Inhibition of the MAP3 kinase Tpl2 protects rodent and human ß-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4.

Proinflammatory cytokines exert cytotoxic effects on ß-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of ß-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated ß-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in ß-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E ß-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects ß-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced ß-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic ß-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 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.

4-Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion.

4-Hydroxyisoleucine, a peculiar amino acid extracted from fenugreek seeds and never found in mammalian tissues, exhibits interesting insulinotropic activity. To investigate the structural requirements for this stimulating effect, the insulinotropic activity of the major isomer (2S,3R,4S) of 4-hydroxyisoleucine, in the presence of 8. 3 mM glucose, was compared to that of (1) its minor isomer (2R,3R, 4S) (2) its lactone form, (3) classical structurally related amino acids, and (4) synthetic monomethylated analogues. In the isolated, ex vivo, perfused rat pancreas, only the major isomer of 4-hydroxyisoleucine (200 microM) potentiated insulin release. On incubated isolated rat islets, the threshold concentration for a significant increase (P<0.05) in insulin release was 200 microM for (2S,3R,4S) 4-hydroxyisoleucine, 500 microM for (2S,4R) and (2S,4S) gamma-hydroxynorvalines as well as (2S,3S) and (2S,3R) gamma-hydroxyvalines, and 1 mM or more for other congeners. In conclusion, the insulinotropic properties of 4-hydroxyisoleucine, in the micromolar range, are seen only in the presence of the linear major isoform; they also require carbon alpha in S-configuration, full methylation and carbon gamma-hydroxylation.

4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties.

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 microM) 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.

Calcitonin gene-related peptide immunoreactivity in the rat efferent vestibular system during development.

The organization of the efferent fiber network during postnatal development was investigated by immunocytochemical detection of the calcitonin gene-related peptide (CGRP) in rat vestibular receptors from postnatal day 0 (PD 0) to adulthood. CGRP was detected at birth in a few efferent fibers below the sensory epithelia of cristae and maculae. Thereafter, the nerve fibers in the cristae progressively invaded the epithelia with an apex to base gradient from PD 2 to PD 4. There was also a rearrangement of the fibers during maturation of the efferent innervation, such that after reaching the surface of the epithelium, they turned back and moved towards the base of the sensory cells, producing numerous synaptic contacts. Analysis of surface preparations of utricules showed the irregular and asymmetric topographic organization of the efferent fiber network and the extensive, complex distribution of this innervation. The presence and broad distribution of CGRP in the epithelium at critical stages of development and synaptogenesis suggests that it is involved in the maturation of vestibular receptors.

4-Hydroxyisoleucine: a novel amino acid potentiator of insulin secretion.

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.

Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide.

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.

Mechanisms involved in the effect of nitric oxide synthase inhibition on L-arginine-induced insulin secretion.

1. A constitutive nitric oxide synthase (NOSc) pathway negatively controls L-arginine-stimulated insulin release by pancreatic beta cells. We investigated the effect of glucose on this mechanism and whether it could be accounted for by nitric oxide production. 2. NOSc was inhibited by N omega-nitro-L-arginine methyl ester (L-NAME), and sodium nitroprusside (SNP) was used as a palliative NO donor to test whether the effects of L-NAME resulted from decreased NO production. 3. In the rat isolated perfused pancreas, L-NAME (5 mM) strongly potentiated L-arginine (5 mM)-induced insulin secretion at 5 mM glucose, but L-arginine and L-NAME exerted only additive effects at 8.3 mM glucose. At 11 mM glucose, L-NAME significantly inhibited L-arginine-induced insulin secretion. Similar data were obtained in rat isolated islets. 4. At high concentrations (3 and 300 microM), SNP increased the potentiation of arginine-induced insulin output by L-NAME, but not at lower concentrations (3 or 30 nM). 5. L-Arginine (5 mM) and L-ornithine (5 mM) in the presence of 5 mM glucose induced monophasic beta cell responses which were both significantly reduced by SNP at 3 nM but not at 30 nM; in contrast, the L-ornithine effect was significantly increased by SNP at 3 microM. 6. Simultaneous treatment with L-ornithine and L-arginine provoked a biphasic insulin response. 7. At 5 mM glucose, L-NAME (5 mM) did not affect the L-ornithine secretory effect, but the amino acid strongly potentiated the alteration by L-NAME of L-arginine-induced insulin secretion. 8. L-Citrulline (5 mM) significantly reduced the second phase of the insulin response to L-NAME (5 mM) + L-arginine (5 mM) and to L-NAME + L-arginine + SNP 3 microM. 9. The intermediate in NO biosynthesis, NG-hydroxy-L-arginine (150-300 microM) strongly counteracted the potentiation by L-NAME of the secretory effect of L-arginine at 5 mM glucose. 10. We conclude that the potentiation of L-arginine-induced insulin secretion resulting from the blockade of NOSc activity in the presence of a basal glucose concentration (1) is strongly modulated by higher glucose concentrations, (2) is not due to decreased NO production but (3) is probably accounted for by decreased levels of NG-hydroxy-L-arginine or L-citrulline, resulting in the attenuation of an inhibitory effect on arginase activity.