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。.

Diagnosis of nonalcoholic steatohepatitis (NASH) relies on liver biopsy. Noninvasive tools would be useful to target patients to refer for a biopsy. We aimed to determine the diagnostic value of the triglycerides and glucose (TyG) index, an insulin-resistance indicator, to predict NASH.Our study included grade II-III obese patients aged 18-65 years undergoing bariatric surgery and included in the COMET (COllection of MEtabolic Tissues) biobank (NCT02861781). Liver biopsies performed during bariatric surgery were collected from the biobank along with blood derivatives. Biopsies were analysed according to the steatosis, activity and fibrosis (SAF) scoring system to diagnose NASH, nonalcoholic fatty liver disease (NAFLD), and fibrosis. Logistic regression models were performed to identify factors predicting NASH, NAFLD, and fibrosis.Of 238 analysed subjects (mean age 43±12 years, 33.6% men), 29% had type 2 diabetes. Steatosis was present in 67.2%, while NASH and advanced fibrosis (stage F3) were diagnosed in 18.1% and 2.9% respectively. TyG index was independently associated with NASH (odds ratio (OR): 4.7 [95% confidence interval: 2.3;9.5] P0.0001), NAFLD (OR: 2.0 [1.1;3.7] P = 0.03) and stages 2-3 fibrosis (OR: 4.0 [1.5;10.8] P = 0.007). NASH was also predicted by gamma-glutamyl transferase (GGT) with an area under the ROC curve: 0.79 [0.71;0.87 P = 0.04] for GGT and TyG index combined.In our cohort of severely obese patients, TyG index, when associated with GGT level, exhibited high diagnostic performance to predict NASH. Although validation in larger populations is needed, this result may be of considerable clinical value to predict need for liver biopsy.

In a rat model of metabolic syndrome (MS), our previous studies have shown that dietary sodium restriction prevents both metabolic and cardiac damages associated with MS. Our aim is now to investigate whether the beneficial effects of sodium restriction could be mediated by endothelial to mesenchymal (EndoMT) transition in the myocardium thereby preventing cardiac fibrosis.High fructose (60%) Sprague Dawley rats were divided into 2 groups: low sodium (in vitro model of EndoMT, using primary human aortic endothelial (HAoE) cells that were transdifferentiated with TGF-β2 (10ng/ml). We evidenced in HAoE cells the co-expression of Pecam-1 and collagen-1, as a signature of EndoMT. Especially, fibulin 5, one of the genes identified by transcriptomics was found upregulated (13 folds) in the presence of TGF-β2, confirming its potential role in EndoMT. Our study shows that EndoMT is involved in the prevention of cardiac fibrosis by sodium restriction in our rat model of MS. We also confirmed the involvement of ew genes that could be of interest to improve the management of cardiac fibrosis.

Excessive fat consumption leads to the development of ectopic adipose tissues, affecting the organs they surround. Peripancreatic adipose tissue is implicated in glucose homeostasis regulation and can be impaired in obesity. High palm oil consumption’s effects on health are still debated. We hypothesised that crude and refined palm oil high-fat feeding may have contrasting effects on peripancreatic adipocyte hypertrophy, inflammation and lipid oxidation compound production in obese rats. In Wistar rats, morphological changes, inflammation and isoprostanoid production following oxidative stress were assessed in peripancreatic adipose tissue after 12 weeks of diets enriched in crude or refined palm oil or lard (56% energy from fat in each case) versus a standard chow diet (11% energy from fat). Epididymal white and periaortic brown adipose tissues were also included in the study. A refined palm oil diet disturbed glucose homeostasis and promoted lipid deposition in periaortic locations, as well as adipocyte hypertrophy, macrophage infiltration and isoprostanoid (5-F2c-isoprostane and 7(RS)-ST-Δ8-11-dihomo-isofuran) production in peripancreatic adipose tissue. Crude palm oil induced a lower impact on adipose deposits than its refined form and lard. Our results show that the antioxidant composition of crude palm oil may have a protective effect on ectopic adipose tissues under the condition of excessive fat intake.

Introduction et but de l’etude Dans les pays developpes, le syndrome metabolique (SM), associant une hypertension arterielle et une insulinoresistance, concerne 25 a 45 % des hommes de plus de 40 ans, les predisposant a un risque d’AVC multiplie par 3. L’augmentation de ces troubles metaboliques va de concert avec la consommation de sucres en constante augmentation dans les pays developpes. L’hypertension participe a la fibrose du myocarde, ce qui aboutit a une dysfonction diastolique et au developpement d’une hypertrophie ventriculaire gauche. Dans ce cadre, nous avons montre qu’un regime alimentaire pauvre en sel pouvait prevenir les dommages cardiaques d’une hypertension associee a une insulinoresistance dans un modele de rat. Nous avons recemment identifie par transcriptomique dans ce modele un groupe de genes module par la restriction sodee, et dont certains sont plus particulierement impliques dans un processus appele transition endothelio-mesenchymateuse. Notre objectif a ete de montrer que le regime desode regule negativement la transition endothelio-mesenchymateuse (TEndM) dans le cœur des rats atteints de syndrome metabolique, ce qui previent le phenomene de fibrose. Materiel et methodes Notre modele d’etude est le rat Sprague Dawley nourri avec 60 % de fructose. Les rats sont divises en 2 groupes : groupe normosode fructose (NSF) (0,64 % NaCl), et groupe desode fructose (DSF) avec 0,01 % de NaCl dans la nourriture. Au bout de 4 semaines de regime, l’angiotensine II est perfusee (200 ng/kg/min) durant 4 semaines additionnelles. Nous avons explore l’expression de marqueurs de la TEndM par western blot (WB) et leur colocalisation par immunofluorescence dans les ventricules gauches des rats. La confirmation des variations d’expression en transcriptomique a ete realisee par qPCR et par WB. La quantification est realisee par le logiciel Image J. Les tests statistiques utilises sont le test de Student ou de Mann et Whitney. Resultats et analyse statistique Deux proteines mesenchymateuses, l’actine du muscle lisse (αSMA), et la vimentine, voient leur expression significativement diminuee en regime desode (p Conclusion Nous montrons que la transition endothelio-mesenchymateuse est impliquee dans la prevention du phenomene de fibrose par le regime desode dans notre modele de SM. Nous avons de plus identifie et valide 17 genes modules par la restriction sodee dont deux d’entre eux inconnus jusqu’alors. Nous esperons par cette etude apporter un argument supplementaire de l’interet d’un regime alimentaire appauvri en sel dans la prevention des dommages cardiaques au cours du syndrome metabolique.

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.

Central serotonin systems have long been associated with the control of feeding behavior and the modulation of behavioral effects of psychostimulants. 5-HT2C receptors are present in hypothalamic centers such as the arcuate nucleus (ARC), controlling homeostatic regulation of food intake, as well as in the ventral tegmental area (VTA), a region involved in motivation aspects in multiple behaviors, including feeding. In the present study, we investigated whether the 5-HT2C receptors control amphetamine-evoked locomotor activity and regulate food consumption. Localized microinjections into the VTA or the ARC were used to assess the effects of a highly selective 5-HT2C receptor agonist, AR231630, on the locomotor stimulant effect of amphetamine as well as on food intake. AR231630 injected into the VTA, but not into the ARC, dose-dependently reduced locomotor activity elicited by amphetamine. Unexpectedly, intra-ARC injection of AR231630 did not reduce food intake even at the dose of 10 μg, whereas intra-VTA injection of the same dose of AR231630 did. In addition, we showed that pretreatment with the selective 5-HT2C receptor antagonist SB242084 infused into the VTA partially prevented hypophagia induced by peripheral administration of AR231630. We can conclude that 5-HT2C receptor in the VTA, but not in the ARC, participates in both homeostatic and hedonic food intake and brain reward function.

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.

Sodium depletion has a protective effect on target-organ damage in hypertension independent of blood pressure. Here we tested whether chronic dietary sodium restriction may prevent the development of renal alterations associated with insulin resistance by reducing the inflammatory and oxidant state. Rats were fed normal-salt-60% fructose, low-salt-60% fructose, or control normal-salt diet for 12 weeks. Insulin resistance induced by high-fructose diet was associated with an increase in albuminuria, tubular and glomerular hypertrophy, and inflammation of kidney and adipose tissue. The low-salt diet improved insulin sensitivity and prevented kidney damage. These beneficial effects of sodium depletion were associated with a decrease in renal inflammation (macrophage infiltration, IL-6, TNF-α) and oxidative stress (NADPH oxidase activity), and a prevention of histologic changes in retroperitoneal fat induced by high fructose. Thus, dietary salt depletion has beneficial effects on renal and metabolic alterations associated with a high-fructose diet in rats.

The aim of this work was to evaluate the influence of dietary sodium restriction on metabolic and renal changes associated with insulin resistance. At 8 weeks of age, rats received either a diet containing 60% fructose with or without sodium or a standard diet for 12 weeks. The insulin resistance and albuminuria induced by the high fructose diet were associated with a fibrosis and increase in oxidative stress in the kidney. The low salt diet prevented insulin resistance, renal fibrosis and albuminuria induced by the fructose diet. These beneficial effects on the kidney were associated with a decrease in kidney NADPH oxidase activity. Oxidative status is probably one of the major targets of the favourable effect of salt restriction on renal changes associated with insulin resistance, without excluding the involvement of other mechanisms.

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.

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.

Introduction PIN (Protein Inhibitor of Neuronal NO synthase) est une proteine « plateforme » capable d'interagir avec de nombreux partenaires comme la NO synthase neuronale (nNOS) et les proteines du cytosquelette, myosine V et tubuline. Nous avons precedemment demontre que PIN est un modulateur positif de la secretion d'insuline induite par le glucose. Afin de mieux comprendre le role de PIN, nous avons mis au point un peptide inhibiteur, capable de bloquer l'interaction de PIN avec ses partenaires proteiques et rendu cell-permeant grâce a un couplage avec la sequence Tat. Materiels et methodes La secretion d'insuline a ete mesuree dans les cellules β INS-1 en presence de glucose, du peptide inhibiteur ou d'un peptide irrelevant. Pour la microscopie TIRF, les cellules sont transfectees avec le rapporteur NPY-EGFP et la secretion d'insuline induite par 75 mm KCl. Resultats Dans la lignee INS-1, le peptide inhibiteur diminue la secretion d'insuline induite par 2,8, 5,6 et 8,3 mM de glucose, de 33 % a 20 µM, et de 58 % a 40 µM ( p p Conclusion La proteine PIN aurait donc un role dans la migration des vesicules secretoires d'insuline, en modulant la polymerisation de la tubuline. Declaration d’interet Les auteurs declarent ne pas avoir d'interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

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.

Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans.Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy.Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules.Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.

Purpose: The aim of this study was to investigate if the metabolic alterations observed after static magnetic field (SMF) exposure participates in the development of a pre-diabetic state. A comparison study using the insulin resistant animal model, the Zucker rat and the SMF-exposed Wistar rat was carried out. Materials and methods: Zucker rats were compared to Wistar rats either exposed to a 128 mT or 0 mT SMF (sham exposed) and analysed. This moderate-intensity SMF exposure of Wistar rats was performed for 1 h/day during 15 consecutive days. Results: Wistar rats exposed to the SMF showed increased levels of carbohydrate and lipid metabolites (i.e., lactate, glycerol, cholesterol and phospholipids) compared to sham-exposed rats. Zucker rats displayed a normoglycemia associated with a high insulin level as opposed to Wistar rats which presented hyperglycemia and hypoinsulinemia after exposure to the SMF. During the glucose tolerance test, unexposed Zucker rats and Wistar rats exposed to the SMF exhibited a significantly higher hyperglycemia compared to sham-exposed Wistar rats suggesting an impairment of glucose clearance. In muscle, glycogen content was lower and phospholipids content was elevated for both unexposed Zucker rats and Wistar rats exposed to the SMF compared to Wistar rats sham control. Conclusions: This study provides evidence that the metabolic alterations following exposure to a static magnetic field of moderate intensity could trigger the development of a pre-diabetic state.

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; BACKGROUND: Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis. METHODOLOGY: We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats). PRINCIPAL FINDINGS: Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid). CONCLUSIONS/SIGNIFICANCE: We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production.

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.

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.

Introduction Un extrait hydrosoluble (BRE) issu de racine de bardane (Arctium lappa) a ete teste in vivo chez des rats lors de tests de tolerance au glucose ; Les resultats obtenus avaient pu ainsi demontrer l'effet anti-hyperglycemique ce dernier. Des essais in vitro avaient demontre que BRE provoque une diminution de la decharge de glucose par les cellules hepatiques mais aussi de la secretion d'insuline par les cellules pancreatiques INS1. Notre objectif est maintenant de rechercher une (la) cible de BRE dans les deux types cellulaires. La presence d'acide chlorogenique (7 %) dans l'extrait nous a conduit a nous interesser a l'activite de la glucose 6-phosphatase, cible connue de cet acide monocaffeoyl-quinique. Materiels et methodes BRE a ete analyse par LC-MS et par CPG. Les microsomes issus de cellules INS1 et hepatiques traitees ou non par BRE ont ete isoles afin de mesurer l'activite enzymatique de la glucose 6-phosphatase. Resultats L'extrait BRE presente une tres forte concentration en acides caffeoyl-quiniques tel que l'acide chlorogenique (acide monocaffeoyl-quinique) present a 7 %. BRE diminue la decharge de glucose par les cellules hepatiques et inhibe la glucose 6-phosphatase (G6Pase), derniere enzyme de la glycogenolyse responsable de la production de glucose. Cet effet BRE peut etre impute a l'acide chlorogenique present et connu comme inhibiteur de la translocase T1 du complexe G6Pase, responsable de l'apport en substrat. A l'inverse, BRE augmente tres fortement l'activite G6Pase dans les cellules INS1 et provoque une diminution de secretion de l'insuline. Nous montrons que cet effet stimulant de BRE sur l'activite G6Pase n'est pas imputable a l'acide chlorogenique contenu dans l'extrait. Conclusion L'extrait BRE de racine de bardane est apte a inhiber l'activite de la G6Pase hepatique tandis qu'il stimule l'activite G6Pase pancreatique. Nous pouvons envisager la presence d'un compose apte a stimuler l'activite G6Pase non substrat limitant.

Rationnel Lors de l’obesite, les facteurs secretes par le tissu adipeux influencent la fonction et la masse β-cellulaire. Lorsque l’adiposite viscerale augmente, se developpe un tissu adipeux peri-pancreatique (TAPP), qui penetre le pancreas et etablit des contacts avec les ilots de Langerhans. Rebuffat et al (Endocrinology, 2012) ont montre dans un modele d’obesite et d’insulino-resistance que des facteurs secretes par le TAPP, modulent la proliferation des cellules β. Ceci est en faveur d’une biocommunication locale entre le TAPP et la cellule β. Notre objectif est de caracteriser le TAPP en termes de cellules immunitaires et de facteurs inflammatoires secretes lors du diabete de type 2 (DT2). Materiels et methodes Notre etude est realisee chez le rat ZDF un modele animal d’obesite et de DT2. Trois groupes d’animaux sont etudies, rats pre-diabetiques (6 et 10 semaines) et rats diabetiques (12 semaines). La presence de cellules immunitaires infiltrant le TAPP est etudiee par immunohistochimie a l’aide du marqueur macrophagique ED1. Le profil d’expression des cytokines secretees par les TAPP est analyse par la technologie de puce a anticorps (Chemiarray, RD System). Resultats Une augmentation des macrophages infiltrant le PPAT est observees chez les animaux pre-diabetiques. Parmi les cytokines/chimiokines analyses, SICAM-1, IL-1α, IP-10, L-selectine, TIMP-1, TNF et VEGF sont secretees par le TAPP independamment du stade de la pathologie. En revanche, CINC- 1, 2, 3, IL-1β, LIX, MIP-1α apparaissent uniquement dans le secretome du TAPP d’animaux pre-diabetiques (10 s). Le niveau de secretion de TIMP-1 est augmente d’un facteur 4. Conclusion Nos resultats montrent que le profil et le niveau d’expression de cytokines/chimiokines secretees par le TAPP varie du stade de pre-diabete au DT2. Ces variations (i) permettraient la mise en place et le maintien de l’inflammation en attirant des cellules immunitaires dans le TAPP et (ii) contribueraient au dysfonctionnement progressif β-cellulaire survenant dans le DT2.

Objectif L’objectif est de comparer l’effet d’un regime desode (DS, Materiels et methodes La pression arterielle (TCP) est mesuree avant et apres AngII. L’index de masse cardiaque (IMCard, poids du cœur/poids du corps) et la taille des cardiomyocytes sont mesures en fin d’etude. L’expression cardiaque de divers genes est determinee par PCR en temps reel (qPCR). La presence de monocytes/macrophages est determinee par immunomarquage et l’expression du CD68. Le facteur de transcription NF-kBp65 et la presence de lymphocytes T CD4 + et CD8 + ainsi que de lymphocytes B (CD20) est mesuree. Resultats L’AngII induit une augmentation de la TCP de 40 ± 5 % en regime NSF et de 20 ± 4 % en regime DSF. L’IMCard est augmente par l’AngII dans les deux regimes NSF et DSF (20 et 10 %, respectivement). L’augmentation de l’aire des cardiomyocytes induite par l’AngII en regime NSF n’est pas observee en regime DSF. L’infiltration macrophagique cardiaque (marquage CD68) et l’expression de CD68 est significativement plus basse en regime desode en absence (9,5 ± 0,8.10−2 vs 7,1 ± 0,5.10−2) et en presence d’AngII (10,6 ± 1,5.10−2 vs 5,6 ± 0,4.10−2). De meme, l’expression de CD4 et de CD8 est reduite en regime desode en absence ou en presence d’AngII. Aucune difference significative n’est retrouvee pour CD20. L’expression de NF-kBp65 est reduite significativement par le regime desode en presence ou absence d’AngII. Conclusion L’effet benefique cardiaque de la restriction sodee est conserve quand la dysfonction metabolique est associee a une hypertension arterielle. L’effet benefique est vraisemblablement lie a la baisse de pression arterielle ainsi qu’a la diminution de l’inflammation et de l’infiltration des cellules de l’immunite (macrophages, cellules T mais pas B) au niveau cardiaque.

Objectif La calcification vasculaire est frequemment observee dans le diabete et dans le syndrome metabolique. Elle est associee a un risque accru de maladies cardio-vasculaires. La capacite a calcifier est evaluee ex-vivo sur des anneaux aortiques de rats dont le metabolisme glucidique est altere. Materiels et methodes L'aorte thoracique est prelevee sur des rats insulinoresistants (Zucker 10 semaines, ZDF 6 semaines et rats regime hypercalorique, cafeteria) ou sur des rats diabetiques ZDF de 13 semaines. Des rats Wistar et Zucker fa/+ servent de temoins. Douze anneaux de chaque aorte sont cultives 14 jours dans un milieu calcifiant (3,8mM de phosphate) ou un milieu controle. Le dosage du calcium est realise a partir du tissu sec avec un kit colorimetrique et exprime en mg/g d'aorte. La localisation du calcium et de la fibrose respectivement par coloration au von Kossa et au rouge sirius est realisee sur coupes histologiques (3–5μm) apres inclusion en paraffine. Une quantification est realisee en pourcentage de l'aire de section. Resultats En absence de phosphate, aucune calcification n'est observee dans les differents groupes. Le phosphate entraine une calcification dans tous les groupes. Elle est significativement plus forte dans les groupes insulinoresistants Zucker et ZDF de 6 semaines (878±141 et 871±60mg/g) comparee au Wistar (300±89mg/g) mais pas au rat Zucker fa/+ (670±80mg/g). Elle est majoree chez le ZDF diabetique âge de 13 semaines (1 390±64mg/g). Elle est la plus forte chez les rats « cafeteria » (1 659±129mg/g). L'histologie confirme ces resultats et montre une localisation differente de la calcification chez le rat « cafeteria ». Le marquage des collagenes a une repartition similaire a celle du von Kossa. Conclusion La calcification vasculaire de gros troncs arteriels est exacerbee en presence d'une dysfonction metabolique, et la difference de localisation chez le rat cafeteria suggere l'existence de mecanismes differents.

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.

Objectif Nous avons montre que la restriction sodee prevenait les lesions renales associees a l’insulinoresistance en partie par un effet anti-inflammatoire. L’objectif de cette etude est d’evaluer l’effet du regime desode sur les atteintes cardiovasculaires induites par un regime riche en fructose, modele d’insulinoresistance chez le rat. Materiels et methodes Les rats recoivent un regime normosode standard (NSS), normosode-60% fructose (NSF), desode-60% fructose (DSF) pendant 12 semaines. La pression arterielle moyenne (PAM), la morphologie et la fonction cardiaques sont evaluees par echocardiographie. L’epaisseur de la paroi carotidienne, la taille des cardiomyocytes et la fibrose sont mesurees histologiquement. La vasorelaxation a l’acethylcholine est evaluee sur des anneaux aortiques. L’inflammation et le stress oxydant cardiaques sont estimes par immunomarquage (ED1 et nitrotyrosine, respectivement). Resultats Le fructose induit une hypertrophie ventriculaire gauche (1,74 ± 0,11 vs 1,63 ± 0,06 mg/g) de type concentrique (RWT : 0,35 ± 0,01 vs 0,29 ± 0,02) avec alteration de la fraction de raccourcissement (FR : 44 ± 2 vs 52 ± 3) sans modifier la PAM (122 ± 4 vs 121 ± 4 mmHg). L’aire de section de la carotide (ASC) est plus grande chez les rats NSF compares aux rats NSS et s’accompagne d’une alteration de la vasorelaxation. Les alterations cardiaques sont associees a une augmentation du collagene (3,2 ± 0,8 vs 1,5 ± 0,2%), a un doublement de l’infiltration macrophagique et a une augmentation du stress oxydant myocardique (nitrotyrosine, 2,3 ± 0,3% vs 1,1 ± 0,3%). Le regime desode previent l’hypertrophie VG (IMVG : 1,41 ± 0,01 mg/g ; RWT : 0,30 ± 0,02) et carotidienne, l’alteration de la fonction systolique (FR : 48 ± 1%) et la fibrose myocardique (Rouge Sirius : 1,40 ± 0,04%) sans influencer la pression arterielle ni la dysfonction endotheliale. Ces effets benefiques de la restriction sodee s’accompagnent d’une diminution de l’infiltration macrophagique (cellules ED1 : 0,4 ± 0,0 par mm2) et du score nitrotyrosine (1,4 ± 0,3%) myocardiques. Conclusion La restriction sodee previent l’hypertrophie cardiovasculaire et la fibrose cardiaque associees a l’insulinoresistance par un effet anti-inflammatoire et anti-oxydant tissulaires.

Introduction Nous avons precedemment demontre que la NO synthase neuronale (nNOS) est impliquee dans le controle de la secretion d’insuline chez le rat. Chez l’homme, nous avons retrouve l’expression de la nNOS dans les cellules b pancreatiques. Notre objectif est donc rechercher si la nNOS, comme chez le rat, est capable de moduler la secretion d’insuline dans les ilots pancreatiques humains. Materiels et methodes La secretion insuline a ete evaluee sur 10 ilots pendant 90 minutes, en presence de de glucose et de differents modulateurs pharmacologiques. Resultats L’inhibition de la nNOS par 10 mm de L-NAME amplifie la secretion d’insuline induite par 8,3 et 16,7 mm de glucose (+ 35 % et + 33 % respectivement, P Conclusion Dans les ilots pancreatiques humains, de faibles niveaux de NO modulent negativement la secretion d’insuline (S-nitrosylation de proteines du metabolisme), tandis qu’a plus fortes concentrations, le NO amplifie la secretion d’insuline, probablement via la nitration de proteines de l’exocytose.

Introduction Nous avons precedemment demontre que la NO synthase neuronale (nNOS) controle la secretion d’insuline grâce a des activites metaboliques et non metaboliques. Cette derniere est liee a l’interaction de la nNOS avec son inhibiteur PIN, egalement chaine legere de la myosine V, au niveau des granules d’insuline. Nous souhaitons determiner si l’interaction nNOS-PIN module la secretion d’insuline et est alteree dans des situations d’hypersecretion d’insuline comme chez le rat Zucker fa/fa. Materiels et methodes Le complexe nNOS/PIN/myosine V a ete etudie par co-immunoprecipitation sur la lignee INS-1. L’interaction nNOS-PIN a ete analysee par la methode Spot et par immunofluorescence. La secretion d’insuline a ete mesuree sur les cellules INS-1 et des ilots de rats Zucker fa/fa et fa/+. Resultats Dans la lignee INS-1, nous avons montre une interaction directe entre la nNOS, PIN et la myosine V ainsi qu’une augmentation du complexe ternaire lorsque PIN est surexprime. Apres analyse des regions de la nNOS impliquees dans l’interaction avec PIN, nous avons pu identifier un peptide mutant qui inhibe l’interaction nNOS-PIN avec un Ki de 0,4 μM. Ce peptide (20 μm), conjugue a la sequence TAT du virus HIV, dissocie l’interaction nNOS-PIN de 47 % dans la lignee INS-1 et diminue de 36 % (p Conclusion Nos resultats demontrent le role du complexe nNOS-PIN dans la modulation de la secretion d’insuline, probablement au niveau de l’exocytose.

Introduction Le muscle squelettique, dont la sensibilite a l’insuline est alteree dans le diabete de type 2, exprime un variant d’epissage de l’isoforme neuronale de la nitric oxyde synthase (nNOS), la nNOSμ. Etant donne le role physiologique de la nNOSμ dans la captation du glucose, notre objectif est de determiner si des anomalies de la nNOSμ musculaire pourraient intervenir dans les etats prediabetiques et les stades precoces du diabete de type 2. Dans ce but, nous avons choisi comme modele, le rat obese Zucker fa / fa , associant une forte insulinoresistance et un hyperinsulinisme caracteristiques d’un etat prediabetique. Materiels et Methodes L’expression de la nNOSμ a ete mesuree par PCR quantitative et par Western blot. L’activite catalytique de la nNOSμ a ete evaluee en mesurant la production de citrulline a partir d’arginine radiomarquee. L’agent MG132 a ete utilise pour inhiber l’activite du proteasome. Enfin, la localisation subcellulaire de la nNOSμ a ete determinee par immunofluorescence a l’aide d’un anticorps specifique. Resultats Chez le rat obese Zucker fa / fa , nous avons trouve une diminution de l’activite catalytique de la nNOSμ d’environ 41 % par rapport aux rats fa /+ controles (P fa / fa , suggerant une degradation proteasomale de la nNOSμ. L’utilisation de l’inhibiteur du proteasome MG132 sur les muscles isoles nous a permis de mettre en evidence une augmentation du taux d’ubiquitination de la nNOSμ chez les rats fa / fa . De plus, en inhibant le proteasome, nous avons pu restaurer significativement (P fa /+. Par immunofluorescence, nous avons confirme la baisse d’expression de la nNOSμ chez le rat fa / fa et observe, au niveau subcellulaire, une perturbation de sa distribution sous-membranaire. Conclusion Des alterations fonctionnelles de la nNOSμ resultant d’une degradation proteasomale accrue observee dans le muscle squelettique des rats obeses fa / fa , pourraient etre impliquees dans l’insulinoresistance musculaire dans certains stades precoces du diabete de type 2.

Introduction L’hyperglycemie chronique induit un stress oxydant implique dans la pathogenie des complications vasculaires du diabete. Nos objectifs etaient d’analyser, chez des patients diabetiques de type 2 (DT2) mal controles sous antidiabetiques oraux (ADO), l’expression d’une enzyme cle du stress oxydant, la NADPH oxydase (NADPHox), et sa regulation apres correction rapide de l’hyperglycemie. Materiels et Methodes L’expression des transcrits de deux sous-unites de la NADPHox, p22 phox (membranaire) et p47 phox (cytosolique), a ete etudiee par Q-PCR (Lightcycler-Roche) dans des monocytes isoles de patients non diabetiques (ND, n = 6), DT2 controles sous ADO (C, n = 6) et DT2 non controles sous ADO (NC, n = 12). Dans ce dernier groupe, l’expression a ete evaluee avant (J0) et apres trois jours (J3) d’insulinotherapie IV normalisant la glycemie. L’expression des transcrits a ete normalisee par la β-2-microglobuline. Resultats Les patients des groupes C et NC sont âges de 56,4 ± 7,9 et 64,0 ± 4,5 ans, ont un DT2 connu depuis 8,3 ± 2,9 et 9,1 ± 3,3 ans et presentent une HbA1c de 7,1 ± 0,4 % et 10,26 ± 1,0 %, respectivement. Dans les monocytes, les niveaux d’expression de la p47 phox sont : 2,14 ± 0,83 (ND) ; 2,57 ± 1,16 (C) ; 3,52 ± 1,86 (NC, J0). Nous observons une tendance a l’augmentation non significative de l’expression de la p47 phox dans le groupe NC a J0 vs . ND et C. En revanche, pour la p22 phox , aucune difference d’expression n’est constatee entre les groupes. Dans le groupe NC, apres normalisation des glycemies (J3), nous observons une diminution significative de l’expression de la p47 phox : 2,57 ± 1,58 vs . 3,52 ± 1,86 (p phox . Conclusion Nos resultats montrent une tendance a l’augmentation de l’expression de la p47 phox de la NADPHox chez des patients DT2 en hyperglycemie chronique et une diminution significative de cette expression lors de la correction rapide de l’hyperglycemie par une insulinotherapie IV. Ces variations d’expression, a confirmer dans des populations plus importantes, sont en faveur de la gluco-dependance du stress oxydant dans le DT2, et suggerent la possibilite de sa suppression rapide lors de la correction glycemique par l’insuline.

Introduction La NO synthase neuronale (nNOS) controle la secretion d’insuline dans la cellule β-pancreatique de rat. Une activite non metabolique de la nNOS implique son interaction avec son inhibiteur endogene PIN au niveau des granules secretoires d’insuline. PIN constitue aussi la chaine legere de la myosine V. En utilisant le modele de rat obese Zucker fa/fa, notre objectif est de determiner si des anomalies de la nNOS pourraient etre impliquees dans le dysfonctionnement de la cellule β-pancreatique observe dans le prediabete. Materiels et methodes L’expression de la nNOS et de PIN a ete etudiee par RT-PCR quantitative et Western Blot. La dimerisation de la nNOS a ete analysee par Western Blot a basse temperature et l’activite catalytique de l’enzyme evaluee en mesurant la production de citrulline radiomarquee. La localisation subcellulaire de nNOS et PIN a ete determinee par immunofluorescence dans les cellules β isolees. Resultats Au niveau proteique, la nNOS est 7 fois plus exprimee dans les ilots de rats fa/fa que de rats fa/+. L’analyse de l’equilibre monomere/dimere revele une quantite de dimeres significativement plus importante chez le rat fa/fa par rapport au rat fa/+. Cependant, l’activite catalytique de la nNOS est diminuee de 25 % chez les rats fa/fa. Nous avons alors analyse l’etat de phosphorylation de la nNOS sur la serine 847 connue pour inactiver l’enzyme. Les ilots de rats fa/fa presentent une augmentation du taux de phosphorylation des monomeres et dimeres de nNOS. Par immunofluorescence, nous avons mis en evidence une colocalisation plus importante de la nNOS avec les granules secretoires d’insuline et son inhibiteur PIN egalement surexprime chez les rats fa/fa. Conclusion Nous avons precedemment demontre que des dimeres de nNOS induits pharmacologiquement sont impliques dans un profil d’hypersecretion d’insuline en reponse au glucose. L’augmentation du taux de dimerisation de la nNOS que nous avons ainsi pu mettre en evidence pourrait etre impliquee dans l’hypersecretion d’insuline presente chez le rat Zucker fa/fa via des interactions de la nNOS avec PIN et la myosine V au niveau des granules d’insuline, plutot qu’a travers une augmentation de l’activite catalytique.

Introduction La NO synthase neuronale (nNOS) peut, chez le rat, moduler a la fois l’amplitude et la cinetique de la secretion d’insuline. Notre objectif est de confirmer son expression dans les cellules β pancreatiques humaines et d’evaluer sa fonctionnalite. Materiels et methodes L’expression de la nNOS a ete etudiee dans des extraits d’ilots humains par RT-PCR quantitative ainsi que par Western Blot. Sa localisation cellulaire a ete precisee par immunofluorescence et microscopie confocale. Enfin, la secretion insulinique a ete evaluee sur 10 ilots pendant 90 minutes, en presence de concentrations croissantes de glucose et d’inhibiteurs pharmacologiques de l’enzyme, le Nω-nitro-L-arginine methyl ester (L-NAME) et le 7-nitroindazole (7-NI), plus selectif de l’isoforme neuronale. Resultats La nNOS est exprimee dans les ilots pancreatiques humains tant au niveau ARN messager qu’au niveau proteique. Elle est preferentiellement cytoplasmique, mais aussi nucleaire et mitochondriale, et co-localise avec les granules de secretion d’insuline. Sur le plan fonctionnel, l’inhibition de l’activite nNOS par le L-NAME et le 7-NI stimule de facon dose-dependante la secretion d’insuline induite par le glucose. A 8,3 mM de glucose, la secretion d’insuline est de 24,3 ± 1,9 ng/mL/10 ilots/90 min et est amplifiee a 29 ± 3,6 et 34,5 ± 3,37 en presence de respectivement 5 mM (non significatif) et 10 mM (p Discussion Des resultats similaires avaient precedemment ete retrouves chez le rat. D’autres experiences associant inhibiteurs de l’activite cytochrome c reductase et donneurs de NO sont necessaires pour preciser le role des differentes activites catalytiques de l’enzyme . Conclusion La nNOS est exprimee dans les cellules β humaines et intervient dans la regulation de la secretion insulinique glucose-dependante.