Patrik Rorsman

Dual effects of glucose on the cytosolic Ca2+ activity of mouse pancreatic β‐cells

The cytosolic Ca2+ activity of mouse pancreatic β‐cells was studied with the intracellular fluorescent indicator quin2. When the extracellular Ca2+ concentration was 1.20 mM, the basal cytosolic Ca2+ activity was 162 ± 9 nM. Stimulation with 20 mM glucose increased this Ca2+ activity by 40%. In the presence of only 0.20 mM Ca2+ or after the addition of the voltage‐dependent Ca2+‐channel blocker D‐600, glucose had an opposite and more prompt effect in reducing cytosolic Ca2+ by about 15%. It is concluded that an early result of glucose exposure is a lowering of the cytosolic Ca2+ activity and that this effect tends to be masked by a subsequent increase of the Ca2+ activity due to influx of Ca2+ through the voltage‐dependent Ca2+ channels.

Galanin and the endocrine pancreas

Galanin is a 29 amino acid peptide, initially isolated from the porcine small intestine. The peptide has been shown to occur in intrapancreatic nerves in close association to the islets. Its effects on islet hormone secretion and its possible mechanisms behind these effects are reviewed. Galanin has been shown to inhibit basal and stimulated insulin secretion both in vivo and in vitro under a variety of experimental conditions. The peptide has also been shown to inhibit somatostatin secretion and the secretion of pancreatic polypeptide (PP). With regard to glucagon secretion, however, results in the literature are not consistent since both stimulatory and inhibitory effects have been reported. A direct interaction with the pancreatic β‐cells has been proposed behind its inhibitory action on insulin secretion, since galanin inhibits insulin secretion from isolated β‐cells from obese, hyperglycaemic, mice. Galanin has thereby also been shown to induce repolarization and to reduce the free Ca2+ concentration, [Ca2+]i. The reduction in [Ca2+]i is probably not due to a direct interference with the voltage‐activated Ca2+ channels, since there is no effect of galanin when these channels are opened by depolarization induced by high concentrations of K+. Instead, preliminary studies indicate that galanin activates the K+ channels that are regulated by ATP, in turn inducing a repolarization‐induced reduction in [Ca2+]i resulting in reduced insulin secretion. However, the possibility that galanin inhibits the insulin secretory mechanism at a step distal to the regulation of cytoplasmic free Ca2+ concentration should not be overlooked.

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism.

The parathyroid hormone (PTH) release and cytosolic Ca2+ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca2+ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca2+ activity. Both the hormone release and the cytosolic Ca2+ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca2+ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca2+ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca2+ activity.

Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINmSF) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101±5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7±0.8 min. In contrast to the action of K+, exposure of the RINmSF cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity . This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in β-cells. Unlike the case in normal β-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINmSF cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINmSF cells exposed to glucose.

Direct measurements of increased free cytoplasmic Ca2+ in mouse pancreatic β-cells following stimulation by hypoglycemie sulfonylureas

The effects of the hypoglycemic sulfonylureas tolbutamide and glibenclamide on free cytoplasmic Ca2+, [Ca2+]i, were compared with that of a depolarizing concentration of K+ in dispered and cultured pancreatic β‐cells from ob/ob mice. [Ca2+]i was measured with the fluorescent Ca2+‐indicator quin2. The basal level corresponded to 150 nM and increased to 600 nM after exposure to 30.9 mM K+. The corresponding levels after stimulation with 1 μM glibenclamide and 100 μM tolbutamide were 390 and 270 nM respectively. K+ depolarization increased [Ca2+]i more rapidly than either of the sulfonylureas. It is suggested that the increased [Ca2+]i obtained after stimulation by sulfonylureas is due to depolarization of the β‐cells with subsequent entry of Ca2+ through voltage‐dependent channels.

Depolarization-independent net uptake of calcium into clonal insulin-releasing cells exposed to glucose

Insulin release, net fluxes of Ca2+, and glucose metabolism were studied in a clonal cell line (RINmSF) established from a transplantable rat islet tumor. The insulin content amounted to only 0.03% of that of the total protein and decreased even further with subsequent passages. The insulin secretion was as high as 10 to 20% of the total hormone content per hour. Insulin release was stimulated by K+ depolarization but not by exposure to glucose. In contrast to this secretory pattern, glucose but not K+ stimulated the net uptake of Ca2+ at micromolar concentrations of the ion. The glucose effect was not mimicked by 20 mM 3-O-methylglucose. It was as pronounced at 1 mM as at 20 mM of the sugar and corresponded to an uptake of 119 fmol cm−2 s−1. Glucose metabolism was typical for tumor cells with a high glycolytic flux and an oxidationtoutilization ratio as low as 0.05–0.15. Maximal oxidative degradation was attained already at l mM. This concentration was also equivalent to the Km for glucose utilization, indicating a substantial left-hand shift of the normal dose-response curve. It is suggested that glucose induces a depolarizationindependent net uptake of Ca2+ by favouring intracellular buffering of the cation.

Calcium and pancreatic β-cell function XI. Modification of 45Ca fluxes by Na+ removal

Abstract Pancreatic islets were isolated from noninbred ob ob mice and used for evaluating how removal of extracellular Na + affects 45 Ca fluxes in the insulin-producing β cells. Isoosmotic replacement of Na + with Li + was less efficient in stimulating the incorporation of intracellular (La 3+ non-displaceable) 45 Ca than substitution with choline + or sucrose. Ample support for the significance of Na + ue5f8Ca 2+ exchange for the net efflux of Ca 2+ was obtained with the observation that Na + deprivation markedly inhibited the 45 Ca washout in a Ca 2+ -deficient medium. The inhibitory action of Na + deficiency exceeded that obtained with 20 m m glucose. The 45 Ca incorporated after removal of Na + differed from that taken in response to glucose in not being exhanged with the Ca 2+ entering the β cells during glucose stimulation and in appearing also in the postmicrosomal supernatant obtained by differential centrifugation.

Direct evidence for opposite effects of D-glucose and D-glyceraldehyde on cytoplasmic pH of mouse pancreatic β-cells

The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH4+ and high concentrations of K+ on cytoplasmic pH (pHi) were investigated in dispersed and cultured pancreatic β-cells fromob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pHi value in resting β-cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pHi with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pHi. The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pHi. In contrast to D-glucose, 10mM of D-glyceraldehyde decreased pHi by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pHi in pancreatic β-cells.

Ca2+ transport in pancreatic beta-cells during glucose stimulation of insulin secretion.

The role of Ca2+ in the regulation of insulin secretion was evaluated using beta-cell-rich pancreatic islets isolated from ob/ob-mice. The glucose stimulation of the secretory activity is supposed to result from accumulation of Ca2+ in the submembrane cytoplasmic space. It is likely that this process reflects the balance between increased entry of Ca2+ into the beta-cells and an enhanced sequestration of Ca2+ in the organelle sinks. The proposed model can explain the cAMP potentiation of glucose-stimulated insulin release with suppression of the mitochondrial Ca2+ uptake. Furthermore, differences in the Ca2+ buffering capacity of the secretory granules may account for other characteristic features of glucose-stimulated insulin release, in particular its biphasic nature and sensitivity to suppression on withdrawal of nutrients.