Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects.

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.     

Mechanism of 3-phenylpyruvate-induced insulin release from isolated pancreatic islets.

3-Phenylpyruvate evoked a monophasic insulin release from perifused mouse islets. L-Phenylalanine was not an insulin secretagogue and was oxidized by islets at a very low rate, suggesting that 3-phenylpyruvate does not trigger insulin release by enhancing production of reducing equivalents. Moreover, allosteric activation of glutamate dehydrogenase does not play a role in 3-phenylpyruvate-induced insulin secretion.     

Inhibition by L-valine and L-norleucine of 3-phenylpyruvate-induced insulin release

Insulin release induced by 3-phenylpyruvate in isolated rat pancreatic islets was inhibited by L-valine, L-norleucine or aminooxyacetate. The inhibitory effect of these three agents coincided with a lesser stimulation by 3-phenylpyruvate of 14CO2 output from islets prelabelled with L-[U-14C] glutamine. Conversely, 3-phenylpyruvate augmented the rate of conversion of L-valine to 2-ketoisovalerate and that of L-norleucine to 2-ketocaproate. However, 3-phenylpyruvate, which increased 2-ketoisovalerate oxidative decarboxylation, inhibited 14CO2 production by islets exposed to D, L-[1-14C] norleucine. These findings reveal that distinct nutrient secretagogues (e.g. 3-phenylpyruvate and L-norleucine), which are each able to stimulate insulin release, may act antagonistically upon the secretory process when used in combination. The present results also emphasize the relevance of both mitochondrial oxidation and intracellular transfer of reducing equivalents as determinants of the secretory response to such nutrients as 3-phenylpyruvate and norleucine.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Regulation of insulin release by ionic and electrical events in B cells

This review article is an attempt to schematize the major alterations in ionic fluxes and B cell membrane potential that underlie the changes in insulin release brought about by glucose and by other stimulators or inhibitors. Glucose metabolism in B cells leads to closure of K channels in the plasma membrane. The resulting decrease in K+ permeability causes depolarization with activation of voltage-dependent Ca channels. An increase in Ca2+ influx ensues, which raises the cytoplasmic concentration of free Ca2+ and ultimately triggers insulin release. Tolbutamide induces a similar sequence of events by a direct action on K channels. In contrast, diazoxide antagonizes the effects of glucose by increasing K+ permeability of the B cell membrane. Among amino acids, leucine largely mimics the effects of glucose, whereas arginine depolarizes the B cell membrane because of its transport in a positively charged form.     

The effects of cesium chloride on insulin release, ionic fluxes and membrane potential in pancreatic B-cells

Cs+ decreases K+ permeability in nerve and muscle cells. Its effects on the pancreatic B-cell function were studied with mouse islets. In the presence of 3 mM glucose, Cs+ substitution for K+ steadily inhibited 86Rb+ efflux and hyperpolarized the B-cell membrane. Addition of Cs+ to a K+-medium also inhibited 86Rb+ efflux, but depolarized the B-cell membrane. None of these changes altered insulin release. Substitution of Cs+ for K+ in a medium containing 10 mM glucose caused a Ca2+-dependent stimulation of insulin release and 45Ca2+ efflux, produced an initial fall and a secondary rise in 86Rb+ efflux and augmented the electrical activity in B-cells. Reintroduction of K+ to the medium was followed by a marked and transient inhibition of insulin release, that was blocked by ouabain and accompanied by an inhibition of 45Ca2+ and 86Rb+ efflux and by a hyperpolarization of the B-cell membrane. Addition of Cs+ to a K+ medium containing 10 mM glucose stimulated insulin release, 45Ca2+ efflux and 86Rb+ efflux. It also increased the electrical activity in B-cells. In the absence of Ca2+, however, Cs+ addition decreased the rate of 86Rb+ efflux. The effects of Cs+ on the B-cell function may be explained by its ability to decrease K+ permeability of the plasma membrane, by its inability to activate the sodium pump, and by a third unidentified effect likely brought about by the accumulation of intracellular Cs+.     

Secretory B-cell activity in insulin dependent maturity-onset diabetic.

In 10 insulin dependent maturity onset diabetics we found elevated basal C-Peptide levels (4.78 +/- 0.5 ng/ml. Normal range 1.1--3.6 ng/ml), which could be suppressed by insulin injection to the same extent as in sulfonylurea treated diabetics could be demonstrated. C-Peptide immunoreactivity in these patients therefore seems to be newly secreted rather than accumulated material. Since adrenalectomized patients could be suppressed in the same way, it is likely, that catecholamines are not the major factor in the mechanism of suppression. Therefore only decrease of bloodsugar levels seems to be accountable for the decrease of C-Petide levels. High C-Petide levels in insulin dependent maturity onset diabetics which cannot be stimulated but suppressed may be explained by a loss of glucoreceptor molecules.     

Secretory granule mediator release and generation of oxidative metabolites of arachidonic acid via Fc-IgG receptor bridging in mouse mast cells.

The releases of beta-hexosaminidase, LTC4, LTB4, and PGD2 after the bridging of Fc gamma R3 were assessed in mouse IL-3-dependent bone marrow-derived progenitor mast cells (BMMC), BMMC maintained in coculture with 3T3 fibroblasts separated by a filter to achieve maturation of the granules toward those of a serosal mast cell (SMC), and SMC that are the prototype of a mouse connective tissue mast cell. Bridging of Fc gamma R on BMMC with the 2.4G2 rat anti-Fc gamma RII/III mAb and anti-rat IgG elicited only 4% net release of beta-hexosaminidase and 4, 2, and 1 ng/10(6) cells of immunoreactive LTC4, LTB4, and PGD2, respectively. Bridging of Fc-IgE receptors (Fc epsilon R) on BMMC yielded 35% net release of beta-hexosaminidase and 9, 4, and 3 ng/10(6) cells of immunoreactive LTC4, LTB4, and PGD2, respectively. BMMC maintained in coculture responded to the bridging of Fc gamma R with statistically significant increases in the net percent release of beta-hexosaminidase to 16% and in the generation of immunoreactive LTC4 to 11 ng/10(6) cells, but without a significant change in the production of either LTB4 or PGD2. Bridging of Fc epsilon R on cocultured mast cells yielded a net percent release of beta-hexosaminidase and lipid mediator amounts and profile similar to those for BMMC. Bridging of Fc gamma R on purified mouse SMC resulted in a maximal net percent release of beta-hexosaminidase of 10% and the generation of 4, 1, and 17 ng/10(6) cells of immunoreactive LTC4, LTB4, and PGD2, respectively; the net percent release of beta-hexosaminidase and PGD2 generation were significantly greater than those obtained from BMMC. The Fc epsilon R-mediated net percent release of beta-hexosaminidase from purified SMC was 34%, with PGD2 being the predominant metabolite of arachidonic acid. That the predominant lipid mediator generated with activation by either Fc gamma R or Fc epsilon R is LTC4 for cocultured mast cells and PGD2 for SMC suggests that the mast cell phenotype rather than the receptor class being bridged determines the lipid mediator profile. The responsiveness to Fc gamma R bridging elicited by coculture of BMMC with fibroblasts in vitro and present in SMC derived in vivo relative to BMMC may relate to the previously measured increases in receptor number per cell, but may also involve the acquisition of an enhanced signal transduction capability, possibly through the increased expression of Fc gamma RIII.     

Selenium, oxidative stress, and health aspects

Metabolic processes which generate oxidants and antioxidants are governed by genetic disposition as well as environmental factors. Changes in lifestyle, including increased environmental pollution, sun exposure, and dietary habits modify the challenge of the organism by reactive oxygen species. Defense mechanisms are reinforced by increasing dietary intake of antioxidants and micronutrients such as vitamins and selenium (Se). Se deficiency has been recognized to promote some disease states. Epidemiological findings link a lowered Se status to neurodegenerative and cardiovascular diseases as well as to increased cancer risk. While evidence exists to suggest that additional selenocompounds would be beneficial in some health conditions, results from future intervention trials are needed to substantiate the argument for increasing Se intake. Several pieces of the puzzle concerning the molecular mechanisms underlying the reactive oxygen species-triggered disease state and intervention by enzymatic antioxidants have been elucidated. A novel concept of protection of stromal cells against the dominating influence of tumor cells in tumor-stroma interaction by selenocompounds and other antioxidants is presented herein, which may translate into therapeutic strategies in chemoprevention of tumor invasion.     

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.     

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na+ on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na+ hyperpolarized the B-cell membrane, inhibited 86Rb+ and 45Ca2+ efflux, but did not affect insulin release. In contrast, Li+ substitution for Na+ depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca2+ efflux and insulin release. Na+ replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca2+ efflux and insulin release. In the absence of Ca2+, 45Ca2+ and 86Rb+ efflux were inhibited and insulin release was unaffected by choline substitution for Na+. Na+ replacement by Li+ in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca2+ efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca2+, Li+ still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na+ plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.     

3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol inhibit leucine-stimulated insulin release from rat pancreatic islets

Individual islets were isolated from rat pancreas to study the effects of tryptophan and its metabolites on leucine-stimulated release of insulin. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol were inhibitors at concentrations below 10 mM whereas tryptophan, kynurenine, kynurenic acid, xanthurenic acid, and anthranilic acid were ineffective inhibitors at concentrations up to 10 mM. A structure-activity analysis of these metabolites demonstrated that vicinal aromatic hydroxy and amino groups with their concomitant electron donating properties are required for inhibition of insulin release. Inhibition of islet insulin release by the three kynurenine metabolites may be involved in the depressed insulin levels found in vitamin B6-deficient rats by other workers.     

Metabolic and ionic coupling factors in amino acid-stimulated insulin release in pancreatic beta-HC9 cells

Fuel stimulation of insulin secretion from pancreatic beta-cells is thought to be mediated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides, and perhaps certain amino acids (AA), as for instance aspartate, glutamate, or glutamine (Q). The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or AA, alone or combined, using (31)P, (23)Na and (1)H NMR technology, respirometry, and biochemical analysis to study the metabolic events that occur in continuously superfused mouse beta-HC9 cells contained in agarose beads and enhanced by the phosphodiesterase inhibitor IBMX. Exposing beta-HC9 cells to high glucose or 3.5 mM of a physiological mixture of 18 AA (AAM) plus 2 mM glutamine caused a marked stimulation of insulin secretion associated with increased oxygen consumption, cAMP release, and phosphorylation potential as evidenced by higher phosphocreatine and lower P(i) peak areas of (31)P NMR spectra. Diazoxide blocked stimulation of IR completely, suggesting involvement of ATP-dependent potassium (K(ATP)) channels in this process. However, levels of MgATP and MgADP concentrations, which regulate channel activity, changed only slowly and little, whereas the rate of insulin release increased fast and very markedly. The involvement of other candidate coupling factors was therefore considered. High glucose or AAM + Q increased pH(i). The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/P(i) x ADP) in fuel-stimulated IR. Intracellular Na+ levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornanecarboxylic acid (which activates glutamate dehydrogenase) decreased beta-cell Na levels. Stimulation of beta-cells by glucose in the presence of AAM + Q (0.5 mM) was associated with rising cellular concentrations of glutamate and glutamine and strikingly lower aspartate levels. Methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q-induced IR and associated changes in glutamine and aspartate but did not prevent the accumulation of glutamate. The results of this study demonstrate again that an increased phosphate potential and a functional K(ATP) channel are essential for metabolic coupling during fuel-stimulated insulin release but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet.     

Effects of a beta 3-adrenoceptor agonist, BRL 26830A, on insulin and glucagon release in mice.

The beta 3-adrenoceptor agonist, BRL 26830A, which is not inhibited by either beta 1 or beta 2-selective antagonists, has been shown to possess anti-obesity and anti-diabetic actions. However, the effects of this agent on insulin and glucagon release have not yet been substantiated. Therefore, we tested the hypothesis that BRL 26830A promotes insulin and glucagon secretion via beta 3 receptors on pancreatic islet B and A cells. In ICR mice fasted for 48 h, BRL 26830A significantly stimulated insulin secretion from 5 min after administration, markedly decreased blood glucose levels from 30 min after administration, and significantly increased glucagon secretion from 30 min after administration. The administration of a non-selective beta-receptor antagonist, at a dose of 50 mg/kg, 30 min prior to BRL 26830A injection completely abolished the effects induced by BRL 26830A. However, the administration of a beta 1-selective antagonist at doses of 50 or 100 mg/kg did not produce any significant effects. On the action of BRL 26830A, whereas the administration of a beta 2-selective antagonist at 50 mg/kg, a near maximal effective dose, partially abolished the effects of BRL 26830A. BRL 26830A had no effect on insulin, glucagon, or glucose levels in streptozocin (STZ) diabetic mice fasted for 48 h. These results suggest that, in mice, BRL 26830A may promote insulin secretion mainly via beta 3 receptors and partially via beta 2 receptors on pancreatic-islet B cells, and that glucagon may be secreted as the result of hypoglycemia induced by this agent.     

Secretory processes, carbohydrate and lipid metabolism in isolated mouse hepatocytes. Aspects of regulation by glucagon and insulin.

The procedure of Berry and Friend for isolation of intact hepatocytes has been adapted to mouse livers. The ultrastructure of these cells was satisfactorily preserved. Isolated mouse hepatocytes secreted proteins and triacylglycerols. These secretory processes were inhibited by colchicine, indicating a likely involvement of the microtubular system for their normal occurrence. Ultracentrifugation of medium incubated with hepatocytes, followed by electrophoresis and electron microscopic examination of the floating fraction (density less than 1.006) allowed to conclude that secreted triacylglycerols were very low density lipoproteins. Glycogenolysis and lipogenesis were stimulated or inhibited, respectively, by low concentrations of glucagon (10(-10) M). Other metabolic parameters were influenced by the hormone but were less sensitive to its action. Inhibition of lipogenesis by glucagon was associated with a decrease in acetyl CoA carboxylase activity. This decrease does not appear to be related to intracellular fatty acyl-CoA accumulation secondary to hepatic lipase activation by the hormone. Insulin was effective alone or counteracted glucagon effects on lipogenesis or glycogenolysis only when exposure of cells to collagenase was held minimal. This suggests that, during isolation of hepatocytes, insulin receptors may, for unknown reasons, be more fragile than those of glucagon.     

Iron release, oxidative stress and erythrocyte ageing

Iron, to be redox cycling active, has to be released from its macromolecular complexes (ferritin, transferrin, hemoproteins, etc.). Iron is released from hemoglobin or its derivatives in a nonprotein-bound, desferrioxamine-chelatable form (DCI) in a number of conditions in which the erythrocytes are subjected to oxidative stress. Such conditions can be related to toxicological events (haemolytic drugs) or to physiological situations (erythrocyte ageing, reproduced in a model of prolonged aerobic incubation), but can also result from more subtle circumstances in which a state of ischemia-reperfusion is imposed on erythrocytes (e.g., childbirth). The released iron could play a central role in oxidation of membrane proteins and senescent cell antigen (SCA) formation, one of the major pathways for erythrocyte removal. Iron chelators able to enter cells (such as ferrozine, quercetin, and fluor-benzoil-pyridoxal hydrazone) prevent both membrane protein oxidation and SCA formation. The increased release of iron observed in beta-thalassemia patients and newborns (particularly premature babies) suggests that fetal hemoglobin is more prone to release iron than adult hemoglobin. In newborns the release of iron in erythrocytes is correlated with plasma nonprotein-bound iron and may contribute to its appearance.     

K+ channel openers and insulin release.

Recent in vivo and in vitro experiments suggested that the smooth muscle relaxation mediated by diverse pharmacologic agents resulted from K+ channel opening. Pinacidil, cromakalim, nicorandil, RP 49356, minoxidil sulfate and diazoxide belong to this new group of smooth muscle relaxants: the "K+ channel openers". Because modifications in the K+ permeability are known to represent a critical event in the insulin-releasing process, numerous studies have been performed in order to examine the putative effects of K+ channel openers on B-cell function. The aim of the present review is to summarize these experimental data which are sometimes divergent.     

Chronic insulin treatment causes insulin resistance in 3T3-L1 adipocytes through oxidative stress

Insulin resistance and hyperinsulinemia are commonly present in obesity and pre-diabetes, and hyperinsulinemia is both a marker and a cause for insulin resistance. However, the molecular link between hyperinsulinemia and insulin resistance remains elusive. The present study examined the effect of chronic insulin treatment on the reactive oxygen species (ROS) production, insulin signalling and insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The results showed that chronic insulin treatment significantly increased the intracellular generation of superoxide anion, hydrogen peroxide and hydroxyl radical. ROS induced by chronic insulin treatment inhibited insulin signalling and glucose uptake, induced endoplasmic reticulum (ER) stress and JNK activation. Furthermore, these effects were reversed by antioxidants N-acetylcysteine, superoxide dismutase or catalase. These results suggested that ROS, ER stress and JNK pathway are involved in insulin resistance induced by chronic insulin treatment. Therefore, oxidative stress could be a potential interventional target for hyperinsulinemia-induced insulin resistance and related diseases.