Permeabilized Islets Research Articles

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Diphenylhydantoin Suppresses Glucose-Induced Insulin Release by Decreasing Cytoplasmic H+ Concentration in Pancreatic Islets

Diphenylhydantoin (DPH), which is clinically used in the treatment of epilepsy, inhibits glucose-induced insulin release from pancreatic islets by a mechanism that remains unknown. In the present study, DPH is shown to suppress glucose-induced insulin release concentration-dependently. In dynamic experiments, 20 microm DPH suppressed 16.7 mm glucose-induced biphasic insulin release. DPH also suppressed insulin release in the presence of 16.7 mm glucose, 200 microm diazoxide, and 30 mm K+ without affecting the intracellular Ca2+ concentration. DPH suppressed ATP content and mitochondrial membrane hyperpolarization in the presence of 16.7 mm glucose without affecting glucose utilization, glucose oxidation, and reduced nicotinamide adenine dinucleotide phosphate fluorescence. DPH increased cytoplasmic pH in the presence of high glucose, but the increase was abolished under Na+ -deprived conditions and HCO3- -deprived conditions, suggesting that Na+ and HCO3- transport across the plasma membrane are involved in the increase in cytoplasmic pH by DPH. Alkalization by adding NH4+ to the extracellular medium also suppressed insulin release, ATP content, and mitochondrial membrane hyperpolarization. Because ATP production from the mitochondrial fraction in the presence of substrates was decreased by increased pH in the medium, DPH suppresses mitochondrial ATP production by reducing the H+ gradient across mitochondrial membrane. Using permeabilized islets, the increase in pH was shown to decrease Ca2+ efficacy at a clamped concentration of ATP in the exocytotic system. Taken together, DPH inhibits glucose-induced insulin secretion not only by inhibiting mitochondrial ATP production, but also by reducing Ca2+ efficacy in the exocytotic system through its alkalizing effect on cytoplasm.

Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose.

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca(2+) concentration, because even in the absence of ATP at low Ca(2+), the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an alpha-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca(2+), suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.

Depolarizing Stimuli Reduce Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Islets of Langerhans

Elevations in intracellular Ca2+ ([Ca2+]i) initiate insulin secretion from pancreatic β-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca2+]i. We have investigated the mechanisms underlying the Ca2+ desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca2+/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca2+-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca2+-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for β-cell function.

An endogenous peptide isolated from the gut, NK-lysin, stimulates insulin secretion without changes in cytosolic free Ca 2+ concentration

We have recently isolated and cloned a novel endogenous peptide from pig intestine, NK-lysin (NKL). In the present study we show that NKL (1–100 nM) potently and reversibly stimulates insulin secretion in rat pancreatic islets and in the β-cell line HIT T15. This effect of NKL was not accompanied by changes in cytoplasmic free calcium concentration. The stimulatory activity of NKL on insulin release was also observed in permeabilized islets under Ca 2+-clamped conditions. Preincubation of HIT T15 cells with NKL for 1 h or 24 h did not influence cell viability. Possible mechanisms of insulinotropic activity of NKL are discussed.

Signaling and sites of interaction for RX-871024 and sulfonylurea in the stimulation of insulin release.

The objective of this study was to compare effects of RX-871024, a compound with imidazoline structure, and the sulfonylurea glibenclamide, representatives of two groups of ATP-dependent potassium channel (KATP) blockers, on insulin secretion and cytoplasmic free calcium concentration ([Ca2+]i). Furthermore, we studied the interaction of the compounds on these two parameters. The experiments were performed in the perfused rat pancreas, isolated rat pancreatic islets, and dispersed beta-cells. At maximal effective concentrations of the compounds, RX-871024 had a more pronounced insulinotropic effect than glibenclamide, but the increase in [Ca2+]i was similar. Glibenclamide enhanced the insulinotropic effect of suboptimal concentrations of RX-871024 at 3.3 and 16.7 mM glucose. Notably, glibenclamide and RX-871024 also stimulated insulin secretion under Ca(2+)-clamped conditions, i.e., during plasma membrane depolarization with KCl and glucose or in permeabilized islets. The magnitudes of insulin stimulation under the latter types of conditions were similar for both compounds. It is concluded that RX-871024 and the sulfonylurea glibenclamide promote insulin secretion by two mechanisms, namely closure of KATP channels and a direct stimulation of exocytosis. At a similar increase in [Ca2+]i, the maximal stimulatory effect of RX-871024 on insulin secretion was stronger than that of glibenclamide, implying that RX-871024 also affects insulin secretion by a signal transduction pathway that is not activated by glibenclamide.

Hexokinase and glucokinase activity in cross-linked and permeabilized pancreatic islets

The activities of hexokinase and glucokinase were measured in cross-linked and permeabilized rat pancreatic islets. After exposure to dimethyl suberimidate (20 mM) and digitonin (0.4 mM), the activity of hexokinase represented about half of that found in homogenates of freshly isolated islets. The K m of hexokinase for d-glucose and the K i for its inhibition by d-glucose-6-phosphate were similar, however, in the cross-linked and permeabilized islets and in homogenates of freshly isolated islets. Glucokinase activity also was documented in the cross-linked and permeabilized islets, it being less sensitive than hexokinase activity to inhibition by d-glucose-6-phosphate. At a high concentration of d-glucose (16.7 mM), the phosphorylation of the hexose failed to be increased by d-fructose-1-phosphate, whether in the absence or presence of d-glucose-6-phosphate. These findings indicate that the intrinsic properties of hexokinase isoenzymes are preserved in cross-linked islets, but suggest that the cross-linking of proteins prevents the activation of glucokinase by its regulatory protein.

Glucose-6-phosphatase activity in islets from ob/ob and lean mice and the effect of dexamethasone

In previous studies we demonstrated a much greater rate of glucose cycling (glucose—>glucose-6-P—>glucose) in islets from ob/ob mice than from lean litter mates. Cycling was further augmented by dexamethasone treatment. To determine whether these findings could be accounted for by increased islet glucose-6-phosphatase activity, we have now measured that enzyme's activity in permeabilized and sonicated islets and in islet microsomes. Activity in permeabilized islets from ob/ob mice was 19 times more than from lean litter mates (17.7 +/- 2.9 vs. 0.9 +/- 0.2 pmol/islet/min). Activity was 6 times higher when calculated per microgram of protein or microgram of DNA. Treatment of ob/ob mice with dexamethasone (25 micrograms/daily for 3 days) increased activity 2- to 3-fold. Activities were about twice as much in sonicated as permeabilized islets. There was no difference between glucose-6-phosphatase activity in microsomes prepared from islets of ob/ob and from lean mice, and the activity was relatively low. Thus, permeabilized islets can be used to determine glucose-6-phosphatase activity and study its regulation. The higher glucose cycling in islets of ob/ob mice and its stimulation by dexamethasone can be attributed to increased glucose-6-phosphatase activity.

The mechanism of arachidonic acid-induced insulin secretion from rat islets of Langerhans

The stimulation of rat pancreatic islets by glucose leads both to the secretion of insulin, and the production of arachidonic acid (AA). We have previously shown that exogenous AA can stimulate insulin secretion and that this secretion was not dependent upon extracellular Ca 2+ nor upon the activation of protein kinase C. We have now demonstrated that AA-induced insulin secretion was a saturable and reversible process. AA-stimulated insulin secretion was slow in onset from intact islets but immediate from electrically permeabilized islets. In permeabilized islets AA-induced insulin secretion was not dependent on changes in intracellular Ca 2+ or ATP and was not inhibited by noradrenaline. These results suggest that AA affects insulin secretion at a late stage in the exocytotic process.

Effects of okadaic acid on insulin secretion from rat islets of Langerhans

We have studied the effects of the phosphatase inhibitor, okadaic acid, on insulin secretion and protein phosphorylation in intact and electrically permeabilized pancreatic islets. Okadaic acid inhibited glucose-induced insulin secretion from intact islets, although this effect was probably non-specific since similar effects were obtained using 1-nor-okadaone, a virtually inactive analogue of okadaic acid. In permeabilized islets, okadaic acid enhanced basal and cyclic-AMP-induced insulin secretion and protein phosphorylation. These results indicate that protein phosphatases may play a role in the regulation of insulin release.

Ca2(+)-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2(+)-induced secretory response is accompanied by loss of Ca2(+)-induced protein phosphorylation.

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

Effects of IP3, spermine, and Mg2+ on regulation of Ca2+ transport by endoplasmic reticulum and mitochondria in permeabilized pancreatic islets

Effects of IP3, spermine, and Mg2+ on regulation of Ca2+ transport by endoplasmic reticulum and mitochondria in permeabilized pancreatic islets

Proinsulin processing in electrically permeabilized rat islets of Langerhans

Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3.5-7.4. Conversion was optimum at pH 4.5-5.5 and was reduced, but not abolished, at pH 7.4. Ca2+ (10 microM) and 4 beta-phorbol 12-myristate 13-acetate (500 nM), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.

Dual Mechanism Involved in the Hydrolysis of Polyphosphoinositides in Rat Pancreatic Islets

We investigated insulin secretion and inositol phosphate formation in intact and permeabilized rat pancreatic islets, the objective being to elucidate mechanisms of activation of phospholipase-C in pancreatic islets. The intact islets prelabeled with myo-[3H]inositol were incubated in Krebs-Ringer bicarbonate buffer containing 10 mM LiCl and 1 mM myoinositol. Glucose, alpha-ketoisocaproate (KIC), and sulfated cholecystokinin (CCK8S) increased insulin secretion and formation of [3H]inositol phosphate, [3H]inositol bisphosphate, and [3H] inositol trisphosphate. Mannoheptulose, a glucokinase inhibitor, inhibited glucose-induced insulin secretion and [3H]inositol phosphate formation; however, it did not inhibit KIC- and CCK8S-induced secretion and formation. Both glucose- and KIC-induced insulin secretion and [3H]inositol phosphate formation were blocked by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation in the mitochondria. The islets prelabeled with myo-[3H]inositol were permeabilized by digitonin and then incubated in intracellular mimicking medium containing 1 microM Ca2+ and 2.5 mM ATP. Glucose had no effect on [3H]inositol phosphate formation in the permeabilized islets, and CCK8S increased the formation of [3H]inositol phosphates. Thus, phospholipase-C in pancreatic islets is activated not only via ligand-receptor interaction in the plasma membrane in the case of hormone stimulation, but also by metabolic product(s) in the case of fuel stimulation.

Glyceraldehyde phosphate: An insulin secretagogue with possible effects on inositol phosphate formation in pancreatic islets

The insulinotropic action of glucose, the most potent physiologic insulin secretagogue, involves its metabolism. However, no glucose metabolite has ever been identified as a key intermediate. We tested the abilities of a number of glucose metabolites to stimulate insulin release from pancreatic islets. Of all of these metabolites, glyceraldehyde 3-phosphate was the most potent insulin secretagogue. In numerous experiments over 3 years, insulin release by 4 m m glyceraldehyde phosphate ranged from 50 to 200% of that initiated by 16.7 m m glucose—a near-maximal insulin stimulus. At concentrations of 1 and 4 m m, glyceraldehyde phosphate was even more potent than the known secretagogues glucose and glyceraldehyde. Glucose metabolites were also tested for their ability to stimulate inositol tris-, bis-, and monophosphate formation by permeabilized islets. Only glyceraldehyde phosphate stimulated inositol phosphate formation and this stimulation occurred at concentrations of glyceraldehyde phosphate which could be present in the β cell under physiologic conditions ( K 0.5 = 25 μM). The current results are consistent with the idea that glyceraldehyde phosphate is a key insulinotropic glucose metabolite that might act directly (or rather directly via a receptor) on the phospholipase C that forms inositol trisphosphate in the plasma membrane.

Role of intracellular mediators in glucagon secretion: studies using intact and electrically permeabilized rat islets of Langerhans

The roles of calcium, cyclic AMP (cAMP), activation of protein kinase C (PKC) and the effect of ATP on glucagon secretion were investigated in intact and permeabilized rat islets of Langerhans, Ca2+ (10 nM-10 microM) stimulated glucagon secretion from electrically permeabilized islets in a dose-dependent manner. Forskolin and cAMP stimulated secretion from intact and permeabilized islets respectively, the latter at both sub-stimulatory (50 nM) and stimulatory (10 microM) Ca2+ concentrations. The tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) increased secretion from both intact and permeabilized islets. In the latter, PMA increased glucagon release at both Ca2+ concentrations, the effect being enhanced at the stimulatory Ca2+ concentration, over and above that caused by Ca2+ alone. Reduction of ATP content by incubation with the metabolic inhibitor 2,4-dinitrophenol resulted in an increased basal release of glucagon from intact islets, whilst arginine-induced glucagon secretion was abolished in both intact and permeabilized islets. Ca2+-induced glucagon secretion required MgATP in the permeabilized islets of Langerhans. These results suggest that Ca2+ acts as an initiator of glucagon secretion, whilst cAMP and activation of PKC may exert their effect as modulators of secretion. ATP is required for glucagon secretion in electrically permeabilized islets and is necessary for arginine-induced glucagon secretion in both intact and permeabilized islets.

Exogenous arachidonic acid promotes insulin release from intact or permeabilized rat islets by dual mechanisms. Putative activation of Ca2+ mobilization and protein kinase C.

A number of indirect studies suggest a role for endogenous arachidonic acid (AA) in pancreatic islet function. To probe the effects of this fatty acid, AA and other polyunsaturated fatty acids were exogenously provided in Ca2+-free medium to avoid the formation of insoluble or impermeant Ca2+-arachidonate complexes. Concentrations of AA of greater than or equal to 3 microM induced potent and sustained but reversible 45Ca efflux from prelabeled intact (or digitonin-permeabilized) islets; AA also induced insulin release at somewhat higher concentrations. Other unsaturated fatty acids (erucic, oleic, linoleic, linolenic, dihomo-gamma-linolenic, eicosapentaenoic, docosahexaenoic acids) were generally less active than AA itself, indicating a structure-function relationship. The effects of AA were saturable, were inhibitable by cooling, and were not accompanied in parallel by 51Cr release or trypan blue retention, suggesting a nontoxic mechanism. At low concentrations (3.3-16 microM), at which AA does not stimulate insulin release, AA-induced 45Ca efflux was not reduced by pretreatment with ionomycin (to deplete membrane-bound Ca2+ stores), suggesting stimulation of Ca2+ extrusion through the plasma membrane. At higher concentrations (greater than or equal to 25 microM), at which AA promotes insulin release, further Ca2+ efflux was stimulated, which was blunted by pretreatment with ionomycin (as well as by trifluoperazine). Conversely, pretreatment with AA obliterated the effects of ionomycin (3 microM) on cellular Ca2+ mobilization. Thus, AA also mobilizes Ca2+ from intracellular organelles, leading to a rise in free cytosolic Ca2+ (as previously reported). AA-induced 45Ca efflux and insulin release were independent of the presence of extracellular Na+ and did not require the oxygenation of AA. Dose-response curves comparing 45Ca efflux and insulin secretion suggested that AA also stimulates hormone release by at least one other mechanism in addition to Ca2+ mobilization. This second stimulatory effect of AA could be seen in digitonin-permeabilized islets, where changes in cytosolic free Ca2+ concentration were vitiated by EGTA-containing buffers. Such secretion was also saturable and was inhibited by cooling or by spermine (which inhibits protein kinase C in the islet). Furthermore, AA-induced secretion from either intact or permeabilized islets was blunted by prolonged pretreatment of islets with a phorbol ester to deplete them of protein kinase C. Thus, exogenous arachidonic acid seems to be a complete secretagogue, having stimulatory effects both on Ca2+ mobilization and Ca2+-related secretory processes, putatively the activation of protein kinase C.

Exogenous arachidonic acid promotes insulin release from intact or permeabilized rat islets by dual mechanisms. Putative activation of Ca2+ mobilization and protein kinase C

Exogenous arachidonic acid promotes insulin release from intact or permeabilized rat islets by dual mechanisms. Putative activation of Ca2+ mobilization and protein kinase C

Protein phosphorylation in electrically permeabilized islets of Langerhans. Effects of Ca2+, cyclic AMP, a phorbol ester and noradrenaline.

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.

Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion.

The metabolism of D-glucose is believed to initiate and regulate insulin secretion by islet beta-cells, although the identity of the metabolite which couples glucose metabolism to the cellular events involved in insulin secretion is unknown. An alternative hypothesis involves the presence of a glucoreceptor for which there has been no biochemical evidence. We have investigated whether glucose recognition by the beta-cell is coupled to phospholipase C. We have used digitonin-permeabilized, [3H]inositol-prelabeled islets to study glucose and carbachol activation of phospholipase C. In this model, carbachol recognition by its muscarinic receptor was coupled to phospholipase C activation. D-Glucose (but not L-glucose) also stimulated phospholipase C activity in these permeabilized islets. This effect was not due to glucose metabolism since glucose 6-phosphate did not affect phospholipase C activity and since phosphorylation of [3H]glucose was not detectable in digitonin-permeabilized islets. Glucose had no effect on the myo-inositol-1,4,5-trisphosphate-5-phosphatase or 3-kinase activities. In the absence of agonist, free Ca2+ concentrations between 0.1 and 1 microM (as determined with a Ca2+-specific electrode) did not influence phospholipase C activity. Stimulation of phospholipase C activity by either carbachol or glucose required Ca2+ in the submicromolar range and was optimal at 0.5 microM free Ca2+.myo-Inositol-1,3,4,5-tetrakisphosphate production from permeabilized islets was synergistically augmented by Ca2+ (0.5-10 microM) and glucose. Phospholipase C activity in islets is therefore not directly activated by free Ca2+ concentrations in the submicromolar range. Furthermore, glucose per se activates phospholipase C activity independently of glucose metabolism. A working hypothesis based on these findings is that glucose is recognized by a site which is coupled to phospholipase C in islets.