Phenotypic characteristics of early Wolfram syndrome.

BACKGROUND: Wolfram Syndrome (WFS:OMIM 222300) is an autosomal recessive, progressive, neurologic and endocrinologic degenerative disorder caused by mutations in the WFS1 gene, encoding the endoplasmic reticulum (ER) protein wolframin, thought to be involved in the regulation of ER stress. This paper reports a cross section of data from the Washington University WFS Research Clinic, a longitudinal study to collect detailed phenotypic data on a group of young subjects in preparation for studies of therapeutic interventions. METHODS: Eighteen subjects (ages 5.9-25.8, mean 14.2 years) with genetically confirmed WFS were identified through the Washington University International Wolfram Registry. Examinations included: general medical, neurologic, ophthalmologic, audiologic, vestibular, and urologic exams, cognitive testing and neuroimaging. RESULTS: Seventeen (94%) had diabetes mellitus with the average age of diabetes onset of 6.3 ± 3.5 years. Diabetes insipidus was diagnosed in 13 (72%) at an average age of 10.6 ± 3.3 years. Seventeen (94%) had optic disc pallor and defects in color vision, 14 (78%) had hearing loss and 13 (72%) had olfactory defects, eight (44%) had impaired vibration sensation. Enuresis was reported by four (22%) and nocturia by three (17%). Of the 11 tested for bladder emptying, five (45%) had elevated post-void residual bladder volume. CONCLUSIONS: WFS causes multiple endocrine and neurologic deficits detectable on exam, even early in the course of the disease. Defects in olfaction have been underappreciated. The proposed mechanism of these deficits in WFS is ER stress-induced damage to neuronal and hormone-producing cells. This group of subjects with detailed clinical phenotyping provides a pool for testing proposed treatments for ER stress. Longitudinal follow-up is necessary for establishing the natural history and identifying potential biomarkers of progression.

Early brain vulnerability in Wolfram syndrome.

Wolfram Syndrome (WFS) is a rare autosomal recessive disease characterized by insulin-dependent diabetes mellitus, optic nerve atrophy, diabetes insipidus, deafness, and neurological dysfunction leading to death in mid-adulthood. WFS is caused by mutations in the WFS1 gene, which lead to endoplasmic reticulum (ER) stress-mediated cell death. Case studies have found widespread brain atrophy in late stage WFS. However, it is not known when in the disease course these brain abnormalities arise, and whether there is differential vulnerability across brain regions and tissue classes. To address this limitation, we quantified regional brain abnormalities across multiple imaging modalities in a cohort of young patients in relatively early stages of WFS. Children and young adults with WFS were evaluated with neurological, cognitive and structural magnetic resonance imaging measures. Compared to normative data, the WFS group had intact cognition, significant anxiety and depression, and gait abnormalities. Compared to healthy and type 1 diabetic control groups, the WFS group had smaller intracranial volume and preferentially affected gray matter volume and white matter microstructural integrity in the brainstem, cerebellum and optic radiations. Abnormalities were detected in even the youngest patients with mildest symptoms, and some measures did not follow the typical age-dependent developmental trajectory. These results establish that WFS is associated with smaller intracranial volume with specific abnormalities in the brainstem and cerebellum, even at the earliest stage of clinical symptoms. This pattern of abnormalities suggests that WFS has a pronounced impact on early brain development in addition to later neurodegenerative effects, representing a significant new insight into the WFS disease process. Longitudinal studies will be critical for confirming and expanding our understanding of the impact of ER stress dysregulation on brain development.

Balance impairment in individuals with Wolfram syndrome.

AIM: Wolfram syndrome (WFS), a rare neurodegenerative disorder, is characterized by early onset insulin-dependent diabetes mellitus, optic atrophy, deafness, diabetes insipidus, and neurological abnormalities. Although previously unreported, we hypothesized that neurological complications may be detectable in relatively early stages of the disease. As the cerebellum and brainstem seem particularly vulnerable in WFS, we focused on balance functions critically dependent on these regions. The primary goal of this investigation was to compare balance in young individuals with WFS, in relatively early stages of the disease, to an age-matched cohort using a clinically applicable test. METHOD: Balance was assessed via the mini-BESTest in 13 children, adolescents and young adults with WFS and 30 typically developing age-matched individuals. RESULTS: A significant difference was observed between groups in balance as well as in three of four subcomponents of the mini-BESTest and in two timed tasks related to balance. Mini-BESTest scores were correlated with age among typically developing individuals. In the WFS group, mini-BESTest scores were related to overall motor dysfunction, but not age. INTERPRETATION: Impairments in balance in WFS may occur earlier in the disease process than previously recognized and appear to be related to overall neurological progression rather than chronological age. Recognizing balance impairments and understanding which balance systems contribute to balance deficits in those with WFS may allow for development of effective patient-centered treatment paradigms.

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.

Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia.

BACKGROUND: Glucose modulates beta-cell mass and function through an initial depolarization and Ca(2+) influx, which then triggers a number of growth regulating signaling pathways. One of the most important downstream effectors in Ca(2+) signaling is the calcium/Calmodulin activated serine threonine phosphatase, calcineurin. Recent evidence suggests that calcineurin/NFAT is essential for beta-cell proliferation, and that in its absence loss of beta-cells results in diabetes. We hypothesized that in contrast, activation of calcineurin might result in expansion of beta-cell mass and resistance to diabetes. METHODOLOGY/PRINCIPAL FINDINGS: To determine the role of activation of calcineurin signaling in the regulation of pancreatic beta-cell mass and proliferation, we created mice that expressed a constitutively active form of calcineurin under the insulin gene promoter (caCn(RIP)). To our surprise, these mice exhibited glucose intolerance. In vitro studies demonstrated that while the second phase of Insulin secretion is enhanced, the overall insulin secretory response was conserved. Islet morphometric studies demonstrated decreased beta-cell mass suggesting that this was a major component responsible for altered Insulin secretion and glucose intolerance in caCn(RIP) mice. The reduced beta-cell mass was accompanied by decreased proliferation and enhanced apoptosis. CONCLUSIONS: Our studies identify calcineurin as an important factor in controlling glucose homeostasis and indicate that chronic depolarization leading to increased calcineurin activity may contribute, along with other genetic and environmental factors, to beta-cell dysfunction and diabetes.

Gene-gene interactions lead to higher risk for development of type 2 diabetes in an Ashkenazi Jewish population.

BACKGROUND: Evidence has accumulated that multiple genetic and environmental factors play important roles in determining susceptibility to type 2 diabetes (T2D). Although variants from candidate genes have become prime targets for genetic analysis, few studies have considered their interplay. Our goal was to evaluate interactions among SNPs within genes frequently identified as associated with T2D. METHODS/PRINCIPAL FINDINGS: Logistic regression was used to study interactions among 4 SNPs, one each from HNF4A[rs1884613], TCF7L2[rs12255372], WFS1[rs10010131], and KCNJ11[rs5219] in a case-control Ashkenazi sample of 974 diabetic subjects and 896 controls. Nonparametric multifactor dimensionality reduction (MDR) and generalized MDR (GMDR) were used to confirm findings from the logistic regression analysis. HNF4A and WFS1 SNPs were associated with T2D in logistic regression analyses [P<0.0001, P<0.0002, respectively]. Interaction between these SNPs were also strong using parametric or nonparametric methods: the unadjusted odds of being affected with T2D was 3 times greater in subjects with the HNF4A and WFS1 risk alleles than those without either (95% CI = [1.7-5.3]; P

Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells.

Wolfram syndrome is an autosomal-recessive disorder characterized by insulin-dependent diabetes mellitus, caused by nonautoimmune loss of beta cells, and neurological dysfunctions. We have previously shown that mutations in the Wolfram syndrome 1 (WFS1) gene cause Wolfram syndrome and that WFS1 has a protective function against ER stress. However, it remained to be determined how WFS1 mitigates ER stress. Here we have shown in rodent and human cell lines that WFS1 negatively regulates a key transcription factor involved in ER stress signaling, activating transcription factor 6alpha (ATF6alpha), through the ubiquitin-proteasome pathway. WFS1 suppressed expression of ATF6alpha target genes and repressed ATF6alpha-mediated activation of the ER stress response element (ERSE) promoter. Moreover, WFS1 stabilized the E3 ubiquitin ligase HRD1, brought ATF6alpha to the proteasome, and enhanced its ubiquitination and proteasome-mediated degradation, leading to suppression of ER stress signaling. Consistent with these data, beta cells from WFS1-deficient mice and lymphocytes from patients with Wolfram syndrome exhibited dysregulated ER stress signaling through upregulation of ATF6alpha and downregulation of HRD1. These results reveal a role for WFS1 in the negative regulation of ER stress signaling and in the pathogenesis of diseases involving chronic, unresolvable ER stress, such as pancreatic beta cell death in diabetes.

LKB1 regulates pancreatic beta cell size, polarity, and function.

Pancreatic beta cells, organized in the islets of Langerhans, sense glucose and secrete appropriate amounts of insulin. We have studied the roles of LKB1, a conserved kinase implicated in the control of cell polarity and energy metabolism, in adult beta cells. LKB1-deficient beta cells show a dramatic increase in insulin secretion in vivo. Histologically, LKB1-deficient beta cells have striking alterations in the localization of the nucleus and cilia relative to blood vessels, suggesting a shift from hepatocyte-like to columnar polarity. Additionally, LKB1 deficiency causes a 65% increase in beta cell volume. We show that distinct targets of LKB1 mediate these effects. LKB1 controls beta cell size, but not polarity, via the mTOR pathway. Conversely, the precise position of the beta cell nucleus, but not cell size, is controlled by the LKB1 target Par1b. Insulin secretion and content are restricted by LKB1, at least in part, via AMPK. These results expose a molecular mechanism, orchestrated by LKB1, for the coordinated maintenance of beta cell size, form, and function.

Presenilins, Notch dose control the fate of pancreatic endocrine progenitors during a narrow developmental window.

Canonical Notch signaling is thought to control the endocrine/exocrine decision in early pancreatic progenitors. Later, RBP-Jkappa interacts with Ptf1a and E12 to promote acinar differentiation. To examine the involvement of Notch signaling in selecting specific endocrine lineages, we deregulated this pathway by targeted deletion of presenilin1 and presenilin2, the catalytic core of gamma-secretase, in Ngn3- or Pax6-expressing endocrine progenitors. Surprisingly, whereas Pax6(+) progenitors were irreversibly committed to the endocrine fate, we discovered that Ngn3(+) progenitors were bipotential in vivo and in vitro. When presenilin amounts are limiting, Ngn3(+) progenitors default to an acinar fate; subsequently, they expand rapidly to form the bulk of the exocrine pancreas. gamma-Secretase inhibitors confirmed that enzymatic activity was required to block acinar fate selection by Ngn3 progenitors. Genetic interactions identified Notch2 as the substrate, and suggest that gamma-secretase and Notch2 act in a noncanonical titration mechanism to sequester RBP-Jkappa away from Ptf1a, thus securing selection of the endocrine fate by Ngn3 progenitors. These results revise the current view of pancreatic cell fate hierarchy, establish that Ngn3 is not in itself sufficient to commit cells to the endocrine fate in the presence of Ptf1a, reveal a noncanonical action for Notch2 protein in endocrine cell fate selection, and demonstrate that acquisition of an endocrine fate by Ngn3(+) progenitors is gamma-secretase-dependent until Pax6 expression begins.

Population-specific risk of type 2 diabetes conferred by HNF4A P2 promoter variants: a lesson for replication studies.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) in the P2 promoter region of HNF4A were originally shown to be associated with predisposition for type 2 diabetes in Finnish, Ashkenazi, and, more recently, Scandinavian populations, but they generated conflicting results in additional populations. We aimed to investigate whether data from a large-scale mapping approach would replicate this association in novel Ashkenazi samples and in U.K. populations and whether these data would allow us to refine the association signal. RESEARCH DESIGN AND METHODS: Using a dense linkage disequilibrium map of 20q, we selected SNPs from a 10-Mb interval centered on HNF4A. In a staged approach, we first typed 4,608 SNPs in case-control populations from four U.K. populations and an Ashkenazi population (n = 2,516). In phase 2, a subset of 763 SNPs was genotyped in 2,513 additional samples from the same populations. RESULTS: Combined analysis of both phases demonstrated association between HNF4A P2 SNPs (rs1884613 and rs2144908) and type 2 diabetes in the Ashkenazim (n = 991; P < 1.6 x 10(-6)). Importantly, these associations are significant in a subset of Ashkenazi samples (n = 531) not previously tested for association with P2 SNPs (odds ratio [OR] approximately 1.7; P < 0.002), thus providing replication within the Ashkenazim. In the U.K. populations, this association was not significant (n = 4,022; P > 0.5), and the estimate for the OR was much smaller (OR 1.04; [95%CI 0.91-1.19]). CONCLUSIONS: These data indicate that the risk conferred by HNF4A P2 is significantly different between U.K. and Ashkenazi populations (P < 0.00007), suggesting that the underlying causal variant remains unidentified. Interactions with other genetic or environmental factors may also contribute to this difference in risk between populations.

Variants in the CD36 gene associate with the metabolic syndrome and high-density lipoprotein cholesterol.

A region along chromosome 7q was recently linked to components of the metabolic syndrome (MetS) in several genome-wide linkage studies. Within this region, the CD36 gene, which encodes a membrane receptor for long-chain fatty acids and lipoproteins, is a potentially important candidate. CD36 has been documented to play an important role in fatty acid metabolism in vivo and subsequently may be involved in the etiology of the MetS. The protein also impacts survival to malaria and the influence of natural selection has resulted in high CD36 genetic variability in populations of African descent. We evaluated 36 tag SNPs across CD36 in the HyperGen population sample of 2020 African-Americans for impact on the MetS and its quantitative traits. Five SNPs associated with increased odds for the MetS [P = 0.0027-0.03, odds ratio (OR) = 1.3-1.4]. Coding SNP, rs3211938, previously shown to influence malaria susceptibility, is documented to result in CD36 deficiency in a homozygous subject. This SNP conferred protection against the MetS (P = 0.0012, OR = 0.61, 95%CI: 0.46-0.82), increased high-density lipoprotein cholesterol, HDL-C (P = 0.00018) and decreased triglycerides (P = 0.0059). Fifteen additional SNPs associated with HDL-C (P = 0.0028-0.044). We conclude that CD36 variants may impact MetS pathophysiology and HDL metabolism, both predictors of the risk of heart disease and type 2 diabetes.

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.

Common variants in WFS1 confer risk of type 2 diabetes.

We studied genes involved in pancreatic beta cell function and survival, identifying associations between SNPs in WFS1 and diabetes risk in UK populations that we replicated in an Ashkenazi population and in additional UK studies. In a pooled analysis comprising 9,533 cases and 11,389 controls, SNPs in WFS1 were strongly associated with diabetes risk. Rare mutations in WFS1 cause Wolfram syndrome; using a gene-centric approach, we show that variation in WFS1 also predisposes to common type 2 diabetes.

A global approach to identify differentially expressed genes in cDNA (two-color) microarray experiments.

MOTIVATION: Currently most of the methods for identifying differentially expressed genes fall into the category of so called single-gene-analysis, performing hypothesis testing on a gene-by-gene basis. In a single-gene-analysis approach, estimating the variability of each gene is required to determine whether a gene is differentially expressed or not. Poor accuracy of variability estimation makes it difficult to identify genes with small fold-changes unless a very large number of replicate experiments are performed. RESULTS: We propose a method that can avoid the difficult task of estimating variability for each gene, while reliably identifying a group of differentially expressed genes with low false discovery rates, even when the fold-changes are very small. In this article, a new characterization of differentially expressed genes is established based on a theorem about the distribution of ranks of genes sorted by (log) ratios within each array. This characterization of differentially expressed genes based on rank is an example of all-gene-analysis instead of single gene analysis. We apply the method to a cDNA microarray dataset and many low fold-changed genes (as low as 1.3 fold-changes) are reliably identified without carrying out hypothesis testing on a gene-by-gene basis. The false discovery rate is estimated in two different ways reflecting the variability from all the genes without the complications related to multiple hypothesis testing. We also provide some comparisons between our approach and single-gene-analysis based methods. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

HNF4A genetic variants: role in diabetes.

PURPOSE OF REVIEW: Variants in the hepatocyte nuclear factor 4alpha (HNF4A) gene play a role in the development of diabetes mellitus. Although genetic variation in and around HNF4A regulatory regions has received considerable attention, the significance of these variants in the common type 2 diabetes varies in the literature. This review will provide a general overview of recent genetic studies involving the evaluation of HNF4A as a contributor to the risk and pathophysiology of diabetes mellitus and related risk factors. RECENT FINDINGS: These studies report newly identified variants, evaluate previously reported polymorphisms that were associated with type 2 diabetes in several distinct populations with maturity-onset diabetes of the young, type 2 diabetes, gestational diabetes, and diabetes related risk factors, and propose a role for HNF4A in insulin secretion via the potassium ATP channel. SUMMARY: HNF4A variants identified so far appear to modestly contribute to predisposition for type 2 diabetes. Continued identification and especially functional characterization of variants, however, will be critical in future studies to enhance our understanding of the metabolic impact of this gene.

Insulin protects islets from apoptosis via Pdx1 and specific changes in the human islet proteome.

Insulin is both a hormone regulating energy metabolism and a growth factor. We and others have shown that physiological doses of insulin initiate complex signals in primary human and mouse beta-cells, but the functional significance of insulin's effects on this cell type remains unclear. In the present study, the role of insulin in beta-cell apoptosis was examined. Exogenous insulin completely prevented apoptosis induced by serum withdrawal when given at picomolar or low nanomolar concentrations but not at higher concentrations, indicating that physiological concentrations of insulin are antiapoptotic and that insulin signaling is self-limiting in islets. Insulin treatment was associated with the nuclear localization of Pdx1 and the prosurvival effects of insulin were largely absent in islets 50% deficient in Pdx1, providing direct evidence that Pdx1 is a signaling target of insulin. Physiological levels of insulin did not increase Akt phosphorylation, and the protective effects of insulin were only partially altered in islets lacking 80% of normal Akt activity, suggesting the presence of additional insulin-regulated antiapoptotic pathways. Proteomic analysis of insulin-treated human islets revealed significant changes in multiple proteins. Bridge-1, a Pdx1-binding partner and regulator of beta-cell survival, was increased significantly at low insulin doses. Together, these data suggest that insulin can act as a master regulator of islet survival by regulating Pdx1.

Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet beta-cell.

Glucose controls islet beta-cell mass and function at least in part through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway downstream of insulin signaling. The Foxo proteins, transcription factors known in other tissues to be negatively regulated by Akt activation, affect proliferation and metabolism. In this study, we tested the hypothesis that glucose regulates Foxo1 activity in the beta-cell via an autocrine/paracrine effect of released insulin on its receptor. Mouse insulinoma cells (MIN6) were starved overnight for glucose (5 mmol/l) then refed with glucose (25 mmol/l), resulting in rapid Foxo1 phosphorylation (30 min, P < 0.05 vs. untreated). This glucose response was demonstrated to be time (0.5-2 h) and dose (5-30 mmol/l) dependent. The use of inhibitors demonstrated that glucose-induced Foxo1 phosphorylation was dependent upon depolarization, calcium influx, and PI3K signaling. Additionally, increases in glucose concentration over a physiological range (2.5-20 mmol/l) resulted in nuclear to cytoplasmic translocation of Foxo1. Phosphorylation and translocation of Foxo1 following glucose refeeding were eliminated in an insulin receptor knockdown cell line, indicating that the glucose effects are mediated primarily through the insulin receptor. Activity of Foxo1 was observed to increase with decreased glucose concentrations, assessed by an IGF binding protein-1 promoter luciferase assay. Starvation of MIN6 cells identified a putative Foxo1 target, Chop, and a Chop-promoter luciferase assay in the presence of cotransfected Foxo1 supported this hypothesis. The importance of these observations was that nutritional alterations in the beta-cell are associated with changes in Foxo1 transcriptional activity and that these changes are predominantly mediated through glucose-stimulated insulin secretion acting through its own receptor.

Diabetes and insulin secretion: the ATP-sensitive K+ channel (K ATP) connection.

The ATP-sensitive K+ channel (K ATP channel) senses metabolic changes in the pancreatic beta-cell, thereby coupling metabolism to electrical activity and ultimately to insulin secretion. When K ATP channels open, beta-cells hyperpolarize and insulin secretion is suppressed. The prediction that K ATP channel "overactivity" should cause a diabetic state due to undersecretion of insulin has been dramatically borne out by recent genetic studies implicating "activating" mutations in the Kir6.2 subunit of K ATP channel as causal in human diabetes. This article summarizes the emerging picture of K ATP channel as a major cause of neonatal diabetes and of a polymorphism in K ATP channel (E23K) as a type 2 diabetes risk factor. The degree of K ATP channel "overactivity" correlates with the severity of the diabetic phenotype. At one end of the spectrum, polymorphisms that result in a modest increase in K ATP channel activity represent a risk factor for development of late-onset diabetes. At the other end, severe "activating" mutations underlie syndromic neonatal diabetes, with multiple organ involvement and complete failure of glucose-dependent insulin secretion, reflecting K ATP channel "overactivity" in both pancreatic and extrapancreatic tissues.

Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice.

Sir2 NAD-dependent deacetylases connect transcription, metabolism, and aging. Increasing the dosage or activity of Sir2 extends life span in yeast, worms, and flies and promotes fat mobilization and glucose production in mammalian cells. Here we show that increased dosage of Sirt1, the mammalian Sir2 ortholog, in pancreatic beta cells improves glucose tolerance and enhances insulin secretion in response to glucose in beta cell-specific Sirt1-overexpressing (BESTO) transgenic mice. This phenotype is maintained as BESTO mice age. Pancreatic perfusion experiments further demonstrate that Sirt1 enhances insulin secretion in response to glucose and KCl. Microarray analyses of beta cell lines reveal that Sirt1 regulates genes involved in insulin secretion, including uncoupling protein 2 (Ucp2). Isolated BESTO islets also have reduced Ucp2, increased ATP production, and enhanced insulin secretion during glucose and KCl stimulation. These findings establish the importance of Sirt1 in beta cell function in vivo and suggest therapeutic interventions for type 2 diabetes.