We took advantage of thè partial protection exerted by suitable dosages of nicotinamide against thè p-cytotoxic effect of Streptozotocin (STZ) to create a new experimental diabetic syndrome in adult rats that appears closer to NIDDM than other available animai models with regard to insulin responsiveness to gincose and sulfonylureas. Among thè various dosages of nicotinamide tested in 3-month-old Wistar rats (100-350 mg/kg body wt), thè dosage of 230 mg/kg, given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded a maximum of animals with moderate and stable nonfasting hyperglycemia (155 ± 3 vs. 121 ± 3 mg/dl in controls; P < 0.05) and 40% preservation of pancreatic insulin stores. We also evaluated liceli function both in vitro and in vivo 4-9 weeks after inducing diabetes. In thè isolated perfused pancreas, insulin response to glucose elevation (5-11 mmol/1) was clearly present, although significantly reduced with respect to controls (P < 0.01). Moreover, thè insulin response to tolbutamide (0.19 mmol/1) was similar to that observed in normal pancreases. Perfused pancreases from diabetic animals also exhibited a striking hypersensitivity to arginine infusìon (7 mmol/1). In rats administered STZ plus nicotinamide, intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness, which were interestingly reversed by tolbutamide administration (40 mg/kg i.v.). In conclusion, this novel NIDDM syndrome with reduced pancreatic insulin stores, which is similar to human NIDDM in that it has a significant response to glucose (although abnormal in kinetics) and preserved sensitivity to tolbutamide, may provide a particularly advantageous tool for pharmacological investigations of new insulinotropic agents.

We took advantage of the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create a new experimental diabetic syndrome in adult rats that appears closer to NIDDM than other available animal models with regard to insulin responsiveness to glucose and sulfonylureas. Among the various dosages of nicotinamide tested in 3-month-old Wistar rats (100-350 mg/kg body wt), the dosage of 230 mg/kg, given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded a maximum of animals with moderate and stable nonfasting hyperglycemia (155 +/- 3 vs. 121 +/- 3 mg/dl in controls; P < 0.05) and 40% preservation of pancreatic insulin stores. We also evaluated beta-cell function both in vitro and in vivo 4-9 weeks after inducing diabetes. In the isolated perfused pancreas, insulin response to glucose elevation (5-11 mmol/l) was clearly present, although significantly reduced with respect to controls (P < 0.01). Moreover, the insulin response to tolbutamide (0.19 mmol/l) was similar to that observed in normal pancreases. Perfused pancreases from diabetic animals also exhibited a striking hypersensitivity to arginine infusion (7 mmol/l). In rats administered STZ plus nicotinamide, intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness, which were interestingly reversed by tolbutamide administration (40 mg/kg i.v.). In conclusion, this novel NIDDM syndrome with reduced pancreatic insulin stores, which is similar to human NIDDM in that it has a significant response to glucose (although abnormal in kinetics) and preserved sensitivity to tolbutamide, may provide a particularly advantageous tool for pharmacological investigations of new insulinotropic agents.

BACKGROUND AND PURPOSEQuercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic b-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting b-cells. Using the INS-1 b-cell line, the effects of quercetin were determined on glucose-or glibenclamide-induced insulin secretion and on b-cell dysfunctions induced by hydrogen peroxide (H2O2). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison. EXPERIMENTAL APPROACHInsulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay. KEY RESULTSQuercetin (20 mmol·L ), protected b-cell function and viability against oxidative damage induced by 50 mmol·L -1 H2O2 and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective. CONCLUSION AND IMPLICATIONSQuercetin potentiated glucose and glibenclamide-induced insulin secretion and protected b-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing b-cell dysfunction associated with diabetes deserves further investigation.

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. Doubleimmunofluorescence studies demonstrated the presence of nNOS in insulin-secreting ␤-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 ␤-cell response to arginine and glucose after nNOS blockade with N Gnitro-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 enzymesubstrate 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 ␤-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 ␤-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in ␤-cell dysregulation involved in certain pathological hyperinsulinic states. T he short-lived free radical gas nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as NO synthases (NOSs). Three NOS isoenzymes, encoded by three separate genes, have been described, including the Ca 2ϩ /calmodulin-dependent and constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS) enzymes and a calmodulin-independent cytokine-inducible NOS (iNOS) enzyme found in various cell types (rev. in 1). The small amounts of NO, produced by the constitutive forms in response to increases in intracellular calcium, play a crucial role in a number of physiological functions, including neurotransmission (2), vascular tone (3) and platelet aggregation (4), whereas the large amounts, produced by iNOS in a calcium-independent manner over prolonged periods of time, are implicated in pathological functions, such as cytotoxicity of activated macrophages (5).Even though the inducible isoform has been cloned in insulin-producing cells after induction by cytokines (6), it is unclear whether pancreatic ␤-cells express a constitutive NOS. Both NADPH-diaphorase (NADPH-d) histochemical staining previously shown to be specific for NOS (7) and immunohistochemical studies using various nNOS antisera yielded apparently conflicting data. Positive NADPH-d and immunoreactive nNOS stainings have been found to be colocalized in most pancreatic endocrine cells (8), a finding not confirmed in other studies...

We report the characterization of a new insulinotropic compound, 4-hydroxyisoleucine. This amino acid has been extracted and purified from fenugreek seeds, which are known in traditional medicine for their antidiabetic properties. 4-Hydroxyisoleucine increases glucose-induced insulin release, in the concentration range of 100 micromol/l to 1 mmol/l, through a direct effect on isolated islets of Langerhans from both rats and humans. The stimulating effect of 4-hydroxyisoleucine was strictly glucose dependent; indeed, ineffective at low (3 mmol/l) or basal (5 mmol/l) glucose concentrations, the amino acid potentiated the insulin secretion induced by supranormal (6.6-16.7 mmol/l) concentrations of glucose. In addition, in the isolated perfused rat pancreas, we could show 1) that the pattern of insulin secretion induced by 4-hydroxyisoleucine was biphasic, 2) that this effect occurred in the absence of any change in pancreatic alpha- and delta-cell activity, and 3) that the more glucose concentration was increased, the more insulin response was amplified. Moreover, 4-hydroxyisoleucine did not interact with other agonists of insulin secretion (leucine, arginine, tolbutamide, glyceraldehyde). Therefore, we conclude that 4-hydroxyisoleucine insulinotropic activity might, at least in part, account for fenugreek seeds' antidiabetic properties. This secretagogue may be considered as a novel drug with potential interest for the treatment of NIDDM.

We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 μM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.

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