[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 07/2012; 7(7):e40604. DOI:10.1371/journal.pone.0040604 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Balance was assessed via the mini-BESTest in 13 children, adolescents and young adults with WFS and 30 typically developing age-matched individuals.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Wolfram syndrome (WS), or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), is a rare autosomal recessive neurodegenerative disorder with a median life expectancy of 30 years and occurs in one in 770,000 live births. To date only five successful pregnancies have been reported among WS subjects worldwide. Here we describe the sixth report of successful pregnancy in a WS patient and the first from India. The subject is still on an insulin pump, now 31 years old and doing well. She developed diabetes at 5 years of age, optic atrophy at 14 years, and diabetes insipidus at 25 years and had a successful delivery in 2007 while on an insulin pump. Sequencing of exonic regions of the WFS1 gene showed five changes, two of which were pathogenic (exon 8). Magnetic resonance imaging of brain showed generalized neurodegenerative changes. The benefits of continuous subcutaneous insulin infusion and that of tight metabolic control in prevention of abortions and fetal malformations in diabetes associated with pregnancy are well documented. The impression of probable pleiotropic action of insulin pumps over and above that of glycemic reduction is gaining momentum. Recent evidence supports use of insulin pumps in alleviating neuropathic pain in diabetes, probably by virtue of its action in minimizing mean amplitude of glycemic excursions not possible with conventional insulin shots. WS is a progressive neurodegenerative disorder, which will probably help us in understanding the positive impact of continuous subcutaneous insulin infusion in prolonging the life span and retarding neuronal damage in WS.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
PLoS ONE 04/2011; 6(4):e18146. DOI:10.1371/journal.pone.0018146 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
PLoS ONE 08/2010; 5(8):e11969. DOI:10.1371/journal.pone.0011969 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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<or=0.0001). Although the univariate association between the TCF7L2 SNP and T2D was relatively modest [P = 0.02], when paired with the HNF4A SNP, the OR for subjects with risk alleles in both SNPs was 2.4 [95% CI = 1.7-3.4; P<or=0.0001]. The KCNJ11 variant reached significance only when paired with either the HNF4A or WFSI SNPs: unadjusted ORs were 2.0 [95% CI = 1.4-2.8; P<or=0.0001] and 2.3 [95% CI = 1.2-4.4; P<or=0.0001], respectively. MDR and GMDR results were consistent with the parametric findings.
These results provide evidence of strong independent associations between T2D and SNPs in HNF4A and WFS1 and their interaction in our Ashkenazi sample. We also observed an interaction in the nonparametric analysis between the HNF4A and KCNJ11 SNPs (P<or=0.001), demonstrating that an independently non-significant variant may interact with another variant resulting in an increased disease risk.
PLoS ONE 03/2010; 5(3):e9903. DOI:10.1371/journal.pone.0009903 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of clinical investigation 02/2010; 120(3):744-55. DOI:10.1172/JCI39678 · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endoplasmic reticulum (ER) stress-mediated cell death has an important role in the pathogenesis of chronic diseases, including diabetes and neurodegeneration. Although proapoptotic programs activated by ER stress have been extensively studied, identification and characterization of antiapoptotic programs that counteract ER stress are currently incomplete. Through the gene expression profiling of beta-cells lacking Wolfram syndrome 1 gene (WFS1), a causative gene for Wolfram syndrome, we discovered a novel antiapoptotic gene of the unfolded protein response (UPR), apoptosis antagonizing transcription factor (AATF). Here, we study the regulation of AATF, identify its target genes, and determine the basis for its antiapoptotic activities in response to ER stress. We show that AATF is induced by ER stress through the PERK-eIF2alpha pathway and transcriptionally activates the v-akt murine thymoma viral oncogene homolog 1 (AKT1) gene through signal transducer and activator of transcription 3 (Stat3), which sustains Akt1 activation and promotes cell survival. Ectopic expression of AATF or a constitutively active form of AKT1 confers on cells resistance to ER stress-mediated cell death, whereas RNAi-mediated knockdown of AATF or AKT1 renders cells sensitive to ER stress. We also discovered a positive crosstalk between the AATF and WFS1 signaling pathways. Thus, WFS1 deficiency or AATF deficiency mediates a self-perpetuating cycle of cell death. Our results reveal a novel antiapoptotic program relevant to the treatment of diseases caused by ER stress-mediated cell death.
Cell death and differentiation 11/2009; 17(5):774-86. DOI:10.1038/cdd.2009.175 · 8.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Genes & development 10/2009; 23(17):2088-101. DOI:10.1101/gad.1800209 · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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]).
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.
[Show abstract][Hide abstract] ABSTRACT: Recently, variants in WFS1 have been shown to be associated with type 2 diabetes. We aimed to examine metabolic risk phenotypes of WFS1 variants in glucose-tolerant people and in individuals with abnormal glucose regulation.
The type 2 diabetes-associated WFS1 variant rs734312 (His611Arg) was studied in the population-based Inter99 cohort involving 4,568 glucose-tolerant individuals and 1,471 individuals with treatment-naive abnormal glucose regulation, and in an additional 3,733 treated type 2 diabetes patients.
The WFS1 rs734312 showed a borderline significant association with type 2 diabetes with directions and relative risks consistent with previous reports. In individuals with abnormal glucose regulation, the diabetogenic risk A allele of rs734312 was associated in an allele-dependent manner with a decrease in insulinogenic index (p = 0.025) and decreased 30-min serum insulin levels (p = 0.047) after an oral glucose load. In glucose-tolerant individuals the same allele was associated with increased fasting serum insulin concentration (p = 0.019) and homeostasis model assessment of insulin resistance (HOMA-IR; p = 0.026). To study the complex interaction of WFS1 rs734312 on insulin release and insulin resistance we introduced Hotelling's T (2) test. Assuming bivariate normal distribution, we constructed standard error ellipses of the insulinogenic index and HOMA-IR when stratified according to glucose tolerance status around the means of each WFS1 rs734312 genotype level. The interaction term between individuals with normal glucose tolerance and abnormal glucose regulation on the insulinogenic index and HOMA-IR was significantly associated with the traits (p = 0.0017).
Type 2 diabetes-associated risk alleles of WFS1 are associated with estimates of a decreased pancreatic beta cell function among middle-aged individuals with abnormal glucose regulation.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Molecular Genetics 07/2008; 17(11):1695-704. DOI:10.1093/hmg/ddn060 · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: 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 (p<0.05) 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.
[Show abstract][Hide abstract] ABSTRACT: 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.