Search

Due to the shortage of multi-organ donors, human pancreatic islet transplantation has now been extended to islets originating from obese subjects. In this study, our aim is to compare the respective sensitivity of human islets from lean vs obese donors to chronic high glucose or high palmitate.Human islets were isolated from pancreases harvested from brain-dead multi-organ donors. Islets were cultured during 72 hours in the presence of moderate (16.7 mmol/L) or high (28 mmoL/L) glucose concentrations, or glucose (5.6 mmoL/L) and palmitate (0.4 mmoL/L), before measurement of their response to glucose.We first observed a greater insulin response in islets from obese donors under both basal and high-glucose conditions, confirming their hyperresponsiveness to glucose. When islets from obese donors were cultured in the presence of moderate or high glucose concentrations, insulin response to glucose remained unchanged or was slightly reduced, as opposed to that observed in lean subjects. Moreover, culturing islets from obese donors with high palmitate also induced less reduction in insulin response to glucose than in lean subjects. This partial protection of obese islets is associated with less induction of inducible nitric oxide synthase in islets, together with a greater expression of the transcription factor forkhead box O1 (FOXO1).Our data suggest that in addition to an increased sensitivity to glucose, islets from obese subjects can be considered as more resistant to glucose and fatty acid excursions and are thus valuable candidates for transplantation.背景: 由于缺乏多器官供体, 人胰岛移植目前已衍生到肥胖者来源的胰岛移植。在这项研究中, 我们旨在比较瘦人和肥胖者的胰岛对慢性高糖或棕榈酸盐的敏感性。 方法: 从脑死亡的多器官捐赠者的胰腺中分离胰岛。胰岛在中等浓度(16.7 mmol/L)或高浓度(28 mmol/L)葡萄糖培养基中, 或是葡萄糖(5.6 mmol/L)和棕榈酸盐(0.4 mmol/L)存在下培养72h, 然后测定其对葡萄糖的反应。 结果: 我们首先观察到肥胖捐赠者的胰岛在基础和高糖条件下都有更大的胰岛素反应, 证实了其对葡萄糖的高反应性。但当来自肥胖捐赠者的胰岛在中高浓度葡萄糖存在的情况下继续培养时, 胰岛对葡萄糖的反应则保持不变或略有降低, 这与在瘦的捐赠者中观察到的相反。此外, 用高棕榈酸培养后的肥胖捐赠者胰岛也比瘦捐献者的对于葡萄糖的胰岛素反应减少得少。这种对肥胖者胰岛的部分保护作用, 与较少诱导一氧化氮合成酶有关, 以及转录因子FOXO1的较高表达相关。 结论: 我们的结果表明, 除了对葡萄糖的敏感性增加外, 肥胖者的胰岛可以被认为对葡萄糖和脂肪酸剧增更有抵抗力, 因此是有价值的移植候选者。.

International audience; More than 160 arginine analogues modified on the C‐terminus via either an amide bond or a heterocyclic moiety (1,2,4‐oxadiazole, 1,3,4‐oxadiazole and 1,2,4‐triazole) were prepared as potential inhibitors of NO synthases (NOS). A methodology involving formation of a thiocitrulline intermediate linked through its side‐chain on a solid support followed by modification of its carboxylate group was developed. Finally, the side‐chain thiourea group was either let unchanged, S‐alkylated (Me, Et) or guanidinylated (Me, Et) to yield respectively after TFA treatment the corresponding thiocitrulline, S‐Me/Et‐isothiocitrulline and N‐Me/Et‐arginine substrate analogues. They all were tested against three recombinant NOS isoforms. Several compounds containing a S‐Et‐ or a S‐Me‐Itc moiety and mainly belonging to both the dipeptide‐like and 1,2,4‐oxadiazole series were shown to inhibit nNOS and iNOS with IC50 in the 1–50 μM range. Spectral studies confirmed that these new compounds interacted at the heme active site. The more active compounds were found to inhibit intra‐cellular iNOS expressed in RAW264.7 and INS‐1 cells with similar efficiency than the reference compounds L‐NIL and SEIT.

Chronic low-grade inflammation and oxidative stress associated with obesity induce molecular changes in extracellular matrix connected to relaxin pathway and fibrosis, the lipidome of various tissues and the level of lipid mediators. Increase of desaturase enzymatic activity and activation of the inflammatory mediator cascades are known to be associated with obesity. Decrease of enzymatic antioxidant defenses is strictly involved. The effects of a 10 days supplementation of encapsulated melon juice concentrate rich in superoxide dismutase were studied in Zucker Fat rats compared with Zucker Lean rats, following relaxin pathways, lipid mediators and fatty acid-based lipidomic analysis in adipose tissue and erythrocytes. Significant ameliorations in obese rats concerned the restored relaxin levels, connected with fibrosis reduction, the reduction of inflammatory mediators and a favorable fatty acid remodeling, with the decrease of unsaturated fatty acids in the lipidome of adipose tissue and erythrocytes becoming closer to the lipidome of normal rats.

International audience; In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (b 0.01% NaCl) for 8 weeks. After 4 weeks, rats were infused or not with angiotensin II (200 ng·kg −1 ·min −1 , sc) for 4 weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry. Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hyper-trophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet. Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling.

National audience; Purinergic P2Y-receptor agonists amplify glucose-induced insulin secretion from pancreatic beta-cells, thus offering new opportunities for the treatment of type 2 diabetes. However, little is known about which subtypes of purinergic P2Y receptors are expressed in these cells. The INS-1 beta-cell line is used as a model of pancreatic beta-cells, expressing most of their properties. Therefore, we investigated the expression of different molecular subtypes in this cell line by means of real time Polymerase Chain Reaction and Western blot. We also performed a characterization of the binding of a prototypic purinergic P2Y agonist, Adenosine-5'-O-(1-[(35)S]thiotriphosphate) (ATP-alpha-[(35)S]), to cell membrane homogenates. The molecular analysis evidenced the presence of five different purinergic P2Y receptor subtypes (P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12)), which were expressed at similar levels. The Western blot analysis allowed detecting corresponding proteins. The binding assay demonstrated a specific ATP-alpha-[(35)S] interaction on high (40%) and low (60%) affinity components. The analysis of ATP-alpha-[(35)S] pharmacological profile on both sites permitted to classify the high affinity binding site as representative of the purinergic P2Y(1) receptor subtype and the low affinity binding site of the P2Y(4) and/or P2Y(6) receptor subtypes. ATP-alpha-S and Adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) exhibited opposite selectivity on high and low affinity binding sites.Although purinergic P2Y(1) receptor, or a P2Y(1)-like subtype, has been generally considered as that implicated in the modulation of glucose-induced insulin release, the present data show that the beta-cell expresses a complex profile of purinergic P2Y receptor subtypes, the functional implication of which remains to be fully elucidated.

We previously showed that pancreatic β-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting β-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nω-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery.

By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.

Adenine nucleotides stimulate insulin secretion by binding to P2 receptors of the pancreatic beta-cells; the stimulus-secretion coupling is not yet clearly established and may depend on the receptor subtype. The aim of the present study was to further investigate the mechanism whereby P2Y receptor agonists enhance glucose-induced insulin secretion. Experiments were performed in rat pancreatic islets and in the INS-1 secreting cell line in the presence of a slightly stimulating glucose concentration (8.3 mmol/l). In isolated islets, the P2Y receptor agonist ADPbetaS (50 micromol/l) induced a significant fivefold increase in the cyclic AMP (cAMP) content, from 43.4+/-3.7 fmol/10 islets in controls to 210.6+/-12.0; it still induced a 4.5-fold increase in cAMP content in the absence of calcium. In another series of experiments, ADPbetaS (50 micromol/l) significantly increased glucose-induced insulin secretion from 7.7+/-0.6 ng/3 islets in controls to 11.2+/-1.0. The adenylyl cyclase inhibitor SQ 22,536 (9-[tetrahydro-2-furanyl]-9 H-purin-6-amine; 100 micromol/l), which was ineffective alone, completely prevented the stimulating effect of ADPbetaS. In a set of experiments in which ADPbetaS increased glucose-induced insulin secretion from 10.0+/-0.7 ng/3 islets to 12.6+/-0.8, the inhibitor of cAMP-dependent protein kinase, TPCK (tos-phe-chloromethylketone; 3 micromol/l), which was ineffective alone, also prevented the stimulating effect of ADPbetaS. In incubated INS-1 cells, the P2Y receptor ligand ATPalphaS increased significantly both the content of cAMP and the release of insulin, in a concentration-dependent manner in the range of 50-150 micromol/l; the insulin release was significantly correlated with the cAMP content. In conclusion, the present results show that P2Y receptor agonists, ADPbetaS and ATPalphaS, amplify glucose-induced insulin secretion by activating beta-cell adenylyl cyclase and the subsequent cAMP/protein kinase A signaling pathway.

Miniglucagon, the COOH-terminal (19-29) fragment processed from glucagon, is a potent and efficient inhibitor of insulin secretion from the MIN 6 β-cell line. Using the rat isolated-perfused pancreas, we investigated the inhibitory effect of miniglucagon on insulin secretion and evaluated the existence of an inhibitory tone exerted by this peptide inside the islet. Miniglucagon dose-dependently inhibited insulin secretion stimulated by 8.3 mol/l glucose, with no change in the perfusion flow rate. A concentration of 1 nmol/l miniglucagon had a significant inhibitory effect on a 1 nmol/l glucagon-like peptide 1 (7-36) amide-potentiated insulin secretion. A decrease in extracellular glucose concentration simultaneously stimulated glucagon and miniglucagon secretion from pancreatic α-cells. Using confocal and electron microscopy analysis, we observed that miniglucagon is colocalized with glucagon in mature secretory granules of α-cells. Perfusion of an anti-miniglucagon antiserum directed against the biologically active moiety of the peptide resulted in a more pronounced effect of a glucose challenge on insulin secretion, indicating that miniglucagon exerts a local inhibitory tone on β-cells. We concluded that miniglucagon is a novel local regulator of the pancreatic islet physiology and that any abnormal inhibitory tone exerted by this peptide on the β-cell would result in an impaired insulin secretion, as observed in type 2 diabetes.

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting beta-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the beta-cell response to arginine and glucose after nNOS blockade with N(G)-nitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that beta-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal beta-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in beta-cell dysregulation involved in certain pathological hyperinsulinic states.

International audience; Hyperglycemia is a well-known inducing factor of oxidative stress through activation of NADPH oxidase. In addition to its plasma glucose lowering effect, insulin may also have antioxidant activity and was shown to downregulate NADPH oxidase expression in vitro. In this study, we show that a short-term (3-day) intravenous insulin infusion in patients with type 2 diabetes induces normalization of both glycemia and mRNA expression of circulating monocyte p47(phox) subunit.

The salivary glands of mammals produce an arrayof biologically active peptides that are releasedinto saliva upon stimulation of glandular exocytosis(1). While the most widely recognized ones aregrowth factors like epidermal growth factor (EGF)and nerve growth factor (NGF), saliva has alsobeen shown to contain peptides such as IGF-Iand -II (2).Insulin has been isolated from a variety ofvertebrate species (3). In all instances, the moleculeis composed of two polypeptide chains (A and B)joined by two disulphide linkages. In most speciesexamined, there appeared to be a unique insulinand a unique gene. However, in three species ofrodents (rat, mouse, and piny mouse) and twospecies of fish (tuna and toadfish) two distinctinsulins have been characterized (4, 5).Moreover, unlike in most vertebrate specieswhere only one ‘ancestral gene’ containing twointrons has been identified, two preproinsulingenes (I and II) have been found in rats (6),di•ering by the absence of the second interferingsequence in preproinsulin I (7). The two non-allelicpreproinsulin genes, sharing a 93% homology intheir coding sequences, are expressed to a similarextent in rat pancreatic b cells (8) but not in extra-pancreatic tissues, where only the ancestral gene isexpressed (9). Our previous observation of insulin-likeimmunoreactivityinratsubmandibularsalivaryglands (SSG) that appeared biologically active(10) prompted us to investigate the possibleexpression of insulin genes in SSG. Our study wassupplemented by analyses performed on the SCA-9mouse immortalized submandibular cell line.Material and methods

Ethnopharmacological relevance In Eurasia folk medicine, roots of chicory ( Cichorium intybus L.) have been reported to exert antidiabetic benefits. In vitro , a natural chicoric acid extract (NCRAE) from Cichorium intybus root has been shown to increase insulin secretion by pancreatic β-cells and glucose uptake by muscle cells. Materials and methods In vitro experiments were designed to compare the effects of two hydroxycinnamic acids, caffeic and ferulic acids, to those obtained with NCRAE (50 and 100 µg.mL −1 ) on the three major tissues implicated in glycemic regulation (pancreas, muscle and liver). In vivo experiments were performed in Wistar rats submitted to a daily intraperitoneal injection of NCRAE (3, 15 or 30 mg kg −1 ) for 4 days. On the fourth day, an intraperitoneal glucose tolerance test (IPGTT; 1 g kg −1 ) was carried out. Results Our results show that the three compounds we used are able each to induce an original response. Caffeic acid mainly promotes a decrease in hepatic glycogenolysis. Ferulic acid elicits a clear increase of insulin release and a reduction of hepatic glycogenolysis. However, this compound induces an inhibition of muscle glucose uptake. NCRAE provokes an increase of insulin release and glucose uptake without any effect on hepatic glycogenolysis. We could also show that none of these compounds implicates hepatic glucose 6-phosphatase in contrast to chlorogenic acid, known as an inhibitor of glucose 6-phosphatase and which is able to decrease glucose output from hepatocytes. Our results point out that NCRAE is able to decrease blood glucose without any effect hepatic effect. Our in vivo experiments bring evidence that 4 daily IP administrations of NCRAE improve IP glucose tolerance in a dose-dependent manner and mainly via an insulin sensitizing effect. Conclusions We conclude that NCRAE presents an antihyperglycemic effect essentially due to a peripheral effect on muscle glucose uptake.

Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect.Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays.nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes.Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.

International audience; Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.

International audience; Background and Aims. Increasing environmental pollution may participate in the growing incidence of metabolic disorders. Static magnetic fields (SMFs) are an emerging environmental health issue due to increased exposure in residential and commercial areas; however, their metabolic effects in serum and skeletal muscle are largely unknown. The aim of this study was to investigate the effect of SMF exposure on glucose and lipid metabolism in serum and skeletal muscles of rats.Methods. Twelve 6-to 7-week-old male Wistar rats were randomly divided into two groups: rats exposed to 128 mT SMF and sham-exposed rats. This moderate-intensity exposure was performed for 1 h/day for 15 consecutive days.Results. Animals exposed to 128 mT SMF displayed significant changes in both glucose (i.e., increases in plasma glucose and lactate and decrease in plasma insulin levels) and lipid (i.e., increases in plasma glycerol, cholesterol and phospholipids but not triglyceride levels) metabolism. During intraperitoneal glucose tolerance tests, SMF-exposed rats displayed significantly higher hyperglycemia compared to sham-exposed rats despite similar insulin levels in both groups. In tissues, SMF exposure induced significant alterations in enzyme activities only in glycolytic muscles and caused a significant decrease in quadriceps and liver glycogen content together with increased phospholipid levels. Conclusions. This study provides evidence that subacute SMF exposure of moderate intensity induces important alterations of glucose and lipid metabolisms, which deserve further investigations to evaluate long-term consequences. Ó

International audience; Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.

International audience; Caffeic acid and chlorogenic acid (CGA), a mono-caffeoyl ester, have been described as potential antidiabetic agents. Using in vitro studies, we report the effects of a dicaffeoyl ester, chicoric acid (CRA) purified from Cichorium intybus, on glucose uptake and insulin secretion. Our results show that CRA and CGA increased glucose uptake in L6 muscular cells, an effect only observed in the presence of stimulating concentrations of insulin. Moreover, we found that both CRA and CGA were able to stimulate insulin secretion from the INS-1E insulin-secreting cell line and rat islets of Langerhans. In the later case, the effect of CRA is only observed in the presence of subnormal glucose levels. Patch clamps studies show that the mechanism of CRA and CGA was different from that of sulfonylureas, as they did not close K(ATP) channels. Chicoric acid is a new potential antidiabetic agent carrying both insulin sensitizing and insulin-secreting properties.

International audience; Extracellular adenosine triphosphate is able to modulate pancreatic beta-cell function, acting on P2 purinergic ionotropic (P2X) and metabotropic (P2Y) receptors. Physiologically, ATP entrains beta-cells into a common rhythm by coordinating Ca(2+) oscillations; it plays a central role in insulin secretion pulsatility. ATP also triggers a positive feedback signal amplifying glucose-induced insulin release, which argues for a potential pharmacological application. ATP has consistently been shown to increase cytoplasmic free calcium concentration, notably in human tissue. Acting on P2X receptors, of which different molecular subtypes are expressed in beta-cells, it leads to a transient insulin release that may involve a closure of K(ATP) channels or a rapidly decaying inward current. Activation of G-protein-coupled P2Y receptors triggers different signalling pathways and amplifies insulin release in a glucose-dependent way. It has recently been shown that pancreatic beta-cells express different molecular subtypes of receptors, which may explain the complex interaction of P2Y ligands on high- and low-affinity binding sites. Despite the complexity of this purinergic pharmacology, consistent pre-clinical data suggest the potential of P2Y receptor agonists as drug candidates for type 2 diabetes.

Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.

Cellulose membrane supported peptide arrays, prepared according to the Spot method, allow the rapid identification and characterization of protein-protein interaction sites. Here, the method was used to screen reactive peptides from different proteins that bind to a single molecule, the PIN protein. PIN possesses two binding grooves, that have been shown to interact with several targets, including neuronal NO synthase, dynein intermediate chain, myosin V, the proapoptotic protein Bim, the scaffolding proteins DAP1alpha and gephyrin, and the transcription factor NRF-1. Arrays of peptides representing sequences of these targets were probed for reactivity with GST-tagged PIN, enabling the precise identification of binding motifs. Binding motifs were then minimized to seven or eight amino acid long peptides: YSKETQT for dynein IC, CDKSTQT for Bim, KDTGIQVD for nNOS, QSVGVQV for DAP1alpha and EDKNTMTD for myosin V. Alascan and substitution analysis provided proof that the Gln residue is critical for the interaction and cannot be easily replaced. Positions -1 and +1, just flanking the pivotal Gln, are also important; they consist of hydrophobic residues (Thr, Val) that could only be replaced by hydrophobic or aromatic amino acids. Position -4 is also critical for binding, with its Asp or Ser being replaceable to some extent. Alignment of sequences of proteins known to bind PIN shows that the most frequent amino acids in the motif are DKGTQT, consistent with the Spot results. We postulate that the degenerate character of binding to PIN is based on the propensity of several sequences to adopt a beta-strand conformation that allows the Gln residue to position itself in the PIN channel and on the conformational breathing of the PIN binding groove.

International audience; We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1β takes part and controls β-cell function. In the present study, using the INS-1 rat pancreatic β-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1β signaling and biological effects on insulin secretion, β-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1β leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic β-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for β-cell secretory function. In the same conditions, IL-1β treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1β inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1β concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1β anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1β deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.

We provide immunocytochemical evidence that the neuronal isoform of constitutive NO synthase (cNOS) is expressed in the rat insulinoma cell line INS-1. Furthermore, using Nω-nitro- l -arginine methyl ester ( l -NAME), a pharmacological inhibitor of cNOS activity, we show that this enzyme is implicated in the modulation of insulin secretion in INS-1 cells. Indeed, in the presence of 2.8 mM glucose, l -NAME induced a specific and dose-dependent increase in insulin release, suggesting that cNOS exerts an inhibitory tone on basal insulin secretion. Moreover, l -arginine, the physiological substrate of cNOS, significantly reduced the marked enhancing effect of l -NAME on insulin release and to a lesser extent, at low concentrations, that of 10 mM KCl. l -NAME also potentiated the insulin secretion stimulated by 5.5 and 8.3 mM glucose, but in this case, its effect was not reduced by l -arginine. In conclusion, our data show that the neuronal isoform of cNOS exerts a negative modulation on insulin secretion in INS-1 cells, confirming the previous results obtained in the isolated perfused rat pancreas or pancreatic islets.

Synaptotagmins are two C2 domain-containing transmembrane proteins. The function of calcium-sensitive members in the regulation of post-Golgi traffic has been well established whereas little is known about the calcium-insensitive isoforms constituting half of the protein family. Novel binding partners of synaptotagmin 11 were identified in β-cells. A number of them had been assigned previously to ER/Golgi derived-vesicles or linked to RNA synthesis, translation and processing. Whereas the C2A domain interacted with the Q-SNARE Vti1a, the C2B domain of syt11 interacted with the SND1, Ago2 and FMRP, components of the RNA-induced silencing complex (RISC). Binding to SND was direct via its N-terminal tandem repeats. Our data indicate that syt11 may provide a link between gene regulation by microRNAs and membrane traffic.