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.     

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.     

Manganese accumulation in pancreatic beta-cells and its stimulation by glucose.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets microdissected from ob/ob mice. The islet content of endogenous manganese was 80 mumol/kg dry wt., which is about half as much as found in the exocrine pancreas. The initial uptake was characterized by two components, with approximate Km values of 35 microM and 3.7 microM respectively. After 60 min of incubation with 0.25 mM-Mn2+, the intracellular concentration of manganese corresponded to an almost 25-fold accumulation compared with that of the extracellular medium. When exposed to 20 mM-D-glucose, the islets retained more manganese, owing to suppression of its mobilization. The glucose inhibition of efflux was prompt and reversible, as indicated from direct recordings of manganese in a perifusion medium. D-Glucose was an equally potent inhibitor of efflux in the presence of 15 microM- and 1.28 mM-Ca2+. The inhibitory action disappeared when metabolism was suppressed by adding 0.1 mM-N-ethylmaleimide or by lowering the temperature from 37 degrees C to 2 degrees C. At a concentration of 0.25 mM, Mn2+ abolished the insulin-releasing action of D-glucose, exerting only moderate suppression of its metabolism. The addition of Mn2+ resulted in inhibition of basal insulin release in the presence of 1.28 mM-Ca2+, but not in a Ca2+-deficient medium. The studies indicate that the previously observed phenomenon of glucose inhibition of 45Ca efflux has a counterpart in the suppression of manganese mobilization from the pancreatic islets. With the demonstration of a pronounced glucose inhibition of manganese efflux, it is evident that Mn2+ may represent a useful tool for exploring the mechanism of glucose-induced retention of calcium in the pancreatic beta-cells.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Palmitic acid stimulates glucose incorporation in the adipocyte by a mechanism likely involving intracellular calcium

The effect of palmitic acid on basal and insulin-stimulated incorporation of glucose into rat adipocytes was studied. Palmitic acid (2.40 mM) stimulated basal as well as insulin-stimulated glucose incorporation in rat adipocytes three and twofold, respectively. Similar degrees of stimulation of basal glucose oxidation by palmitate were also observed. The ability of palmitic acid to stimulate glucose uptake was additive with respect to the stimulation induced by insulin and was proportional to the palmitic acid concentration between 0.15 mM and 2.40 mM. Stimulation of glucose incorporation by palmitic acid was inhibited by preincubating the cells with quin2-AM, which accumulates intracellularly yielding the trapped chelator form. quin2, which binds intracellular Ca2+.The concentration of quin2-AM required for half-maximal inhibition of palmitic acid stimulated glucose incorporation was 3.8 +/- 1.2 microM (mean +/- SEM). The inhibition of palmitic acid-stimulated glucose incorporation by quin2-AM (10 microM) was overcome by incubating cells with the Ca2+ ionophore, A23187, in the presence of extracellular Ca2+ (2.6 mM). Chelation of extracellular Ca2+ with EGTA did not significantly affect the magnitude of palmitic acid-stimulated glucose incorporation. Dantrolene (12.5-100 microM) failed to affect basal or palmitic acid-stimulated glucose incorporation. These findings suggest that palmitic acid stimulates incorporation of glucose in the adipocyte by a mechanism dependent upon intracellular but not extracellular Ca2+.     

Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

The role of Ca2+ on insulin release has been studied by the use of ionophore A23187. The ionophore complexes divalent cations and permits Ca2+ entry into cells by acting as a carrier in the plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the 3rd day of culture. With Ca2+ in the incubation medium the ionophore induced sustained insulin release even in the absence of glucose. Optimal effects of the ionophore were observed at 3 and 10 mug per ml in the presence of 0.3 to 1.0 mM Ca-2+. Under these conditions the insulin release was greater than that caused by 16.7 mM glucose. A graded response was observed to changes in Ca-2+ concentration from 0.1 to 1.0 mM Ca-2+. Higher Ca-2+ concentrations caused a large amount of insulin to be released promptly, but the release was not sustained. Mg-2+ and Sr-2+ were not found to substitute for Ca-2+. Ba-2+ at 0.3 mM stimulated insulin release even in the absence of ionophore. Cyclic adenosine 3':5'-monophosphate was able to increase ionophore-induced insulin release. The alpha-adrenergic effect of epinephrine to inhibit insulin release was not observed in the presence of Ca-2+ and the ionophore, and a stimulatory effect of epinephrine was seen. This unusual stimulatory effect of epinephrine was blocked by propranolol indicating a beta-adrenergic mechanism for epinephrine. It is concluded that Ca-2+, which plays an essential role in the stimulus-secretion coupling, can alone initiate and cause sustained insulin release.     

Glucose-induced early changes in cytoplasmic calcium of pancreatic beta-cells studied with time-sharing dual-wavelength fluorometry

The cytoplasmic calcium concentration (Ca2+i) was measured in suspensions of fura-2-loaded mouse pancreatic beta-cells by recording the 340:380 nm fluorescence excitation ratio. Exposure to 20 mM glucose resulted in an initial reduction and later increase of Ca2+i irrespective of preincubation in medium containing 0.5 or 1.28 mM Ca2+ and 0 or 3 mM glucose. When elevating the Ca2+ concentration to 5 or 10 mM only 5 min before raising glucose to 20 mM, the sugar-induced reduction of Ca2+i became more pronounced like the subsequent increase. However, when the Ca2+ concentration was increased from 1.28 to 10 mM 2 min after stimulation with glucose, there was a sudden pronounced Ca2+i transient, which was followed by a decrease and a slower secondary rise. After preincubation in 20 mM glucose the glucose-induced initial reduction of Ca2+i was only seen in a Ca2+-deficient medium. Reintroduction of the sugar in the presence of extracellular Ca2+ resulted in an immediate rise of Ca2+i, the rapidity of which depended on the transmembrane Ca2+ gradient. The results emphasize the role of a saturable beta-cell pool of Ca2+ in glucose-induced reduction of Ca2+i and indicate that the first phase of insulin release depends on an influx of extracellular Ca2+.     

Effects of Cd2+ and two cadmium organic complexes on isolated rat liver mitochondria

Effects of Cd2+ and two complexes of bivalent cadmium with 1,3-bis(4-chlorbenzylidenamino)-guanidine and anabasine on ion permeability of the inner membrane and respiration of isolated rat liver mitochondria were studied. Starting from 5 microM, Cd2+ decreased state 3 and DNP-stimulated respiration of mitochondria and increased their state 4 respiration. At 30 microM, Cd2+ decreased state 4 respiration. The complexes, particularly complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine, inhibited the mitochondrial respiration at lower concentration of Cd2+. Nonenergized mitochondria incubated in media containing 125 mM of NH4NO3 or KNO3 showed more pronounced swelling in experiments with 10 microM of the complexes than with Cd2+. The complexes produced swelling of the mitochondria energized by 5 mM of succinate and incubated in medium containing 25 mM K-acetate and 100 mM sucrose. Uptake of 137-Cs by succinate-energized mitochondria in the presence of 10(-8) M of valinomycin was substantially decreased in experiments with 10 microM of the complexes than with Cd2+. Ruthenium red (7.5 microM) prevented this effect with 10 microM of complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine and especially complex of Cd2+ with anabasine and Cd2+. These results indicate that the cadmium organic complexes affect respiration and perturb ion permeability significantly stronger than Cd2+.     

Insulin secretagogues induce Ca(2+)-like changes in cytoplasmic Mg2+ in pancreatic beta-cells

The effects of insulin secretagogues on the cytoplasmic Mg2+ concentration ([Mg2+]i) of pancreatic beta-cells were studied in suspensions and in individual beta-cells using dual-wavelength fluorometry and the indicator mag-fura-2. Average [Mg2+]i was in the 800-900 microM range in a medium containing 3 mM glucose. When the sugar concentration was raised to 20 mM, the cells reacted with an initial lowering of [Mg2+]i followed by an increase. The sugar apparently also stimulated leakage of the Mg2+ indicator. Addition of 100 microM tolbutamide or raising the K+ concentration by 25 mM caused relatively rapid increases of [Mg2+]i. Methoxyverapamil prevented the [Mg2+]i-increasing actions of glucose, K+ and tolbutamide. The greatest change in [Mg2+]i was obtained when beta-cells were exposed to 100 microM carbachol. In this case there was a more than 10% lowering, which was reversed upon removal of the agonist. Measurements of [Mg2+]i are important not only for understanding fluctuations of this ion, but may also aid to elucidate the mechanisms involved in the regulation of cytoplasmic Ca2+.     

Insulin action in isolated fat cells. I. Effects of divalent cations on the stimulation by insulin of glucose uptake.

The effects of divalent cations, in particular Ca2+ and Mg2+, on glucose uptake by rat isolated fat cells in the presence and absence of insulin have been studied. EDTA (disodium salt) was used to deplete the bovine serum albumin present in the incubation medium of endogenous divalent cations prior to incubation with the cells, but was not present in the incubation medium during the incubation of the cells. The removal of Ca2+ and Mg2+ from the incubation medium did not affect the basal glucose uptake, but abolished the ability of insulin to stimulate glucose uptake by the cells. Addition of 25 microM MgCl2 or CaCl2 to the incubation medium restored a significant insulin stimulation, and this stimulation was maximal when 0.1 mM MgCl2 or CaCl2 had been added. SrCl2 and BaCl2 were also effective in restoring the insulin stimulation, but did not substitute fully for Ca2+ and Mg2+ in the incubation medium. Possible explanation for these observations are discussed.     

The interaction between manganese and calcium fluxes in pancreatic beta-cells.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets isolated from ob/ob mice. The efflux from preloaded islets was estimated from the amounts remaining after 30 min of subsequent test incubations in the absence of Mn2+. An increase in the extracellular Mg2+ concentration promoted the Mn2+ efflux and removal of Na+ from a Ca2+-deficient medium had the opposite effect. Addition of 25 mM-K+ failed to affect Mn2+ outflow as did 3-isobutyl-1-methylxanthine and dibutyryl cyclic AMP. Whereas tolbutamide caused retention of manganese, the ionophore Br-X537A promoted an efflux. D-Glucose was equally potent in retaining the islet manganese when the external Ca2+ concentration ranged from 15 microM to 6.30 mM. Subcellular-fractionation experiments indicated a glucose-stimulated incorporation of manganese into all fractions except the microsomes. The effect was most pronounced in the mitochondrial fraction, being as high as 164%. The glucose-induced uptake of intracellular 45Ca was abolished in the presence of 0.25 mM-Mn2+. When added to medium containing 2.5 mM-Mn2+, glucose even tended to decrease 45Ca2+ uptake. The inhibitory effect of Mn2+ was apparent also from a diminished uptake of 45Ca into all subcellular fractions. The efflux of 45Ca2+ was markedly influenced by Mn2+ as manifested in a prominent stimulation followed by inhibition. In addition to demonstrating marked interactions between fluxes of Mn2+ and Ca2+, the present studies support the view that the glucose inhibition of the efflux of bivalent cations from pancreatic beta-cells is accounted for by their accumulation in the mitochondria.     

Testosterone rapidly stimulates insulin release from isolated pancreatic islets through a non-genomic dependent mechanism.

The action of testosterone on the 45Ca2+ uptake and insulin secretion was studied in short-term experiments using isolated pancreatic islets of Langerhans. Testosterone (1 microM) stimulated 45Ca2+ uptake within 60 seconds of incubation on similar proportion than tolbutamide. Also, the hormone rapidly increased insulin release (34%; 180 seconds) on the presence of non-stimulatory concentrations of glucose (3 mM). Impermeant testosterone-BSA significantly stimulated the secretion of insulin to a lower percentage (10%). The action of the hormone is specific--neither 17beta-E2 nor progesterone stimulated insulin secretion in the presence of 3 mM glucose. The action of testosterone on insulin secretion was dose-dependent, and at rat plasma physiological concentrations (25 nM), stimulus was 17% (p < 0.05). In conclusion, in isolated pancreatic islets experiments, physiological concentration of testosterone rapidly stimulate insulin secretion and 45Ca2+ uptake through a membrane bound mechanism.     

Studies on the interaction of staphylococcal delta-haemolysin with isolated islets of Langerhans.

delta-Haemolysin, a small surface-active polypeptide purified from the culture media of Staphylococcus aureus, was observed to stimulate the release of insulin from isolated rat islets of Langerhans. This effect was dose-dependent and saturable, with the half-maximal response elicited by a delta-haemolysin concentration of 10 micrograms/ml. Stimulation of insulin release by delta-haemolysin (10 micrograms/ml) was not dependent on the presence of glucose in the incubation medium, but was augmented by increasing concentrations of the sugar. The release of insulin in response to delta-haemolysin could be inhibited by depletion of extracellular Ca2+ or by adrenaline (epinephrine) (10 microM) and was readily reversible when delta-haemolysin was removed from the medium. In addition, the response was potentiated by incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). These observations suggest that delta-haemolysin induced a true activation of the beta-cell secretory mechanism. Stimulation of islets of Langerhans with delta-haemolysin was found to be associated with a modest increase in intracellular cyclic AMP levels, although the adenylate cyclase activity of islet homogenates was not increased by delta-haemolysin. delta-Haemolysin was observed to induce a dose-dependent net accumulation of 45Ca2+ by islet cells and to stimulate the efflux of 45Ca2+ from preloaded islets. The efflux of 45Ca2+ was modest in size and short-lived, but dramatically increased in medium depleted fo 40Ca2+. Incubation in the presence of verapamil augmented delta-haemolysin-induced 45Ca2+ efflux and insulin secretion. delta-Haemolysin was found to be a potent 45Ca2+-translocating ionophore in an artificial system. This response was dose-dependent and could be augmented by verapamil. In addition, phosphatidylcholine (25 micrograms/ml) was found to inhibit both delta-haemolysin induced 45Ca2+ translocation and insulin release in a precisely parallel manner. These studies suggest that the ability of delta-haemolysin to stimulate insulin release may be due, in part, to the facilitation of Ca2+ entry into the beta-cells of islets of Langerhans, mediated directly by an ionophoretic mechanism.     

Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.     

The effects of alpha- and beta-adrenergic agents, Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 microM) and 1 microM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 microM), 50 microM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 microM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 microM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 microM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 microM) and epinephrine (1 microM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 microM but maximal concentrations of epinephrine (e.g., 1 microM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 microM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 microM) was added. Similarly, removal of 0.1 microM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either alpha or beta receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Glucose has regulatory effects on insulin release also in the virtual absence of extracellular Ca2+.

The glucose effect on insulin release in a Ca(2+)-deficient medium was analyzed in perifusion experiments with aggregates of cells prepared by dispersal of the beta-cell-rich pancreatic islets of ob/ob-mice. Hyperosmolar additions of 20 mM D-glucose or its poorly metabolized transport analogue 3-0-methyl-D-glucose resulted in 50% suppression of the secretory rate. However, after isosmolar additions of the sugars, replacing non-penetrating sucrose, there was a stimulation of insulin release. Whereas D-glucose was less effective than 3-O-methyl-D-glucose in stimulating insulin release after isosmolar addition, the opposite was found for the enhanced secretory response obtained when the sugars were excluded from the perifusion medium. The studies indicate that D-glucose has regulatory actions on insulin release also in the virtual absence of extracellular Ca2+. This effect is not only due to osmolar influences but involves also direct suppression of the secretory activity probably mediated by the metabolism of the sugar.     

Effect of calcium ions on the processing of pro-opiomelanocortin by bovine intermediate lobe pro-opiomelanocortin-converting enzyme.

The effect of Ca2+ on the extent and pattern of processing of pro-opiomelanocortin and an N-terminal fragment by a purified pituitary secretory vesicle, soluble aspartic endoprotease, was studied. Ca2+ stimulated the first cleavage of pro-opiomelanocortin by pro-opiomelanocortin-converting enzyme to yield 21-23 kDa adrenocorticotropin and beta-lipotropin, but its effect was minimal. The production of adrenocorticotropin from the 21-23 kDa intermediate was stimulated approximately 2.3-fold in the presence of 10 mM Ca2+, and processing of beta-lipotropin to beta-endorphin was stimulated about 1.3-1.4-fold by 5-10 mM Ca2+. The production of gamma-melanotropin-immunoreactive material from bovine N-pro-opiomelanocortin(1-77) was stimulated approximately 1.3-fold at both 100 microM and 1.5-2.0 mM Ca2+. Further characterization of the gamma-melanotropin-immunoreactive material by HPLC demonstrated that the major products were gamma 3-[Lys]melanotropin and gamma 3-melanotropin at both Ca2+ concentrations. These results indicate that pro-opiomelanocortin-converting enzyme is stimulated by Ca2+.     

Calcium and pancreatic beta-cell function. I. Stimulatory effects of pentobarbital on insulin release.

Islets microdissected from ob/ob-mice were exposed to 3mM pentobarbital in media which were normal or deficient in Ca2+. This treatment resulted in marked decrease of the islet content of cyclic AMP recorded in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Pentobarbital had a dual effect on insulin release. In addition to being a potent inhibitor of glucose-stimulated insulin release in media containing 2.56 mM Ca2+ it increased the amounts of insulin released in high glucose media deficient in Ca2+. There was a transient stimulation with ordinary concentrations of Ca2+ and 3mM glucose whtn the media also contained 3-isobutyl-1-methylxanthine. The stimulatory effect of pentobarbital persisted after replacing part of the Ca2+ in the beta-cell membrane with lanthanum ions and it could not be mimicked by lowering the oxygen tension of the incubation medium. It is suggested that pentobarbital stimulation of insulin release is the result of a specific action of the drug on the distribution of Ca2+ within the pancreatic beta-cells.