Intracellular pH and the stimulus-secretion coupling in insulin-producing RINm5F cells.
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ABSTRACT:
SUBMITTER: Juntti-Berggren L
PROVIDER: S-EPMC1133123 | biostudies-other | 1992 Oct
REPOSITORIES: biostudies-other
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Project description:The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.
| S-EPMC1149756 | biostudies-other
Project description:1. Pancreatic islet insulin secretion and 45Ca uptake showed similar responses to variation in the extracellular concentration of 4-methyl-2-oxopentanoate with a threshold at 4 mM and a maximal response at a 25 mM concentration. 2. Islet respiration, acetoacetate production and rates of substrate utilization, oxidation and amination all changed as a simple hyperbolic function of 4-methyl-2-oxopentanoate concentration and exhibited a maximal response at 25 mM. 3. The responses of ATP content, [ATP]/[ADP] ratio, adenylate energy charge and [NADH]/[NAD+] ratio were also hyperbolic in nature but were maximally elevated at lower concentrations of the secretagogue. The islet [NADPH]/[NADP+] ratio, however, was tightly correlated with parameters of metabolic flux, 45Ca uptake and insulin release. 4. NH4+ and menadione, agents that promote a more oxidized state in islet NADP, did not affect islet ATP content or the rates of [U-14C]4-methyl-2-oxopentanoate oxidation or amination, but markedly inhibited islet 45Ca uptake and insulin release. 5. It is proposed that changes in the redox state of NADP and Ca transport may serve as mediators in the stimulus-secretion coupling mechanism of insulin release induced by 4-methyl-2-oxopentanoate.
| S-EPMC1161765 | biostudies-other
Project description:Glucagon-like peptide-1-based (GLP-1-based) therapies improve glycemic control in patients with type 2 diabetes. While these agents augment insulin secretion, they do not mimic the physiological meal-related rise and fall of GLP-1 concentrations. Here, we tested the hypothesis that increasing the number of intestinal L cells, which produce GLP-1, is an alternative strategy to augment insulin responses and improve glucose tolerance. Blocking the NOTCH signaling pathway with the ?-secretase inhibitor dibenzazepine increased the number of L cells in intestinal organoid-based mouse and human culture systems and augmented glucose-stimulated GLP-1 secretion. In a high-fat diet-fed mouse model of impaired glucose tolerance and type 2 diabetes, dibenzazepine administration increased L cell numbers in the intestine, improved the early insulin response to glucose, and restored glucose tolerance. Dibenzazepine also increased K cell numbers, resulting in increased gastric inhibitory polypeptide (GIP) secretion. Using a GLP-1 receptor antagonist, we determined that the insulinotropic effect of dibenzazepine was mediated through an increase in GLP-1 signaling. Together, our data indicate that modulation of the development of incretin-producing cells in the intestine has potential as a therapeutic strategy to improve glycemic control.
| S-EPMC4382243 | biostudies-literature
Project description:Stimulus-secretion coupling (SSC) in endocrine cells remains underappreciated as a subject for the study/teaching of general physiology. In the present article, we review key new electrophysiological, electrochemical, and fluorescence optical techniques for the study of exocytosis in single cells that have made this a fertile area for recent research. Based on findings using these techniques, we developed a model of SSC for adrenal chromaffin cells that blends features of Ca(2+) entry-dependent SSC (characteristic of neurons) with G protein receptor-coupled, Ca(2+) release-dependent, and second messenger-dependent SSC (characteristic of epithelial exocrine cells and nucleated blood cells). This model requires two distinct pools of secretory graunules with differing Ca(2+) sensitivities. We extended this model to account for SSC in a wide variety of peripheral and hypothalamic/pituitary-based endocrine cells. These include osmosensitive magnocellular neurosecretory cells releasing antidiuretic hormone, stretch-sensitive atrial myocytes secreting atrial natriuretic peptide, K(+)-sensitive adrenal glomerulosa cells secreting aldosterone, Ca(2+)-sensitive parathyroid chief cells secreting parathyroid hormone, and glucose-sensitive beta- and alpha-cells of pancreatic islets secreting insulin and glucagon, respectively. We conclude this article with implications of this approach for pathophysiology and therapeutics, including defects in chief cell Ca(2+) sensitivity, resulting in the hyperparathyroidism of renal disease, and defects in biphasic insulin secretion, resulting in diabetes mellitus.
| S-EPMC3747786 | biostudies-literature
Project description:1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.
| S-EPMC1164811 | biostudies-other
Project description:Insulin receptors on RINm5F cell membranes (an insulin-producing rat pancreatic cell line) were studied. To study the insulin receptor alpha-subunit, 125I-labelled photoreactive insulin was covalently bound to the membranes in the absence or presence of unlabelled insulin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions showed specific labelling of an Mr 130 000 protein. The receptor beta-subunit was studied by using a cell-free phosphorylation assay. Analysis under reducing conditions showed a phosphoprotein of Mr 95 000 whose level of phosphorylation was selectively increased by insulin, and which was specifically immunoprecipitated by antibodies to the insulin receptor. Further, covalent hormone-receptor complexes purified with anti-insulin antibodies were able to undergo autophosphorylation, indicating the existence of operational receptor subunit arrangements. RINm5F cell insulin receptors (and, by analogy, possibly those of native B-cells) thus display structural and functional integrity comparable with those of conventional insulin target cells.
| S-EPMC1144788 | biostudies-other
Project description:In pancreatic ?-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca(2+) influx, which triggers insulin exocytosis. The mitochondrial Ca(2+) uniporter (MCU) mediates Ca(2+) uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal ?-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca(2+) rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca(2+). Suppression of the putative Ca(2+)/H(+) antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca(2+)-induced matrix acidification. These results demonstrate that MCU-mediated Ca(2+) uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.
| S-EPMC4326818 | biostudies-literature
Project description:1. When pancreatic islets were exposed to a K(+)-free medium, the intracellular concentration of K(+) was decreased and that of Na(+) increased. 2. In the K(+)-deprived islets, the utilization of [5-(3)H]glucose, output of lactic acid and oxidation of [U-(14)C]-glucose were decreased by about 30-40% below the control values found at normal extracellular K(+) concentration (5.0mm). However, the oxidation of [U-(14)C]pyruvate was unaffected. 3. The omission of extracellular K(+) little affected the production of (14)CO(2) from islets prelabelled with [U-(14)C]palmitate and incubated in the absence of glucose, despite the fact that K(+) deprivation significantly increased the ATP concentration and ATP/ADP concentration ratio in the glucose-deprived islets. 4. At normal K(+) concentration, glucose increased the concentrations of phosphoenolpyruvate, NAD(P)H and ATP in the islets. In the glucose-stimulated islets, the concentration of phosphoenolpyruvate, but not that of either NAD(P)H or ATP, was higher in the absence than in the presence of extracellular K(+). In islet homogenates, the activity of pyruvate kinase (EC 2.7.1.40) was stimulated by K(+) (optimal activity at 100-150mm-K(+)) and inhibited by Na(+) (except at very low K(+) concentrations). 5. K(+) could be replaced by NH(4) (+), Rb(+), Cs(+) or Na(+) to maintain, at least to some extent, pyruvate kinase activity in islet homogenates. Addition of Rb(+) or Cs(+), but not NH(4) (+), to K(+)-deprived media also increased [U-(14)C]glucose oxidation by intact islets. 6. The omission of K(+) did not cause any obvious anomaly in the apparent dependency of (45)Ca(2+) net uptake on NAD(P)H concentration in the islets. 7. These data suggest that the coupling between metabolic and ionic events in the islet cells involves feedback mechanisms through which glucose oxidation may be modulated by cationic factors.
| S-EPMC1161518 | biostudies-other
Project description:1. In isolated pancreatic islets, pyruvate causes a shift to the left of the sigmoidal curve relating the rate of insulin release to the ambient glucose concentration. The magnitude of this effect is related to the concentration of pyruvate (5--90 mM) and, at a 30 mM concentration, is equivalent to that evoked by 2 mM-glucose. Pyruvate also enhances insulin release in the presence of fructose, leucine and 4-methyl-2-oxopentanoate. 2. In the presence of glucose 8 mM), the secretory response to pyruvate is an immediate process, displaying a biphasic pattern. 3. The insulinotropic action of pyruvate coincides with an inhibition of 45Ca efflux and a stimulation of 45Ca net uptake. The relationship between 45Ca uptake and insulin release displays its usual pattern in the presence of pyruvate. 4. Exogenous pyruvate rapidly accumulates in the islets in amounts close to those derived from the metabolism of glucose. The oxidation of [2-14C]pyruvate represents 64% of the rate of [1-14C]pyruvate decarboxylation and, at a 30 mM concentration, is comparable with that of 8 mM-[U-14C]glucose. 5. When corrected for the conversion of pyruvate into lactate, the oxidation of 30 mM-pyruvate corresponds to a net generation of about 314 pmol of reducing equivalents/120 min per islet. 6. Pyruvate does not affect the rate of glycolysis, but inhibits the oxidation of glucose. Glucose does not affect pyruvate oxidation. 7. Pyruvate (30 mM) does not affect the concentration of ATP, ADP and AMP in the islet cells. 8. Pyruvate (30 mM) increases the concentration of reduced nicotinamide nucleotides in the presence but not in the absence of glucose. A close correlation is seen between the concentration of reduced nicotinamide nucleotides and the net uptake of 45Ca. Menadione inhibits the effect of pyruvate on insulin release, without altering its rate of oxidation. 9. Pyruvate, like glucose, modestly stimulates lipogenesis. 10. Pyruvate, in contrast with glucose, markedly inhibits the oxidation of endogenous nutrients. The latter effect accounts for the apparent discrepancy between the rate of pyruvate oxidation and the magnitude of its insulinotropic action. 11. Dichloroacetate fails to affect glucose oxidation and glucose-stimulated insulin release. 12. It is concluded that the effect of pyruvate to stimulate insulin release depends on its ability to increase the concentration of reduced nicotinamide nucleotides in the islet cells.
| S-EPMC1186223 | biostudies-other
Project description:Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing ?-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, ?-cell proliferation, and some not previously linked to insulin production or ?-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.
| S-EPMC4012477 | biostudies-literature