Successful cultivation of isolated islets of Langerhans without attachment: relationship between Glucose- and theophylline-induced insulin release and insulin content in rats islets after cultivation.

The effect of various inhibitors of insulin secretion such as mannoheptulose (20 mM), atropine (1 mM), diphenylhydantoin (20 microng/ml), high concentration of Mg++ (5.3 mM) in the presence of 20 mM glucose (control) on insulin content and secretion from collagenase-isolated rat pancreatic islets was studied in vitro by cultivation of islets up to 5 or 9 days in glass Petri dishes without attachment. In a following short-term incubation for 60 min the glucose-induced insulin release without and with theophylline (5 mM) was investigated. Islets cultivated at 5 mM glucose and at 20 mM glucose with the inhibitors mannoheptulose or atropine lost the responsiveness to glucose and theophylline whereas such islets cultivated at 20 mM glucose alone or with diphenylhydantoin (DPH) or 5.3 mg Mg++ showed a stimulation of insulin secretion by glucose and theophylline. Compared, however, with freshly isolated islets all cultivated islets were restricted in their maximal glucose response and this defect was not evoked alone by quantitative changes in islet insulin content. Nevertheless, culture conditions which facilitate a net increase of insulin (content and release) during cultivation influenced also positively the glucose-induced insulin release without and with 5 mM theophylline in the following short-term experiments.     

Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.     

Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release.

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent on extracellular Ca2+ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the beta-cell glucose-sensor mechanism.     

Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent of extracellular Ca²⁺ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the β-cell glucose-sensor mechanism.     

Respiration and insulin release in mouse pancreatic islets: Effects of l-leucine and 2-ketoisocaproate in combination with d-glucose and l-glutamine

In order to further evaluate the importance of B-cell metabolism for the stimulation of insulin release, respiration and insulin release were studied in mouse pancreatic islets. Leucine and 2-ketoisocaproate stimulated insulin release during an initial 1-h period, whereas there was no stimulation during two subsequent 1-h periods. This effect was in contrast to that of 16.7 mM glucose, which was a potent stimulator through all the 3 h. Furthermore, the presence of glucose (5.6 mM) or glutamine together with either leucine or 2-ketoisocaproate enhanced the insulin release and prolonged the stimulation. When the kinetics of islet respiration were studied both leucine and 2-ketoisocaproate exerted an initial stimulation on the O₂ uptake which, however, was short-lived (less than 30 min). The presence of 5.6 mM glucose strongly delayed the respiratory retardation seen after the initial stimulation. Similarly, glutamine enhanced the leucine- and 2-ketoisocaproate-stimulated respiratory rates and prevented the respiratory retardation otherwise observed. Leucine (20 mM) and 2-ketoisocaproate (10 and 20 mM) stimulated the oxidation of glucose (5.6 mM). It is concluded that there is a strong correlation between respiratory stimulation and the enhancement of insulin release and that leucine and 2-ketoisocaproate depend on the presence of endogenous fuels for their ability to stimulate islet functions in vitro.     

Accumulation of cadmium in pancreatic beta cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd2+ and the effects of this ion on secretory activity and metabolism were investigated in beta cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 mumol/kg dry wt. After 60 min of incubation in a Ca2+-deficient medium containing 2.5 microM Cd2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca2+. The incorporation of Cd2+ was stimulated either by raising the concentration of glucose to 20 mM or K+ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K+. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd2+ efflux into a Ca2+-deficient medium. Concentrations of Cd2+ up to 2.5 microM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd2+ concentration was above 10 microM. Basal insulin release was stimulated by 5 microM Cd2+. At a concentration of 160 microM, Cd2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the beta cell uptake of Cd2+ is facilitated by the activation of voltage-dependent Ca2+ channels. Apparently, the accumulation of Cd2+ mimics that of Ca2+ also involving a component of intracellular sequestration promoted by glucose.     

The effect of N-acylglucosamines on the biosynthesis and secretion of insulin in the rat.

The effects of N-acylglucosamines on insulin release have been studied. N-Acylglucosamines stimulated insulin release from rat islets in vitro only if a sub-stimulatory concentration of glucose was also present, and this secretory response was abolished by mannoheptulose. In perifused islets the rapidity of the secreotry response to N-acetyl-D-glucosamine was similar to that observed with D-glucose. Increasing acyl-chain length from N-acetyl- to N-hexanoyl-D-glucosamine impaired the secretory response; however, N-dichloroacetyl-D-glucosamine was a more potent stimulator of release than was N-acetyl-D-glucosamine. Polymers of N-acetyl-D-glucosamine containing two to six monomers linked alpha1-4 did not stimulate insulin release; glucosamine linked to dextran via a propionyl or hexanoyl spacer group was also without insulin-releasing ability. N-Acylglucosamines were also effective in eliciting insulin release in vivo when injected into conscious rats. At the dose used (86 mumol), N-acetylgucosamine elicited a rapid rise in plasma-insulin concentration; N-butyrylglucosamine was less effective, and there was little or no response to N-hexanoylglucosamine. The response to N-dichloroacetyl-glucosamine was greater than that to N-acetylglucosamine; an increase in plasma insulin concentration could be elicited by N-dichloroacetylglucosamine at a dose (17 mumol) at which neither glucose nor N-acetylglucosamine was effective. The secretory response to acetylglucosamine is not mediated by conversion into glucose. Rates of (pro)-insulin biosynthesis by rat islets have been measured (Pro)-insulin biosynthesis was stimulated by glucose, and this response was abolished by mannoheptulose. N-Acetylglucosamine also stimulated (pro)-insulin biosynthesis; this effect of N-acetylglucosamine did not require the presence of glucose, and was not abolished by mannoheptulose. It is concluded that there are differences in signal reception and/or transduction for the processes of insulin biosynthesis and release.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets.

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Metabolic control of the potassium permeability in pancreatic islet cells

The K(+) permeability of pancreatic islet cells was studied by monitoring the efflux of (86)Rb(+) (used as tracer for K(+)) from perifused rat islets and measuring the uptake of (42)K(+). Glucose markedly and reversibly decreased (86)Rb(+) efflux from islet cells and this effect was antagonized by inhibitors of the metabolic degradation of the sugar, i.e. mannoheptulose, iodoacetate, glucosamine and 2-deoxyglucose. Among glucose metabolites, glyceraldehyde reduced the K(+) permeability even more potently than did glucose itself; pyruvate and lactate alone exhibited only a small effect, but potentiated that of glucose. Other metabolized sugars, like mannose, glucosamine and N-acetylglucosamine, also decreased (86)Rb(+) efflux from islet cells. Fructose was effective only in the presence of glucose. Non-metabolized sugars like galactose, 2-deoxyglucose and 3-O-methylglucose had no effect. The changes in K(+) permeability by agents known to modify the concentrations of nicotinamide nucleotides, glutathione or ATP in islet cells were also studied. Increasing NAD(P)H concentrations in islet cells by pentobarbital rapidly and reversibly reduced (86)Rb(+) efflux; exogenous reduced glutathione produced a similar though weaker effect. By contrast, oxidizing nicotinamide nucleotides with phenazine methosulphate or Methylene Blue, or oxidizing glutathione by t-butyl hydroperoxide increased the K(+) permeability of islet cells. Uncoupling the oxidative phosphorylations with dicumarol also augmented (86)Rb(+) efflux markedly. In the absence of glucose, but not in its presence, methylxanthines reduced (86)Rb(+) efflux from the islets; such was not the case for cholera toxin or dibutyryl cyclic AMP. Glucose and glyceraldehyde had no effect on (42)K(+) uptake after a short incubation (10min), but augmented it after 60min; the effect of glucose was suppressed by mannoheptulose and not mimicked by 3-O-methylglucose. The results clearly establish the importance of the metabolic degradation of glucose and other substrates for the control of the K(+) permeability in pancreatic islet cells and support the concept that a decrease in the K(+) permeability represents a major step of the B-cell response to physiological stimulation.     

Interrelationship of islet metabolism, adenosine triphosphate content and insulin release

The oxidation of some exogenous substrates and their effects on ATP content and insulin release in mouse pancreatic islets were measured. The ATP concentration of islets incubated without exogenous substrate shows a gradual decrease, which can be prevented by glucose or mannose (20mm) or leucine (2.5mm); d-glyceraldehyde (5mm) is as effective as glucose (5mm); fructose or N-acetylglucosamine (20mm), pyruvate (10mm) and dl-3-hydroxybutyrate (2mm) are less effective; galactose (20mm), acetate (10mm), octanoate (2mm) and succinate (10mm) have no ATP-maintaining ability. Islets oxidize glucose, mannose, glyceraldehyde, leucine and, less readily, N-acetylglucosamine and glucosamine; galactose, however, is poorly metabolized. Mannoheptulose inhibits the oxidation of glucose but not of glyceraldehyde. Insulin release, measured over a 2h incubation, is stimulated by glucose, mannose, leucine, glyceraldehyde or glucosamine but not by fructose or N-acetylglucosamine. The latter, however, potentiates the effects of glucose or glyceraldehyde (5mm) or leucine (2.5mm) on release; the potentiating effects are inhibited by mannoheptulose, which also blocks glucose-, but not glyceraldehyde- or leucine-stimulated release. In the presence of glucose (20mm), metabolic inhibitors depress insulin release and islet ATP content in parallel. However, rates of insulin release and ATP content measured after incubation with various combinations of exogenous substrates do not appear to be correlated. Sulphonylureas stimulate insulin release but decrease islet ATP concentrations. These results provide further evidence of a close association between the metabolic activity of exogenous substrates and their ability to initiate insulin release. Glucoreceptor models are formulated in the light of these observations and discussed.     

Effects of galnon, a non-peptide galanin-receptor agonist, on insulin release from rat pancreatic islets

Galanin is a neurotransmitter peptide that suppresses insulin secretion. The present study aimed at investigating how a non-peptide galanin receptor agonist, galnon, affects insulin secretion from isolated pancreatic islets of healthy Wistar and diabetic Goto-Kakizaki (GK) rats. Galnon stimulated insulin release potently in isolated Wistar rat islets; 100 microM of the compound increased the release 8.5 times (p<0.001) at 3.3 mM and 3.7 times (p<0.001) at 16.7 mM glucose. Also in islet perifusions, galnon augmented several-fold both acute and late phases of insulin response to glucose. Furthermore, galnon stimulated insulin release in GK rat islets. These effects were not inhibited by the presence of galanin or the galanin receptor antagonist M35. The stimulatory effects of galnon were partly inhibited by the PKA and PKC inhibitors, H-89 and calphostin C, respectively, at 16.7 but not 3.3 mM glucose. In both Wistar and GK rat islets, insulin release was stimulated by depolarization of 30 mM KCl, and 100 microM galnon further enhanced insulin release 1.5-2 times (p<0.05). Cytosolic calcium levels, determined by fura-2, were increased in parallel with insulin release, and the L-type Ca2+-channel blocker nimodipine suppressed insulin response to glucose and galnon. In conclusion, galnon stimulates insulin release in islets of healthy rats and diabetic GK rats. The mechanism of this stimulatory effect does not involve galanin receptors. Galnon-induced insulin release is not glucose-dependent and appears to involve opening of L-type Ca2+-channels, but the main effect of galnon seems to be exerted at a step distal to these channels, i.e., at B-cell exocytosis.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

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+.     

Insulin release in pancreatic islets by a glycolytic and a Krebs cycle intermediate: contrasting patterns of glyceraldehyde phosphate and succinate

Glyceraldehyde phosphate, a glycolytic intermediate, and succinic acid (as its methyl ester to make it permeable to the cell), a citric acid cycle intermediate, were the only glucose metabolites of many recently tested that stimulated insulin release. The effects of these two "new" insulin secretagogues on several pancreatic islet parameters were compared. Glyceraldehyde phosphate stimulated all of the insulin it released during the first 5 min after islets were exposed to it, and its maximum effect on calcium uptake was observed at 5 min. Monomethyl succinate stimulated insulin release mostly during the last 30 min of a 1-h incubation and its maximum effect on calcium uptake was at 60 min after it was applied to islets. Monomethyl succinate-induced insulin release, but not glyceraldehyde phosphate-induced insulin release, was inhibited by metabolic inhibitors (antimycin A, rotenone, cyanide, FCCP, fluoride, and iodoacetamide). This is consistent with the idea that monomethyl succinate is hydrolyzed to succinate which is metabolized intramitochondrially. The effects of glyceraldehyde suggest that glucose signals the first phase of insulin release by an agonist-like mechanism that originates in the cytosol and requires minimal energy. The effects of monomethyl succinate suggest that the signal for the second phase of glucose-induced insulin release originates in the mitochondrion and requires a large amount of energy.     

Glucose stimulates and potentiates islet amyloid polypeptide secretion by the B-cell.

Islet amyloid polypeptide (IAPP) has been shown to be actively secreted by the pancreatic B-cell along with insulin. To determine whether the modulation of B-cell IAPP secretion is similar to that of insulin, we assessed IAPP release in response to glucose at 4 different concentrations (1.67, 5.5, 8.8 and 16.7 mM) and to non-glucose secretagogues at different glucose concentrations in a neonatal rat islet monolayer culture preparation. Glucose alone stimulated IAPP and insulin secretion in a dose dependent fashion with maximal release for both peptides occurring at 8.8 mM. B-cell secretion of IAPP in response to arginine, isobutylmethylxanthine or both together was potentiated by increasing glucose concentrations from 1.67 to 16.7 mM. This same pattern of glucose potentiation was observed for insulin secretion. The data indicate that the pattern of peptide responses of cultured neonatal B-cells to glucose is similar for both IAPP and insulin release. Furthermore, the data suggest that glucose is capable of potentiating B-cell secretion of both IAPP and insulin.     

Vanadate stimulation of insulin release in normal mouse islets.

The effects of vanadate (Na3VO4) on pancreatic B-cell function were studied in normal mouse islets. Vanadate did not affect basal insulin release but potentiated the effect of 7-30 mM glucose at concentrations of 0.1-1 mM. This effect was progressive and slowly reversible. It was abolished by omission of extracellular Ca2+ but unaffected by blockers of adrenergic or muscarinic receptors. Comparison of the changes in membrane potential, 86Rb efflux and 45Ca efflux that vanadate and ouabain produced in B-cells made it possible to exclude the hypothesis that vanadate increases insulin release by blocking the sodium pump. Vanadate was also without effect on cAMP levels. On the other hand, it markedly changed the characteristics of the Ca(2+)-dependent electrical activity and of the oscillations of cytoplasmic Ca2+ recorded in B-cells stimulated by 15 mM glucose. In the steady state, Ca2+ influx was increased by vanadate, and this resulted in a rise in cytoplasmic Ca2+. The exact mechanisms underlying these changes could not be established but a blockade of K channels was excluded. In the presence of LiCl, vanadate markedly increased inositol phosphate levels in islet cells. This effect was attenuated but not suppressed by omission of Ca2+. A small increase in inositol bisphosphate was still produced by vanadate in the absence of LiCl. These results suggest that vanadate both stimulates phosphoinositide breakdown and inhibits inositol phosphate degradation. In conclusion, vanadate does not induce insulin release, but markedly potentiates the stimulation by glucose. This property is not due to an inhibition of the sodium pump or to a rise in cAMP concentration. It results from a complex interplay between changes in B-cell membrane potential, phosphoinositide metabolism and Ca2+ handling.     

Regulation of immunoreactive-insulin release from a rat cell line (RINm5F).

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.     

The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets.

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.