1gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic β-cells are selectively lost from the patient’s islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses.Design and methods: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic β-cell-derived MIN6 cells, and Akita mouse-derivedIns296Y/Yinsulinoma cells. The humanWFS1gene promoter-luciferase reporter analysis was also conducted.Result: Islet β-cells were the major site ofWfs1expression. This expression was also found in δ-cells, but not in α-cells.WFS1expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increasedWFS1mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells.WFS1gene expression was also increased inIns296Y/Yinsulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. TheWFS1gene promoter-luciferase reporter system revealed that the humanWFS1promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active inIns296Y/Ycells thanIns2wild/wildcells.Conclusion:Wfs1expression, which is localized to β- and δ-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link betweenWfs1and ER stress.

To clarify heterogeneity in Japanese adult-onset type 1 diabetes, we analyzed the HLA-DR and -DQ haplotypes, depending on the clinical phenotype, and compared them with those in childhood-onset type 1 diabetes (CO). The patients in a previously reported Ehime Study were divided into subgroups by the mode of onset of diabetes: 68 acute-onset type 1 diabetic patients (AO) and 28 slowly progressive type 1 diabetic patients (SO). HLA haplotypes were compared with those of 80 CO patients and 190 control subjects. Two major susceptible HLA haplotypes in the Japanese, DRB1*0405-DQB1*0401 (DR4) and DRB1*0901-DQB1*0303 (DR9), were significantly increased in the AO and CO groups, but only DR9 was increased in the SO group. AO subjects had a higher frequency of DR9 than CO subjects. Accordingly, the DR9:DR4 frequency increased with increasing age of onset. Another susceptible haplotype, DRB1*0802-DQB1*0302 (DR8), was involved only in the CO group. Analysis of haplotype combinations revealed that DR4 and DR9 had significant dosage effects on the AO and CO groups (P < 0.0001), but only DR9 had such an effect in the SO group (P < 0.03). These results suggest differences in the contribution of HLA class II haplotypes to susceptibility of type 1 diabetes depending on the clinical phenotype and also indicate that HLA class II haplotypes may be associated with the onset age of type 1 diabetes.

To test the hypothesis that leptin secreted from adipose tissue is a mediator linking obesity and pancreatic islet hypertrophy, we examined the effects of leptin on proliferative and apoptotic responses in rat islet cells. Rat pancreatic islets were isolated and incubated with 0, 1, 5, or 75 nM leptin for 24 h under serum-deprived conditions. Cell viability was assessed with 2,5-diphenyltetrazolium bromide and trypan blue dye exclusion tests. Cell proliferation and apoptosis were evaluated with 5-bromo-2'-deoxyuridine incorporation into DNA and DNA ladder formation, respectively. Incubation for 24 h with 1 and 5 nM leptin, the concentrations observed in obese subjects, increased the viability of isolated pancreatic islet cells. Five nanomolar concentrations of leptin did not stimulate 5-bromo-2'-deoxyuridine incorporation into incubated islet cells, indicating no influence on cell proliferation, but did inhibit DNA ladder formation, a hallmark of cell apoptosis. Moreover, 5 nM leptin reduced the triglyceride content and suppressed inducible nitric oxide synthase mRNA expression in incubated islets. These results suggest that leptin increased viable cell numbers via suppression of apoptosis in isolated pancreatic islet cells under these experimental conditions. This mechanism might account at least in part for an obesity-induced increase in pancreatic beta-cell mass.

Troglitazone is one of the thiazolidinediones, a new class of oral antidiabetic compounds that are ligands of peroxisome proliferator–activated receptor-γ. This study on vascular endothelial growth factor (VEGF), also known as vascular permeability factor, was prompted by our clinical observation that the characteristics of troglitazone-induced edema were very similar to those caused by vascular hyperpermeability. When Japanese diabetic patients were screened for plasma VEGF, we found levels to be significantly (P < 0.001) increased in troglitazone-treated subjects (120.1 ± 135.0 pg/ml, n = 30) compared with those treated with diet alone (29.2 ± 36.1 pg/ml, n = 10), sulfonylurea (25.8 ± 22.2 pg/ml, n = 10), or insulin (24.6 ± 19.0 pg/ml, n = 10). Involvement of troglitazone in increased VEGF levels was further supported by the plasma VEGF levels in five patients before treatment (20.2 ± 7.0 pg/ml), after 3 months of troglitazone treatment (83.6 ± 65.9 pg/ml), and 3 months after discontinuation (28.0 ± 11.6 pg/ml). We further demonstrated that troglitazone, as well as rosiglitazone, at the plasma concentrations observed in patients, increased VEGF mRNA levels in 3T3-L1 adipocytes. VEGF is an angiogenic and mitogenic factor and is currently considered the most likely cause of neovascularization and hyperpermeability in diabetic proliferative retinopathy. Although increased VEGF may be beneficial for subjects with macroangiopathy and troglitazone is currently not available for clinical use, vascular complications, especially diabetic retinopathy, must be followed with great caution in subjects treated with thiazolidinediones.