[Oxydative stress and beneficial effect of sodium restriction on kidney damage associated with insulin resistance in rats]. / Stress oxydant et effets bénéfiques rénaux de la restriction sodée dans l'insulinorésistance chez le rat.

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.

Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

Alteration of beta-cell constitutive NO synthase activity is involved in the abnormal insulin response to arginine in a new rat model of type 2 diabetes.

We have previously obtained a new type 2 diabetic syndrome in adult rats given streptozotocin and nicotinamide, characterized by reduced beta-cell mass, partially preserved insulin response to glucose and tolbutamide and excessive responsiveness to arginine. We have also established that the neuronal isoform of constitutive NO synthase (nNOS) is expressed in beta-cells and modulates insulin secretion. In this study, we explored the kinetics of glucose- and arginine-stimulated insulin release in perifused isolated islets as well as the effect of N-omega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, to get insight into the possible mechanisms responsible for the arginine hypersensitivity observed in vitro in this and other models of type 2 diabetes. A reduced first phase and a blunted second phase of insulin secretion were observed upon glucose stimulation of diabetic islets, confirming previous data in the isolated perfused rat pancreas. Exposure of diabetic islets to 10 mM arginine, in the presence of 2.8 mM glucose, elicited a remarkable monophasic increment in insulin release, which peaked at 639 +/- 31 pg/islet/min as compared to 49 +/- 18 pg/islet/min in control islets (P << 0.01). The addition of L-NAME to control islets markedly enhanced the insulin response to arginine, as expected from the documented inhibitory effect exerted by nNOS activity in normal beta-cells, whereas it did not further modify the insulin secretion in diabetic islets, thus implying the occurrence of a defective nNOS activity in these islets. A reduced expression of nNOS mRNA was found in the majority but not in all diabetic islet preparations and therefore cannot totally account for the absence of L-NAME effect, that might also be ascribed to post-transcriptional mechanisms impairing nNOS catalytic activity. In conclusion, our results provide for the first time evidence that functional abnormalities of type 2 experimental diabetes, such as the insulin hyper-responsiveness to arginine, could be due to an impairment of nNOS expression and/or activity in beta-cells.

P2Y receptor activation enhances insulin release from pancreatic beta-cells by triggering the cyclic AMP/protein kinase A pathway.

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.

A constitutive nitric oxide synthase modulates insulin secretion in the INS-1 cell line.

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 N omega-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.

A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion.

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.

Preproinsulin I and II mRNA expression in adult rat submandibular glands.

Mammalian salivary glands are known to produce a number of biologically active peptides. The aim of this study was to extend our previous results showing the presence of a biologically active insulin-like immunoreactive peptide in rat salivary glands. In rodents, where two nonallelic and functional insulin genes are expressed, the co-expression of both genes seems to be limited to beta-cells of pancreatic islets or to embryologic developmental processes. We have investigated the expression of insulin genes in rat submandibular glands and in a murine immortalized submandibular cell line, SCA-9. For this purpose, total RNAs were isolated and submitted to reverse transcription. The cDNAs obtained were amplified by a nested polymerase chain reaction using rat preproinsulin I and II primers. Our data show that both preproinsulin I and II mRNAs are expressed in adult rat submandibular glands as well as in the SCA-9 cell line. The identification of salivary gland rat preproinsulin I and II was confirmed by direct sequencing. These results provide, for the first time, evidence for the expression of both preproinsulin I and II mRNA in an extra-pancreatic tissue from adult rodents.