The stimulus-secretion coupling of amino acid-induced insulin release

Summary Aminooxyacetate, an inhibitor of cytosolic transamination reactions, inhibited insulin release evoked by either 2-ketoisocaproate or L-leucine in rat pancreatic islets incubated in the presence of L-glutamine or L-asparagine. As a rule, aminooxyacetate also inhibited the oxidation of these nutrient secretagogues and impaired the respiratory response of the islets to the combinations of nutrients. However, the oxidative and secretory response to the combination of L-leucine and L-glutamine was less severely affected by aminooxyacetate than that evoked by the three other combinations of exogenous nutrients. These findings reinforce the view that the stimulus-secretion coupling of insulin release in response to L-leucine and 2-ketoisocaproate in association with either L-glutamine or L-asparagine tightly depends on the oxidation of these nutrient secretagogues, on their effect upon O₂ uptake and, within limits, on the intracellular site of generation of reducing equivalents in the pancreatic islet cells.This paper is the 16th in a series.     

Does leucine- and norleucine-induced insulin release depend on amino acid aminotransferase activity?

In the absence of another exogenous nutrient, L-leucine but not L-norleucine stimulates insulin release from rat pancreatic islets, although the corresponding keto acids, 2-ketoisocaproate and 2-ketocaproate, are equally potent secretagogues. Such a situation cannot be ascribed to the preferential transamination of L-leucine as compared to L-norleucine in islet homogenates. Indeed, in the presence of a suitable activator of glutamate dehydrogenase, L-leucine and L-norleucine stimulate secretion to the same extent. It is concluded that the rate of transamination of these amino acids in intact islet cells depends on the availability of a 2-keto acid partner rather than on the assayed amino acid aminotransferase activity.     

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.     

The stimulus-secretion coupling of amino acid-induced insulin release synergistic effects of L-glutamine and 2-keto acids upon insulin secretion

1. L-Glutamine markedly enhances insulin release evoked in rat pancreatic islets by 2-ketoisocaproate or 2-ketocaproate. L-Glutamine exerts a lesser enhancing action in the presence of 2-ketovalerate or 2-ketoisovalerate, which are themselves poor insulin secretagogues. L-Glutamine fails to affect insulin release in the presence of 2- ketobutyrate, pyruvate and β-hydroxybutyrate. 2. The relase of insulin evoked by the combination of L-glutamine and 2-ketoisocaproate represents a sustained phenomenon. It coincides with a stimulation of ⁴⁵Ca net uptake by the islets, and is inhibited in the absence of extracellular Ca²⁺ and presence of either menadione or epinephrine. 3. L-Valine inhibits insulin releaseevoked by either 2-ketoisocaproate or 2-ketocaproate, whether in the presence or absence of L-glutamine, but does not abolish the enhancing action of L-glutamine. L-Valine fails to affect insulin release evoked by the combination of L-leucine and L-glutamine. 4. L-Isoleucine also inhibits 2-keto acid-induced insulin release. However, in contrast to L-valine, L-isoleucine fails to affect or slightly augments insulin release in the simultaneous presence of L-glutamine and either 2-ketoisocaproate or 2-ketocaproate. 5. L-Leucine causes a dose-related enhancement of insulin release evoked by the combination of 2-ketoisocaproate and L-glutamine. Likewise, in the presence of L-glutamine, L-leucine and 2-ketocaproate act synergistically upon insulin release. 6. The hypothesis is advances that the enhancing action of L-glutamine upon 2-keto acid-stimulated insulin release depends on the availability of the 2-keto acid to act as a partner in the conversion of L-glutamate derived from exogenous L-glutamine to 2-ketoglutarate by transamination reaction, rather than being attributable to activation of glutamate dehydrogenase as observed in islets exposed to both L-glutamine and L-leucine.     

Stimulation of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid

An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [³H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.     

The stimulus-secretion coupling of amino acid-induced insulin release. Insulinotropic action of L-alanine

Available information on the fate and insulinotropic action of L-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na(+) co-transport in the insulinotropic action of L-alanine. In the present study conducted in islets prepared from normal adult rats, L-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or L-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of L-[U-14C]alanine was unaffected by D-glucose, but inhibited by L-leucine. Inversely, L-alanine decreased the oxidation of D-[U-14C]glucose, but failed to affect L-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na(+) co-transport in the secretory response to L-alanine cannot be ignored.     

Stimulation of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid

An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [³H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction of L-asparagine and L-leucine in pancreatic islets

Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. L-Asparagine augmented the oxidation of L-leucine, an effect possibly attributable to activation of 2-ketoisocaproate dehydrogenase. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

IRS-1 tyrosine phosphorylation reflects insulin-induced metabolic and mitogenic responses in 3T3-L1 pre-adipocytes

We determined the involvement of Tyr-1158 within the regulatory loop of the insulin receptor (IR) in the generation of insulin-specific responses in situ. For this purpose chimeric receptors with an epidermal growth factor (EGF) receptor extracellular domain and an IR cytoplasmic domain (EIR) were constructed, which allow activation of the cytoplasmic IR domain without activation of endogenous wt-IRs. Tyr-1158 of the chimera EIR was exchanged for Phe, creating a mutant chimeric receptor (EIR-Y1158F). Chimeric receptors were expressed in 3T3-L1 pre-adipocytes, which do not show insulin-specific responses upon EGF stimulation. We found that pre-adipocytes expressing EIR-Y1158F were impaired in their ability to stimulate glycogen synthesis and DNA synthesis upon maximal stimulation with EGF. EIR-Y1158F was impaired in its ability to phosphorylate insulin receptor substrate (IRS)-1 and induce downstream signals of IRS-1 phosphorylation, such as the association of IRS-1 with phosphatidyl-inositol-3'-kinase and the activation of protein kinase B (Akt). In contrast with the phosphorylation of IRS-1, the phosphorylation of IRS-2 and extracellular regulated protein kinase-1/-2 was normal in EIR-Y1158F expressing cells. These observations suggest that the level of IRS-1 phosphorylation rather than the level of IRS-2 phosphorylation mediates insulin-induced glycogen synthesis and DNA synthesis in 3T3-L1 pre-adipocytes.     

Molecular mechanisms of the alternative lipogenic function of insulin

The review discusses the hypothesis that a major function of insulin is to stimulate triglyceride accumulation in adipose tissue and glycogen synthesis in the liver and muscles. Malfunction of insulin decreases triglyceride storage in adipose tissue, while its extreme activation induces obesity. In either case, low-molecular-weight lipid metabolites, such as oxybutyrates, ketobutyrates, ketone bodies, etc., increase in content in peripheral tissues and are utilized as a preferable substrate in energy production, thus reducing the glucose uptake in cells. Leptin inhibits the lipogenic function of insulin and prevents lipid accumulation, while leptin deficiency or a decrease in leptin activity increases the lipid production and induces obesity. Lipodystrophy decreases leptin secretion by adipocytes and facilitates the lipogenic effect of insulin, but insulin does not stimulate the triglyceride accumulation in adipose tissue in the absence of subcutaneous fat. Lipid metabolites accumulate in peripheral organs and induce lipoatrophic diabetes mellitus. The hypothesis of the alternative mechanisms of insulin functioning is consented with the data obtained in mice with a targeted knockout of the insulin receptor gene in individual organs (muscles, adipose tissue, etc.) and transgenic animals with restored expression of the gene.     

Ultrastructural changes of the pancreatic beta-cell and the insulin secretion by islets from lactating and non-lactating rats

Islets isolated from lactating rats, as compared to islets from non-lactating rats, release less insulin when incubated in the absence of exogenous nutrient or presence of either D-glucose (11.1 mM) or the association of L-leucine and L-glutamine (10.0 mM each). The insulin content of the islets is not different in lactating and non-lactating rats. The volume density of the dark granules in the beta-cells is not at variance in both groups. However the volume density of the light (pale) granules is significantly lower in the lactating rats. The reduced amount of light granules is in keeping with the reduced secretory capacity of the beta-cells from lactating rats.     

Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.

This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.     

Insulin release from a cloned hamster B-cell line (HIT-T15). The effects of glucose, amino acids, sulphonylureas and colchicine

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.     

Mechanism of 3-phenylpyruvate-induced insulin release. Secretory, ionic and oxidative aspects.

1. 3-Phenylpyruvate caused a dose-related stimulation of insulin release from rat pancreatic islets deprived of exogenous nutrient or incubated in the presence of 5.6 or 8.3 mM-D-glucose. 2. 3-Phenylpyruvate inhibited insulin release evoked by high concentrations of D-glucose (16.7 or 27.8 mM) or 4-methyl-2-oxopentanoate (10.0 mM). This inhibitory effect appeared to be attributable to impairment of 2-oxo-acid transport into the mitochondria, with resulting inhibition of D-glucose, pyruvate or 4-methyl-2-oxopentanoate oxidation. 3. 3-Phenylpyruvate failed to affect the oxidation of, and secretory response to, L-leucine, and did not augment insulin release evoked by a non-metabolized analogue of the latter amino acid. 4. L-Glutamine augmented 3-phenylpyruvate-induced insulin release. The release of insulin evoked by the combination of 3-phenylpyruvate and L-glutamine represented a sustained phenomenon, abolished in the absence of extracellular Ca2+ or the presence of menadione and potentiated by theophylline. 5. Whether in the presence or in the absence of L-glutamine, the secretory response to 3-phenylpyruvate coincided with an increase in O2 uptake, a decrease in K+ conductance, a stimulation of both Ca2+ inflow and 45Ca2+ net uptake and an increase in cyclic AMP content. 6. It is concluded that the release of insulin induced by 3-phenylpyruvate displays features classically encountered when the B-cell is stimulated by nutrient secretagogues, and is indeed attributable to an increase in nutrient catabolism.     

Role of divalent metals in the activation and regulation of insulin receptor tyrosine kinase

Multiple equilibrium equations were solved to separate the individual effects of ionic divalent metals, free nucleotides and their chelated species on insulin receptor tyrosine kinase (IRTK). Basal IRTK is activated by divalent metal cations when present in excess of that required for substrate formation, indicating the presence of a divalent cation-dependent regulatory site on the kinase. The activatory order for basal activity was Mn2+ greater than Co2+ greater than Mg2+ and Ca2+ = 0. The insulin-dependent activation of IRTK was minimal in the absence of excess free divalent metal, even when the concentration of MnATP or MgATP substrate present exceeded the apparent Km of the kinase. The activatory order for insulin-dependent activation of IRTK changed to Mg2+ greater than Mn2+ and Co2+ = 0. The titration of the MnCl2 saturation response at several concentrations of MgCl2 revealed that the insulin-dependent response of IRTK increases as a function of increasing MgCl2, while basal activity was unaffected. This enhancement of the responsiveness to insulin in the presence of both cations was not due to differing affinities of the kinase for substrate, as evidenced by nearly identical apparent Km values for MnATP and MgATP. The Mg2+-dependent increase in the response of the kinase to insulin may be due to Mg2+ inducing a stronger coupling between receptor and kinase than that observed with Mn2+ alone. The plotting of the effect of several concentrations of free divalent metals on substrate saturation curves revealed that an increase in either of the reactants increased the affinity of the insulin-activated kinase for the other respective reactant. Accordingly, free divalent metal and metal-ATP substrate interact with IRTK in a mutually inclusive manner. CaCl2 saturation curves in the presence of constant MnCl2 and increasing MgCl2 showed that the affinity of IRTK for Ca2+ decreases and the affinity for CaATP increased with increasing Mg2+. Our data suggests that IRTK contains three sites for interaction with divalent metal cations: a MeATP (active) site, a regulatory site, and a metal-dependent site acting to couple the receptor with the kinase.     

Insulin stimulates the tyrosine phosphorylation of a 165 kDa protein that is associated with microsomal membranes of rat adipocytes

The beta-subunit of the insulin receptor possesses an insulin-stimulatable protein tyrosine kinase activity. It has been widely postulated that this activity may mediate the transduction of the insulin signal by phosphorylation of cellular substrates involved in the mechanism of insulin action. We have identified, by immunoblotting with antiphosphotyrosine antibodies, a 165 kDa protein in rat adipocytes that is rapidly phosphorylated in response to insulin. Phosphorylation of this protein (pp165) occurs within 5-10 s of exposure to 10 nM insulin, suggesting that it may be a direct substrate for the insulin receptor. This protein was recovered in an intracellular membrane that fractionates with the low-density microsomes. Using discontinuous sucrose density-gradient centrifugation, pp165-containing vesicles were separated from other vesicles of the low-density microsomes including the glucose transporter-containing vesicles, indicating that pp165 is probably not a regulatory component of the vesicles that translocate glucose transporters in response to insulin. However, pp165 may be involved in conveying receptor activation at the cell surface to an intracellular site of insulin action.     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation.

We have studied the phosphatidylinositol 3-kinase (PtdIns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (alpha PY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (Ruderman, N., Kapeller, R., White, M.F., and Cantley, L.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). Stimulation was maximal within 1 min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total alpha PY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the PtdIns 3-kinase, but in all cases the presence of PtdIns 3-kinase in alpha PY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IRA1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in alpha PY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr1146 in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in alpha PY-precipitable PtdIns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR delta 960) displayed normal in vivo autophosphorylation but failed to stimulate the PtdIns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR delta CT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in alpha PY immunoprecipitates. These data suggest that the PtdIns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the PtdIns 3-kinase furthermore suggests that the PtdIns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.     

LysB3, GluB29] insulin: a novel insulin analog with enhanced beta-cell protective action

Insulin receptor substrate (IRS)-2 has been implicated in the promotion of beta-cell survival. Here we tested the hypothesis that the novel analog [LysB3, GluB29] insulin (insulin glulisine, IG) might mediate an enhanced beta-cell protective effect due to its unique property of preferential IRS-2 phosphorylation. We assessed IRS activation by IG and its anti-apoptotic activity against cytokines or palmitic acid in comparison to insulin, insulin analogs, and insulin-like growth factor (IGF)-I using INS-1 cells. IG induced a prominent IRS-2 activation without significant IRS-1 stimulation. The marked cytokine- and fatty acid-induced apoptosis was strongly (55-60%) inhibited by IG both at the level of caspase 3 activation and nucleosomal release, with only 15% inhibition of apoptosis by regular insulin. At 1nM, insulin, insulin aspart, and insulin lispro were much less effective compared to IG. In conclusion, the prominent anti-apoptotic activity of insulin glulisine might serve to counteract autoimmune- and lipotoxicity-induced beta-cell destruction.     

Effects of second messengers on serine/threonine protein phosphatases in insulin-secreting cells

Reversible protein phosphorylation is an important and versatile mechanism by which cells transduce external signals into biological responses. Cellular levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). Compared with protein kinases, however, serine/threonine PPases have received less attention. In the present study, the effects of certain insulin secretagogues and intracellular second messengers, known to stimulate or inhibit insulin secretion, on the activities of cation-independent serine/threonine PPases were investigated in insulin-secreting RINm5F insulinoma cells. Raising cellular cAMP through adenylyl cyclase activation and phosphodiesterase inhibition in intact cells, evoked inhibitory effects on PPase activities. The addition of a nitric oxide donor, cyclic nucleotides, or proinflammatory prostaglandins to RINm5F cell homogenates at widely different concentrations did not affect type-1 or -2A PPase activities. Phosphatidyl serine seemingly activated PPase-1, while inactivating PPase-2A. A protein kinase C-activating phorbol ester produced the opposite results when added to RINm5F cell homogenates. These studies suggest that several known intracellular second messengers are without effect on beta-cell PPase activities. However, phosphatidyl serine and protein kinase C activation, whose activity is transiently increased by glucose, may promote insulin release through PPase inactivation, likely contributing to the increase in phosphorylation state that occurs after stimulation of insulin release. Thus, inhibition of protein dephosphorylation may be a novel regulatory mechanism, assisting in activation of the stimulus-secretion coupling in insulin-producing cells.