Comprehensive overview of disease models for Wolfram syndrome: toward effective treatments.

Wolfram syndrome (OMIM 222300) is a rare autosomal recessive disease with a devastating array of symptoms, including diabetes mellitus, optic nerve atrophy, diabetes insipidus, hearing loss, and neurological dysfunction. The discovery of the causative gene, WFS1, has propelled research on this disease. However, a comprehensive understanding of the function of WFS1 remains unknown, making the development of effective treatment a pressing challenge. To bridge these knowledge gaps, disease models for Wolfram syndrome are indispensable, and understanding the characteristics of each model is critical. This review will provide a summary of the current knowledge regarding WFS1 function and offer a comprehensive overview of established disease models for Wolfram syndrome, covering animal models such as mice, rats, flies, and zebrafish, along with induced pluripotent stem cell (iPSC)-derived human cellular models. These models replicate key aspects of Wolfram syndrome, contributing to a deeper understanding of its pathogenesis and providing a platform for discovering potential therapeutic approaches.

Selective proteasome degradation of C-terminally-truncated human WFS1 in pancreatic beta cells.

Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1 proteins by studying the stability of full-length wild-type (WT) WFS1, a missense mutant P724L, and two C-terminally truncated mutants, W837X and Y652X. We compared their stability by overexpressing them in MIN6 and HEK293T cells. The C-terminally truncated mutants W837X and Y652X are degraded more rapidly than the missense P724L mutant or wild-type WFS1 in MIN6 cells. In contrast, Y652X is more stable than WT or other mutant WFS1 proteins in HEK293T. In conclusion, we found that C-terminally truncated WFS1 mutants are selectively degraded in a cell type-specific manner.

Hachimijiogan, a traditional herbal medicine, modulates adipose cell function and ameliorates diet-induced obesity and insulin resistance in mice.

Hachimijiogan (HJG) has originally been used to ameliorate a variety of symptoms associated with low ambient temperatures. However, its pharmacological action in metabolic organs remains unclear. We hypothesized that HJG may modulate metabolic function and have a potential therapeutic application to metabolic diseases. To test this hypothesis, we investigated metabolic action of HJG in mice. Male C57BL/6J mice chronically administered with HJG showed a reduction in adipocyte size with increased transcription of beige adipocyte-related genes in subcutaneous white adipose tissue. HJG-mixed high-fat diet (HFD)-fed mice showed alleviation of HFD-induced weight gain, adipocyte hypertrophy, liver steatosis with a significant reduction in circulating leptin and Fibroblast growth factor 21 despite no changes in food intake or oxygen consumption. Feeding an HJG-mixed HFD following 4-weeks of HFD feeding, while a limited effect on body weight, improved insulin sensitivity with a reversal of decreased circulating adiponectin. In addition, HJG improved insulin sensitivity in the leptin-deficient mice without significant effects on body weight. Treatment with n-butanol soluble extracts of HJG potentiated transcription of Uncoupling protein 1 mediated by ß3-adrenergic agonism in 3T3L1 adipocytes. These findings provide evidence that HJG modulates adipocyte function and may exert preventive or therapeutic effects against obesity and insulin resistance.

A case of multiple glucagonomas with no clinical manifestations of excess glucagon despite hyperglucagonemia.

Herein we report the case of a patient with multiple glucagonomas that have been precisely described with endoscopic ultrasound. A 36-year-old woman was referred to our hospital for computed tomography investigation of multiple pancreatic masses. Physical examination was unremarkable; on contrast-enhanced computed tomography, mass lesions were evident in the head, body, and tail of the pancreas. The mass in the pancreatic head was poorly demarcated and exhibited a faint contrast effect, the one in the pancreatic body was a cystic lesion, and the one in the pancreatic tail was hypervascular. Blood investigations showed that serum glucagon was abnormally high at 7670 pg/ml; glucose tolerance was not impaired. There was no family history that suggested multiple endocrine neoplasia type 1 or von Hippel-Lindau disease. Endoscopic ultrasound revealed that there were additional masses, which were scattered isoechoic to hyperechoic lesions a few millimeters in size. Ultrasound-guided fine needle biopsy of the lesion in the pancreatic tail resulted in a diagnosis of a neuroendocrine tumor. Based on these pathologic findings, we performed a total pancreatectomy. A large number of nodules with tumor cells were evident in all cut surfaces of the surgical specimen. Immunostaining was positive for chromogranin A and glucagon, and glucagonoma was therefore diagnosed. It is conceivable that attenuated glucagon action could have contributed to the development of the multiple glucagonomas.

Gsk-3-Mediated Proteasomal Degradation of ATF4 Is a Proapoptotic Mechanism in Mouse Pancreatic ß-Cells.

Endoplasmic reticulum (ER) stress is a key pathogenic factor in type 1 and 2 diabetes. Glycogen synthase kinase 3 (Gsk-3) contributes to ß-cell loss in mice. However, the mechanism by which Gsk-3 leads ß-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-3ß. 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 ßTrCP, 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 ß-cell death under ER stress conditions.

Deficiency of WFS1 leads to the impairment of AVP secretion under dehydration in male mice.

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 ß-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.

A patient with sudden hearing loss induced by propylthiouracil.

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.

Importance of Intestinal Environment and Cellular Plasticity of Islets in the Development of Postpancreatectomy Diabetes.

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 ß-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 ß-cell areas were predictors of PPDM. Furthermore, in DP patients, α-cell and ß-cell areas were associated with ALDH1A3 expression in islets. CONCLUSIONS: We postulate that a greater removal of ß-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.

Islet cell dedifferentiation is a pathologic mechanism of long-standing progression of type 2 diabetes.

Dedifferentiation has been implicated in ß 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 ß 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 ß cell failure during the course of diabetes progression in humans.

Activation of GLP-1 receptor signalling alleviates cellular stresses and improves beta cell function in a mouse model of Wolfram syndrome.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

Interorgan Crosstalk Contributing to ß-Cell Dysfunction.

Type 2 diabetes mellitus (T2DM) results from pancreatic ß-cell failure in the setting of insulin resistance. In the early stages of this disease, pancreatic ß-cells meet increased insulin demand by both enhancing insulin-secretory capacity and increasing ß-cell mass. As the disease progresses, ß-cells fail to maintain these compensatory responses. This involves both extrinsic signals and mediators intrinsic to ß-cells, which adversely affect ß-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 ß-cell dysfunction and cellular loss. In this review, we discuss current knowledge of interorgan communications underlying ß-cell failure during the progression of T2DM.

[Wolfram syndrome: clinical features, molecular genetics of WFS1 gene].

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.

Wolfram syndrome in the Japanese population; molecular analysis of WFS1 gene and characterization of clinical features.

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.

Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1ß gene expression in pancreatic islet ß-cells.

Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1ß (HIF-1ß) has emerged as a potential determinant of pancreatic ß-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 ß-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.

Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3ß independent of JNK activation.

BACKGROUND: The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic ß-cell death. This study examines the synergistic effects of glucose and FFA on ß-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 3ß (Gsk3ß) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3ß. CONCLUSIONS/SIGNIFICANCE: Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3ß activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet ß-cell Gsk3ß as a potential therapeutic target.

Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

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 ß-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 ß-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 ß-cells, there was a 32% reduction in the intensity in WFS1-deficient ß-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 ß-cells relative to that in wild-type ß-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 ß-cell dysfunction in patients with Wolfram syndrome.

Identification of Glypican3 as a novel GLUT4-binding protein.

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.

Genetic deficiency of glycogen synthase kinase-3beta corrects diabetes in mouse models of insulin resistance.

Despite treatment with agents that enhance beta-cell function and insulin action, reduction in beta-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-3beta (Gsk-3beta). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3beta to regulation of beta-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 beta-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3beta (Gsk-3beta+/-) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced beta-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 beta-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3beta activity associated with a marked reduction of beta-cell proliferation and increased apoptosis. Irs2-/- mice crossed with Gsk-3beta+/- mice preserved beta-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 p27(kip1) that was limiting for beta-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of beta-cell mass in Gsk-3beta+/- Irs2-/- mice was accompanied by suppressed p27(kip1) levels and increased Pdx1 levels. To separate peripheral versus beta-cell-specific effects of reduction of Gsk3beta activity on preservation of beta-cell mass, mice homozygous for a floxed Gsk-3beta allele (Gsk-3(F/F)) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce beta-cell-specific knockout of Gsk-3beta (betaGsk-3beta-/-). Like Gsk-3beta+/- mice, betaGsk-3beta-/- 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 beta-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of beta-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve beta-cells and prevent diabetes onset.

Inhibition of Foxo1 protects pancreatic islet beta-cells against fatty acid and endoplasmic reticulum stress-induced apoptosis.

OBJECTIVE: beta-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 beta-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 micromol/l palmitate or oleate) led to early Jun NH(2)-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 beta-cell survival from fatty acid and ER stress, identifying a potential therapeutic target for preserving beta-cells in type 2 diabetes.