Regulation of insulin secretion by phosphatidylinositol-4,5-bisphosphate.

The role of PIP(2) in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP(2) with PH-PLC-GFP or PIP5KIgamma RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIgamma improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP(2), transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP(2) co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIgamma-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIgamma, while blocking PIP(2) reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP(2) plays a pro-survival role in MIN6B1 cells, excessive PIP(2) production because of PIP5KIgamma over-expression inhibits secretion because of both a defective Arf6/PIP5KIgamma-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIgamma-dependent perturbation of F-actin cytoskeleton remodelling

Increasing GLP-1-induced beta-cell proliferation by silencing the negative regulators of signaling cAMP response element modulator-alpha and DUSP14.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) is a growth and differentiation factor for mature beta-cells and their precursors. However, the overall effect of GLP-1 on increasing beta-cell mass in both in vivo and in vitro conditions is relatively small, and augmenting this effect would be beneficial for the treatment or prevention of type 1 and type 2 diabetes. Here, we searched for cellular mechanisms that may limit the proliferative effect of GLP-1 and tested whether blocking them could increase beta-cell proliferation. RESEARCH DESIGN AND METHODS: We examined GLP-1-regulated genes in beta TC-Tet cells by cDNA microarrays. To assess the effect of some of these gene on cell proliferation, we reduced their expression using small heterogenous RNA in beta-cell lines and primary mouse islets and measured [(3)H]thymidine or 5'-bromo-2'-deoxyuridine incorporation. RESULTS: We identified four negative regulators of intracellular signaling that were rapidly and strongly activated by GLP-1: the regulator of G-protein-signaling RGS2; the cAMP response element-binding protein (CREB) antagonists cAMP response element modulator (CREM)-alpha and ICERI; and the dual specificity phosphatase DUSP14, a negative regulator of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We show that knockdown of CREMalpha or DUSP14 or expression of a dominant-negative form of DUSP14 increased beta-cell line proliferation and enhanced the GLP-1-induced proliferation of primary beta-cells. CONCLUSIONS: Together, our data show that 1) the cAMP/protein kinase A/CREB and MAPK/ERK1/2 pathways can additively control beta-cell proliferation, 2) beta-cells have evolved several mechanisms limiting GLP-1-induced cellular proliferation, and 3) blocking these mechanisms increases the positive effect of GLP-1 on beta-cell mass

Overexpression of regenerative, antioxidant and inflammatory genes in adult diabetic GK islets

National audienceIn the adult GK rat, a spontaneous model of type 2 diabetes, total pancreatic β-cell number is decreased by 60%. This alteration cannot be ascribed to increased β-cell apoptosis but is related to decreased β-cell replication. Moreover, the adult GK pancreas exhibits large islets disrupted by connective tissue. Our objective was therefore to identify genes possibly involved in the β-cell growth phenotype in adult diabetic GK rat. Differential gene expression was evaluated in islets of adult diabetic GK and normal Wistar (W) rats by high density oligonucleotide microarray. Total RNA was extracted from 16 week-old W and GK islets. Biotin-labeled cRNA probes were synthesized and hybridized to Affymetrix RG-U34A oligonucleotide microarrays containing approximately 7,000 rat genes. The arrays were scanned and expression values for the genes were determined using Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 2.0. Expression pattern of reg in GK pancreas was determined by immunohistochemical analysis. No difference in expression levels of genes encoding growth factors for β-cells (GH, IGF1, PDGF, HGF, insulin) and the c-Myc transcription factor was found in Wistar and GK rats. By contrast, several reg-related genes were up-regulated in the GK islets (46-, 12- and 11-fold respectively for reg II, reg I and reg III genes). These results were confirmed by real-time PCR. Expression of several stress genes such as glutathione peroxidase (2.7-fold) , thioredoxine interating protein (5.4-fold) and heat shock protein 70 (2.3-fold) were increased in GK islets. Islets from GK rats also display increased expression of inflammatory genes such as lipocalin 2 (72.8-fold), decorin (4.1-fold) and annexins 1 and 2 genes (8.7- and 2.1-fold). To localize the reg-I protein in pancreas we stained sections of GK and W pancreases. Immunoreactivity was observed only in islets. Reg staining was colocalized with staining for insulin but did not seem restricted to the β-cells.Our data suggest that in GK islets 1) the increased expression of reg family genes whose involvement in β-cell regeneration has been proposed, may be an integral part of the β-cell growth phenotype of GK islets; 2) the lack of increased apoptosis of the β-cells may be related to activation of several stress genes that confer protection against β-cell death; 3) the increased expression of cytokine genes could be related to a local inflammatory response in GK islets; 4) the local inflammatory gene overexpression together with the cytokine environment in GK islets may have an influence on reg gene expression. Our working hypothesis is, that these expression changes reported for the first time in GK islets could represent an acquired adaptation in response to chronic hyperglycemia (glucotoxicity). To learn what genes are more directly involved in the pathology of the GK rats, we are currently analyzing the gene expression profile in young prediabetic GK rat

Mutant Proinsulin That Cannot Be Converted Is Secreted Efficiently from Primary Rat β-Cells via the Regulated Pathway

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called “constitutive-like” pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin

Circulating follistatin is liver-derived and regulated by the glucagon-to-insulin ratio.

Context: Follistatin is a plasma protein recently reported to increase under conditions with negative energy balance, such as exercise and fasting in humans. Currently, the perception is that circulating follistatin is a result of para/autocrine actions from various tissues. The large and acute increase in circulating follistatin in response to exercise suggests that it may function as an endocrine signal. Objective: We assessed origin and regulation of circulating follistatin in humans. Design/Interventions: First, we assessed arterial-to-venous difference of follistatin over the splanchnic bed at rest and during exercise in healthy humans. To evaluate the regulation of plasma follistatin we manipulated glucagon-to-insulin ratio in humans at rest as well as in cultured hepatocytes. Finally, the impact of follistatin on human islets of Langerhans was assessed. Results: We demonstrate that in humans the liver is a major contributor to circulating follistatin both at rest and during exercise. Glucagon increases and insulin inhibits follistatin secretion both in vivo and in vitro, mediated via the secondary messenger cAMP in the hepatocyte. Short-term follistatin treatment reduced glucagon secretion from islets of Langerhans, whereas long-term follistatin treatment prevented apoptosis and induced proliferation of rat beta cells. Conclusions: In conclusion, in humans, the liver secretes follistatin at rest and during exercise, and the glucagon-to-insulin ratio is a key determinant of circulating follistatin levels. Circulating follistatin may be a marker of the glucagon-to-insulin tone on the liver

Hypercholesterolaemia, signs of islet microangiopathy and altered angiogenesis precede onset of type 2 diabetes in the Goto-Kakizaki (GK) rat

International audienceThe adult non-obese Goto–Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals. Blood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates). Systemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms. These data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass