Activin B receptor ALK7 is a negative regulator of pancreatic beta-cell function.
ABSTRACT: All major cell types in pancreatic islets express the transforming growth factor (TGF)-beta superfamily receptor ALK7, but the physiological function of this receptor has been unknown. Mutant mice lacking ALK7 showed normal pancreas organogenesis but developed an age-dependent syndrome involving progressive hyperinsulinemia, reduced insulin sensitivity, liver steatosis, impaired glucose tolerance, and islet enlargement. Hyperinsulinemia preceded the development of any other defect, indicating that this may be one primary consequence of the lack of ALK7. In agreement with this, mutant islets showed enhanced insulin secretion under sustained glucose stimulation, indicating that ALK7 negatively regulates glucose-stimulated insulin release in beta-cells. Glucose increased expression of ALK7 and its ligand activin B in islets, but decreased that of activin A, which does not signal through ALK7. The two activins had opposite effects on Ca(2+) signaling in islet cells, with activin A increasing, but activin B decreasing, glucose-stimulated Ca(2+) influx. On its own, activin B had no effect on WT cells, but stimulated Ca(2+) influx in cells lacking ALK7. In accordance with this, mutant mice lacking activin B showed hyperinsulinemia comparable with that of Alk7(-/-) mice, but double mutants showed no additive effects, suggesting that ALK7 and activin B function in a common pathway to regulate insulin secretion. These findings uncover an unexpected antagonism between activins A and B in the control of Ca(2+) signaling in beta-cells. We propose that ALK7 plays an important role in regulating the functional plasticity of pancreatic islets, negatively affecting beta-cell function by mediating the effects of activin B on Ca(2+) signaling.
Project description:Activins belong to the transforming growth factor (TGF)-? family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2, causing activation of the SMAD1/5/8 pathway. In this study, we aimed to dissect how activin A and activin B homodimers, and activin AB and AC heterodimers activate the two main SMAD branches. We compared the activin-induced signaling dynamics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via the ALK2-SMAD1/5 pathway exhibited greater differences between ligands than signaling via ALK4/ALK7-SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. As SMAD1/5 was also activated by activins in other cell types, we propose that dual specificity is a general mechanism for activin ligands. In addition, we found that the antagonist follistatin inhibited signaling by all the tested activins, whereas the antagonist cerberus specifically inhibited activin B. Taken together, we propose that activins may be considered dual specificity TGF-? family members, critically affecting how activins may be considered and targeted clinically.
Project description:Hyperandrogenism is associated with hyperinsulinemia and insulin resistance in adult females. We tested whether androgens dysregulate pancreatic beta cell function to induce hyperinsulinemia through transcriptional regulation of insulin gene (Ins) in the islets. Adult female Wistar rats implanted with dihydrotestosterone (DHT; 7.5-mg, 90-d release) or placebo pellets were examined after 10 weeks. DHT exposure increased plasma DHT levels by 2-fold similar to that in polycystic ovary syndrome in women. DHT exposure induced hyperinsulinemia with increased HOMA-IR index in fasting state and glucose intolerance and exaggerated insulin responses following glucose tolerance test. DHT females had no change in islet number, size and beta cell proliferation/apoptosis but exhibited significant mitochondrial dysfunction (higher ADP/ATP ratio, decreased mtDNA copy number, increased reactive oxygen production and downregulation of mitochondrial biogenesis) and enhanced glucose-stimulated insulin secretion. Ins expression was increased in DHT islets. In vitro incubation of control islets with DHT dose dependently stimulated Ins transcription. Analysis of Ins1 gene revealed a putative androgen responsive element in the promoter. Chromatin-immunoprecipitation assays showed that androgen receptors bind to this element in response to DHT stimulation. Furthermore, reporter assays showed that the promoter element is highly responsive to androgens. Insulin-stimulated glucose uptake in skeletal muscle was decreased with associated decrease in IRβ expression in DHT females. Our studies identified a novel androgen-mediated mechanism for the control of Ins expression via transcriptional regulation providing a molecular mechanism linking elevated androgens and hyperinsulemia. Decreased IRβ expression in the skeletal muscles may contribute, in part, to glucose intolerance in this model.
Project description:Islet beta-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from rodents in vivo and from islets isolated from rodents or humans suggests that the insulin signaling pathway is physiologically important for glucose sensing. We evaluated whether insulin regulates beta-cell function in healthy humans in vivo. Glucose-induced insulin secretion was assessed in healthy humans following 4-h saline (low insulin/sham clamp) or isoglycemic-hyperinsulinemic (high insulin) clamps using B28-Asp insulin that could be immunologically distinguished from endogenous insulin. Insulin and C-peptide clearance were evaluated to understand the impact of hyperinsulinemia on estimates of beta-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by approximately 40%. C-peptide response also increased, although not to the level predicted by insulin. Insulin clearance was not saturated at hyperinsulinemia, but metabolic clearance of C-peptide, assessed by infusion of stable isotope-labeled C-peptide, increased modestly during hyperinsulinemic clamp. These studies demonstrate that insulin potentiates glucose-stimulated insulin secretion in vivo in healthy humans. In addition, hyperinsulinemia increases C-peptide clearance, which may lead to modest underestimation of beta-cell secretory response when using these methods during prolonged dynamic testing.
Project description:Pancreatic islet beta-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate beta-cell function is of immense clinical relevance. Transforming growth factor (TGF)-beta signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-beta pathway in regulation of insulin gene transcription and beta-cell function. We identify insulin as a TGF-beta target gene and show that the TGF-beta signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucose-stimulated insulin release. Interestingly, Smad3-deficient islets harbor an activated insulin-receptor signaling pathway and TGF-beta signaling regulates expression of genes involved in beta-cell function. Together, these studies emphasize TGF-beta/Smad3 signaling as an important regulator of insulin gene transcription and beta-cell function and suggest that components of the TGF-beta signaling pathway may be dysregulated in diabetes.
Project description:The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.
Project description:AIMS/HYPOTHESIS: Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. METHODS: MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, ?-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca(2+) concentration ([Ca(2+)]c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. RESULTS: Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca(2+)]c response. CONCLUSIONS/INTERPRETATION: We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca(2+)]c.
Project description:Human adipose tissue is a major site of expression of inhibin beta B (INHBB) which homodimerizes to form the novel adipokine activin B. Our aim was to determine if molecules needed for a local action of activin B are expressed in adipose tissue. Microarray analysis showed that adipose tissue expressed activin type I and II receptors and that the expression of activin receptor-like kinase 7 (ALK7) was adipose tissue specific. In obesity discordant siblings from the SOS Sib Pair study, adipose tissue ALK7 expression was higher in lean (n=90) compared to obese (n=90) subjects (p=4 x 10(-31)). Adipose tissue ALK7 expression correlated with several measures of body fat, carbohydrate metabolism and lipids. In addition, ALK7 and INHBB expression correlated but only in lean subjects and in subjects with normal glucose tolerance. We conclude that activin B may have local effects in adipose tissue and thereby influence obesity and its comorbidities.
Project description:Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca(2+)-handling in pancreatic ? cells. Insulin secretion is triggered by elevation in cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) of ? cells. This elevation in [Ca(2+)]cyt leads to activation of Ca(2+)/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca(2+)-release channel implicated in Ca(2+)-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic ?-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in ? cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca(2+) leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca(2+)]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca(2+) pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.
Project description:The cannabinoid 1 receptor (CB1) regulates insulin sensitivity and glucose metabolism in peripheral tissues. CB1 is expressed on pancreatic beta (β)-cells where its functions have not been fully described. We generated a β-cell-specific CB1-knockout (β-CB1-/-) mouse to study the long-term consequences of CB1 ablation on β-cell function in adult mice. β-CB1-/- mice had increased basal- and stimulated-insulin secretion and intra-islet cAMP levels, resulting in primary hyperinsulinemia, as well as increased β-cell viability, proliferation, and islet area. Hyperinsulinemia led to insulin resistance, which was aggravated by a high fat/high glucose diet and weight gain, although β-cells maintained their insulin secretory capacity in response to glucose. Strikingly, islets from β-CB1-/- mice were protected from diet-induced inflammation. Mechanistically we show that this is a consequence of curtailment of oxidative stress and reduced activation of Nlrp3 inflammasome in β-cells. Our data demonstrate CB1 to be a negative regulator of β-cells and a mediator of islet inflammation under conditions of metabolic stress. Overall design: Gene expression profiles from islets from wildtype (β-CB1+/+) (N=4) and a β-cell-specific CB1-knockout (β-CB1-/-) mice (N=3) were examined using gene expression microarray analysis.
Project description:AIMS/HYPOTHESIS:Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. METHODS:We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. RESULTS:In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. CONCLUSIONS/INTERPRETATION:The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.