Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.     

Amounts and distribution of intracellular magnesium and calcium in pancreatic beta-cells

beta-Cell-rich pancreatic islets were incubated for 60-120 min in the presence of 1 mM or 20 mM glucose and analysed with regard to their contents of magnesium and calcium and how these elements were distributed among subcellular fractions. The islets contained 42 mmol magnesium per kg protein with as much as 70 mmol per kg protein in the microsomal fraction. Both the total amount and intracellular distribution of magnesium remained unaffected after raising the glucose concentration of the incubation medium. The islet content of calcium was twice as high as that of magnesium, the mitochondria and secretory granules accounting for most of the calcium in the sedimentable fractions. In both organelles a substantial fraction of calcium was exchangeable as indicated from the incorporation of 45Ca during 90 min of incubation of the islets. When raising the glucose concentration to 20 mM the percentage exchange of calcium increased from 10 to 27 in the mitochondria and from 13 to 28 in the secretory granules. The glucose stimulation of 45Ca uptake was not associated with a statistically significant increase in the total amounts of calcium. However, in addition to stimulating calcium/calcium exchange, it cannot be excluded that glucose also induces a net accumulation of intracellular calcium in the beta-cells.     

Oscillatory control of insulin secretion

Pancreatic beta-cells possess an inherent ability to generate oscillatory signals that trigger insulin release. Coordination of the secretory activity among beta-cells results in pulsatile insulin secretion from the pancreas, which is considered important for the action of the hormone in the target tissues. This review focuses on the mechanisms underlying oscillatory control of insulin secretion at the level of the individual beta-cell. Recent studies have demonstrated that oscillations of the cytoplasmic Ca(2+) concentration are synchronized with oscillations in beta-cell metabolism, intracellular cAMP concentration, phospholipase C activity and plasma membrane phosphoinositide lipid concentrations. There are complex interdependencies between the different messengers and signalling pathways that contribute to amplitude regulation and shaping of the insulin secretory response to nutrient stimuli and neurohormonal modulators. Several of these pathways may be important pharmacological targets for improving pulsatile insulin secretion in type 2 diabetes.     

Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release

Summary Microdissected pancreatic islets from non-inbredob/ob-mice, were cultured for 6 or 7 days in serum-free tissue culture medium 199. The insulin content of the islets decreased 60% during culture in 17 mM or 28 mM glucose and about 70% in the presence of 3.3 mM or 5.6 mM glucose. At the end of a culture period in high glucose, the sum of the insulin in the islet plus that in the culture medium was almost twice as high as the insulin content of fresh islets, indicating an active insulin biosynthesis. The maximal insulin response to glucose after culture in 17 mM or 28 mM glucose was about 40% of that in fresh islets; after culture in 3.3 mM glucose it was 10%. Half-maximal stimulation was observed at a glucose concentration of 5 mM for islets cultured with high glucose as compared to 9 mM for fresh islets. Like glucose, glibenclamide was a more effective insulin stimulator after culture with a high glucose concentration than with a low one. However, leucine-induced insulin release was not affected by the glucose concentration in the preceding culture medium. Whereas potentiation of glucose-stimulated release by arginine or dibutyryl-cAMP was independent of glucose concentration during the culture, theophylline released three times more insulin when the islets had been cultured with high glucose.     

Regulation of parathyroid hormone release in normal and pathological parathyroid cells exposed to modulators of protein kinase C.

Effects of the protein kinase C activating phorbol ester 12-O-tetradecanoyl phorbol 13-acetate and the inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) on parathyroid hormone (PTH) release were studied in normal bovine and pathological human parathyroid cells. An increase of extracellular Ca2+ from 0.5 to 3.0 mmol/l inhibited PTH release by 60% in the bovine cells with half maximal effect (ED50) at 1.31 mmol/l. This inhibition reached less than 50% in the cells from patients with primary and uremic hyperparathyroidism, and the ED50 values were 1.49 and 1.42 mmol/l, respectively. The phorbol ester (0.1 mumol/l) made secretion insensitive to changes of extracellular Ca2+, an action counteracted by H-7 (50 mumol/l) in the bovine cells, whereas H-7 alone had no effects. The phorbol ester and H-7 had opposite actions on regulation of PTH release also from cells from patients with hyperparathyroidism. However, in pathological cells H-7 alone improved Ca2+ inhibition of secretion by stimulating release in low Ca2+ concentrations and decreasing the ED50 values. The magnitude of changes in ED50 values by H-7 increased with the severity of the secretory disturbance of the pathological cells. The results indicate that increased protein kinase C activity may be a factor of importance in the pathophysiology of hyperparathyroidism.     

Defective glucose-dependent cytosolic Ca2+ handling in islets of GK and nSTZ rat models of type 2 diabetes

We examined to what extent the abnormal glucose-dependent insulin secretion observed in NIDDM (non-insulin-dependent diabetes mellitus) is related to alterations in the handling of cytosolic Ca2+ of islets of Langerhans. Using two recognized rat models of NIDDM, the GK (Goto-Kakizaki) spontaneous model and the nSTZ (neonatal streptozotocin) induced model, we could detect several common alterations in the glucose-induced [Ca2+]i cytosolic responses. First, the initial reduction of [Ca2+]i following high glucose (16.7 mM) observed routinely in islets obtained from non-diabetic Wistar rats could not be detected in GK and nSTZ islets. Second, a delayed response for glucose to induce a subsequent 3% increase of [Ca2+]i over basal level was observed in both GK (321+/-40 s, n=11) and nSTZ (326+/-38 s, n=13) islets as compared with Wistar islets (198+/-20 s, n=11), values representing means+/-s.e.m. Third, the rate of increase in [Ca2+]i in response to a high glucose challenge was 25% and 40% lower in GK and nSTZ respectively, as compared with Wistar islets. Fourth, the maximal [Ca2+](i) level reached after 10 min of perifusion with 16.7 mM glucose was lower with GK and nSTZ islets and represented respectively 60% and 90% of that of Wistar islets. Further, thapsigargin, a blocker of Ca2+/ATPases (SERCA), abolished the initial reduction in [Ca2+]i observed in response to high glucose and induced fast [Ca2+]i oscillations with high amplitude in Wistar islets. The latter effect was not seen in GK and nSTZ islets. In these two NIDDM models, several common alterations in glucose-induced Ca2+ handling were revealed which may contribute to their poor glucose-induced insulin secretion.     

Sulfonylurea mimics the effect of glucose in inducing large amplitude oscillations of cytoplasmic Ca2+ in pancreatic beta-cells

The effects of the insulin-releasing sulfonylurea tolbutamide on the cytoplasmic Ca2+ concentration [( Ca2+]i) in individual pancreatic beta-cells or suspensions of beta-cells were analyzed using the probe fura-2 and dual-wavelength fluorometry. Subsequent additions of 1, 10, and 100 microM tolbutamide induced a graded response, ranging from a single [Ca2+]i peak to a sustained increase. These effects depended on the presence of extracellular Ca2+ and were reversed by the hyperglycemic sulfonamide diazoxide. The responses were diminished in the presence of albumin and varied considerably between different cells. Sometimes tolbutamide triggered slow large amplitude oscillations in [Ca2+]i similar to those induced by glucose. The increase in [Ca2+]i during each tolbutamide-induced oscillation was often more rapid than for glucose-induced oscillations. Oscillations or steady state increases in [Ca2+]i induced by glucose were little influenced by tolbutamide. However, subthreshold concentrations of glucose could reactivate [Ca2+]i response to tolbutamide that had declined. Although in several ways the abilities of glucose and tolbutamide to raise [Ca2+]i were similar, the sulfonylurea lacked a [Ca2+]i-lowering component. The latter effect of glucose was so pronounced that an increase of its concentration from 3 to 20 mM caused temporary lowering of [Ca2+]i to the basal level, even during tolbutamide stimulation. The results indicate that closure of the ATP-sensitive K(+)-channels is important for the large amplitude oscillations of [Ca2+]i the appearance of which reflects the balance between entry of Ca2+ through the voltage-dependent channels and its removal from the cytoplasm.     

Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands

This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans.