Verapamil corrects abnormal metabolism of pancreatic islets and insulin secretion in phosphate depletion.

Phosphate depletion (PD) causes impaired insulin secretion and metabolic derangements in pancreatic islets. We studied PD, pair-weighed (PW), and PD and PW rats treated with verapamil (PD-V and PW-V) to examine the mechanisms of these derangements. Cytosolic calcium ([Ca2+]i) in PD islets was higher than that in PW, PD-V, and PW-V islets, and the values in the latter three groups were not different. Both basal and stimulated ATP in PD islets were lower than those in PW, PW-V, or PD-V islets. The maximum velocity (Vmax) of Ca(2+)-ATPase and the Km and Vmax of Na+,K(+)-ATPase were reduced in PD islets. In both PD-V and PW-V, the Vmax of Ca(2+)-ATPase was higher than that in PD, but lower than that in PW. Both initial and second phases of insulin secretion by PD islets were lower than those by PW and PW-V islets. In PD-V rats, insulin secretion was greater than that in PD rats, but only the second phase was significantly higher. The data are consistent with either of the following possibilities: 1) PD causes a change in the permeability of islets, allowing increased entry of Ca2+ into them and a fall in ATP of islets; the latter would impair the activity of both ATPases, leading to reduced Ca2+ extrusion from islets and, hence, an elevation in their [Ca2+]i; or 2) the primary defect in PD is a reduction in the activities of ATPases of islets due to the fall in ATP secondary to phosphorus deficiency. The decreased Ca2+ extrusion that ensues, even in the face of normal Ca2+ entry, will result in high [Ca2+]i. In either of these scenarios the rise in [Ca2+]i would inhibit mitochondrial oxygen consumption and ATP production, further lowering the ATP content of the islets. The higher [Ca2+]i and low ATP of PD underlie the impaired insulin secretion. Verapamil, by blocking normal or augmented Ca2+ entry into the islets, mitigates or prevents the derangements in islet function and metabolism.     

Stimulus-secretion coupling of arginine-induced insulin release: comparison with lysine-induced insulin secretion

L-Lysine, like-L-arginine, L-ornithine, or L-homoarginine, accumulated in rat pancreatic islets and stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin release in islets exposed to D-glucose (7.0 mM). The effect of L-lysine differed from that of the other cationic amino acids by such features as the absence of a threshold concentration for stimulation of insulin release, a much lesser sensitivity of the secretory response to intracellular acidification, and the stimulation of 86Rb net uptake over 60 min of incubation. This coincided with the fact that even in the absence of another exogenous nutrient, L-lysine was well oxidized, augmented NH4+ production, increased both the ATP content and ATP/ADP ratio, caused a time-related decrease in 86Rb fractional outflow, and provoked either a transient (10 mM L-lysine) or sustained (20 mM L-lysine) stimulation of insulin secretion. It is proposed, therefore, that the functional response of the pancreatic B-cell to L-lysine involves not only a biophysical mechanism similar to that responsible for the insulinotropic action of L-homoarginine, but also a significant, albeit modest, metabolic component, which reflects the capacity of L-lysine to act as a fuel in islet cells.     

Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes

Extracellular adenosine triphosphate (ATP) is known to reversibly increase the cation permeability of a variety of freshly isolated and cultured cell types. In this study the effects of extracellular ATP were studied using peripheral blood lymphocytes (PBL) isolated from both normal subjects and from patients with chronic lymphocytic leukemia (CLL). Changes in the permeability to Na+, Rb+, and Li+ ions were measured using conventional isotope and flame photometry techniques. In addition, changes in cytosolic (Ca2+) were fluorimetrically monitored to assess possible changes in net Ca2+ influx. ATP produced a 12-fold increase in 22Na+ influx into CLL cells but only a 3.5-fold increase in this flux in PBL cells. A maximal response was produced by 0.1 mmol/L ATP in the absence of Mg2+, while a twofold molar excess of Mg2+ over ATP abolished the response. ATP had no effect on the passive (ouabain-insensitive) 86Rb+ influx into PBL cells but stimulated this flux by fivefold in the CLL cells. Li+ influx into CLL cells was also stimulated threefold by ATP. Under these same conditions ATP also produced a net increase in total cell Na and a decrease in total cell K in the CLL cells. Exclusion of two normally impermeable dyes, trypan blue and ethidium bromide, was not altered in the ATP-treated CLL cells. Finally, extracellular ATP (3 mmol/L) produced no significant change in the cytosolic (Ca2+) of normal, monocyte-depleted populations of PBL. Conversely, this same concentration of ATP produced a very rapid (complete within 30 seconds) and a significant (an average threefold peak change) increase in the cytosolic (Ca2+) of cell preparations derived from five out of nine CLL patients. In these latter CLL cells, the ATP-induced elevation in cytosolic (Ca2+) appeared to be due to a net increase in Ca2+ influx, since no elevations were observed when the extracellular (Ca2+) was reduced to less than 0.1 mmol/L. These actions of ATP were specific in that equimolar concentrations of other nucleotides were without effect. These data indicate that treatment of CLL lymphocytes with extracellular ATP4 produces large increases in cation permeability. In contrast, there is less or no ATP-induced permeabilization of normal PBL.     

Effects of 5-hydroxytryptamine on rubidium ion fluxes and insulin release in cultured pancreatic islets

We have incubated pancreatic islets isolated from noninbred ob/ob mice and NMRI mice for 3 days with or without 5-hydroxytryptamine (5-HT) in the medium and tested the effect of such long term treatment on subsequent insulin release and 86Rb+ accumulation and efflux. Two tenths millimolars of 5-HT abolished insulin release in response to 20 mM glucose. Two tenths millimolars of 5-HT also diminished the ability of islets to accumulate 86Rb+ and the effect of 10 mM glucose on 86Rb+ efflux. One one-hundredth millimolars of 5-HT had no effect on insulin release or 86Rb+ fluxes. Clearly, islets subjected to 5-HT for 3 days at concentrations that do not elicit demonstrable effects in short term incubations show a reduced secretory response. However, the physiological role of the high affinity uptake system for 5-HT in islet cells [Michaelis-Menten constant (Km) = 1.6 microM] remains unknown.     

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells

To determine the effect of D-glucose on the beta-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Ume?-ob/ob mice in the absence or presence of 1mM ouabain. D-glucose (20mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Ume?-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3-20mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135mM Na+ and 20mM K+). Neither D-glucose (3-20mM) nor L-glucose or 3-O-methyl-D-glucose (20mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7 mM K+). Diphenylhydantoin (150 microM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 microM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of beta-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.     

Galanin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K+ permeability of the B-cell membrane.

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.     

Modulation of beta-cell ouabain-sensitive 86Rb+ influx (Na+/K+ pump) by D-glucose, glibenclamide or diazoxide

The activity of the beta-cell Na+/K+ pump was studied by using ouabain-sensitive (1mM ouabain) 86Rb+ influx in beta-cell-rich islets of Ume?-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive 86Rb+ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at 0 mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of 86Rb+ influx during the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive 86Rb+ uptake in the same manner as glucose. The stimulatory effect showed its apparent maximum at 0.5 microM. Pre-treatment (60 min) of islets with 1 microM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive 86Rb+ influx. The stimulatory effect of glibenclamide and D-glucose were not additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1 microM) was observed on the Na+/K+ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na+/K+ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably as part of the membrane repolarisation process.     

Insulin secretion and the morphological and metabolic characteristics of pancreatic islets of hyperthyroid ob/ob mice.

Thyroxine treatment induced experimental hyperthyroidism in ob/ob mice, inhibited glucose-induced insulin secretion from the isolated perfused ob/ob mouse pancreas, and reduced total pancreas insulin content. In contrast, glucose-induced insulin release from incubated pancreatic islets and insulin content of pancreatic islets from ob/ob mice isolated by freehand microdissection were not reduced after thyroxine treatment when expressed per microgram dry islet. Histological examination of the ob/ob mouse pancreas revealed islets without degenerative lesions of islet cells. Granularity of beta cells was well preserved. The average number of pancreatic islets was unchanged. However, the beta cell area was significantly decreased in relation to the total pancreatic parenchyma after thyroxine treatment. This implies that insulin release and content per pancreatic islet was half of that of the controls. ATP content of islets was slightly reduced. Glucose oxidation and glucose utilization by islets from treated mice were slightly increased. Thyroxine treatment of the animals did not abolish the stimulation of 45Ca2+ uptake by glucose, but it did suppress the potentiating effect of fasting on the stimulatory effect of glucose on 45Ca2+ uptake. The metabolic characteristics of islets from experimentally hyperthyroid mice are those of all hyperthyroid tissues. The results provide no evidence for the view that the effects of thyroxine treatment may be due to disturbed metabolic function or energy deprivation of pancreatic islets. Inhibition of insulin secretion from the pancreas after thyroxine administration is apparently due to a reduction in pancreas insulin content and a diminished pancreatic islet volume. Reduced pancreatic islet volume represents most probably a reduction of individual islet cell volume.     

Evolution of metabolic and functional derangements of pancreatic islets in phosphate depletion.

Phosphate depletion (PD) causes a rise in basal level of cytosolic calcium ([Ca2+]i) of pancreatic islets, a decrease in their basal and stimulated ATP content, a reduction in the maximum velocity (Vmax) of Ca2+ adenosine triphosphatase (ATPase) and Na(+)-K+ ATPase, impaired glucose-induced calcium signal and decreased glucose-induced insulin secretion. The sequence of events that lead to these derangements during the evolution of PD are not defined. The present study examined this issue by measuring the metabolic and functional profile of pancreatic islets weekly during the evolution of PD over a period of 6 weeks, and whether phosphate repletion reverses these abnormalities. The results show that initial abnormalities are a rise in Vmax of Ca2+ ATPase and modest rise in basal [Ca2+]i. This was followed by a fall in basal and stimulated ATP content. With the fall in ATP content, the Vmax of Ca2+ ATPase and Na(+)-K+ ATPase decreases and the rise in [Ca2+]i becomes more pronounced. A decrease in glucose-induced insulin secretion becomes evident with the fall in ATP, the decrease in glucose-induced calcium signal, and/or delta[Ca2+]i/basal[Ca2+]i. All functional and metabolic derangements of the pancreatic islets returned to normal after phosphate repletion. Taken together, our data are consistent with the notion that PD is associated with an initial increase in calcium influx into the islets. This is followed by modest but significant rise in [Ca2+]i which, in turn, would inhibit mitochondrial oxidation and ATP generation leading to a decrease in ATP content. The latter compromises the activity of Ca2+ ATPase and Na(+)-K+ ATPase which are involved, directly or indirectly, in calcium extrusion out of the islets. The increased influx of calcium combined with decreased calcium extrusion is followed by a further rise in basal levels of [Ca2+]i. This sequence of events continues until a steady state is reached and is characterized by reduced basal and stimulated ATP content, reduced Vmax of Ca2+ ATPase and Na(+)-K+ ATPase and elevated basal level of [Ca2+]i. Phosphate repletion reverses all these abnormalities.     

Stimulus-secretion coupling of arginine-induced insulin release. Uptake of metabolized and nonmetabolized cationic amino acids by pancreatic islets

In order to assess the possible role of L-arginine accumulation in islet cells as a determinant of its insulinotropic action, the uptake of L-arginine and other cationic amino acids (L-ornithine, L-homoarginine, D,L-alpha-methylornithine, D,L-alpha-difluoromethylornithine) by rat pancreatic islets was compared to the ionic and secretory responses of the islets to the same amino acids. A tight correlation was found between the net uptake of these amino acids and their capacity to stimulate 86Rb efflux, 45Ca uptake and efflux, and insulin release. In the latter respect, there was little difference between metabolized and nonmetabolized amino acids. Thus, although L-homoarginine and 4-amino-1-guanylpiperidine-4-carboxylic acid failed to act as a substrate for either arginase or amino acid aminotransferase in islet homogenates, they both stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin secretion in intact islets. These findings are compatible with the view that the accumulation of these positively charged amino acids in islet cells represents an essential determinant of their secretory action. Hence, the release of insulin evoked by these amino acids could be due to depolarization of the plasma membrane with subsequent gating of voltage-sensitive Ca2+ channels and/or to some other biophysical effect, as suggested by the persistence of a sizeable secretory response to L-arginine or L-ornithine in islets perifused at a high concentrations of extracellular K+ (50 mM).     

Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx

Mouse pancreatic islets were used to investigate the mechanisms and functional significance of the B cell membrane depolarization by acetylcholine (ACh). At low glucose (3mM), ACh (20 microM) increased 22Na+ influx, and slightly depolarized the B cell membrane but did not induce electrical activity or stimulate 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and barely affected basal insulin release. At a stimulatory concentration of glucose (10 mM), ACh stimulated 22Na+ influx, depolarized the B cell membrane, increased glucose-induced electrical activity, and stimulated 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and strongly potentiated insulin release. Omission of extracellular Ca2+ did not impair ACh stimulation of 22Na+ influx or 86Rb+ efflux, slightly modified the acceleration of 45Ca2+ efflux, and almost completely suppressed the increase in insulin release. Na+ omission (with N-methyl-D-glucamine as substitute) prevented the B cell membrane depolarization and the stimulation of 45Ca2+ influx, largely inhibited the acceleration of 86Rb+ efflux and insulin release, and suppressed the late phase of 45Ca2+ efflux otherwise produced by ACh. On the other hand, ACh stimulation of 3H efflux from islets prelabeled with myo-[2-3H]inositol was not affected by Na+ omission. All effects of ACh were blocked by atropine and unaffected by nicotinic antagonists. It is concluded that activation of muscarinic receptors depolarized the B cell membrane by increasing its permeability to Na+. When the membrane is already depolarized by glucose, this further depolarization augments Ca2+ influx and, hence, potentiates insulin release.     

Prior exposure to high glucose augments depolarization-induced insulin release by mitigating the decline of ATP level in rat islets.

A brief exposure to elevated glucose augments the insulin secretory response of islets to subsequent stimulation. The site of this priming effect of glucose in the mechanism of the regulation of insulin secretion is not completely known, however. Insulin release triggered by a depolarizing concentration of K+ in the presence of basal glucose is markedly enhanced in primed rat islets. To clarify the role of priming on Ca(2+) and ATP efficacy in the exocytotic apparatus, islets were electrically permeabilized to vary the intracellular Ca(2+) and ATP concentrations according to the extracellular medium, and insulin release was evaluated. Ca(2+) and ATP efficacy in Ca(2+)- and ATP-dependent insulin secretion was not affected by priming, and alteration of the intracellular Ca(2+) concentration after depolarization cannot account for the phenomenon. There was no difference in ATP content before depolarization between nonprimed and primed islets. Moreover, the decline in ATP level after depolarization with basal glucose was observed in both primed and nonprimed islets. However, a reduced decline in ATP level in the early phase was observed in primed islets. In addition, oligomycin, a mitochondrial metabolism inhibitor, abolished the difference in ATP level between primed and nonprimed islets, suggesting that mitochondrial ATP production may be linked to the phenomenon.     

cAMP-mediated signaling normalizes glucose-stimulated insulin secretion in uncoupling protein-2 overexpressing beta-cells

We investigated whether an increase in cAMP could normalize glucose-stimulated insulin secretion (GSIS) in uncoupling protein-2 (UCP2) overexpressing (ucp2-OE) beta-cells. Indices of beta-cell (beta-TC-6f7 cells and rodent islets) function were measured after induction of ucp2, in the presence or absence of cAMP-stimulating agents, analogs, or inhibitors. Islets of ob/ob mice had improved glucose-responsiveness in the presence of forskolin. Rat islets overexpressing ucp2 had significantly lower GSIS than controls. Acutely, the protein kinase A (PKA) and epac pathway stimulant forskolin normalized insulin secretion in ucp2-OE rat islets and beta-TC-6f7 beta-cells, an effect blocked by specific PKA inhibitors but not mimicked by epac agonists. However, there was no effect of ucp2-OE on cAMP concentrations or PKA activity. In ucp2-OE islets, forskolin inhibited ATP-dependent potassium (K(ATP)) channel currents and (86)Rb(+) efflux, indicative of K(ATP) block. Likewise, forskolin application increased intracellular Ca(2+), which could account for its stimulatory effects on insulin secretion. Chronic exposure to forskolin increased ucp2 mRNA and exaggerated basal secretion but not GSIS. In mice deficient in UCP2, there was no augmentation of either cAMP content or cAMP-dependent insulin secretion. Thus, elevating cellular cAMP can reverse the deficiency in GSIS invoked by ucp2-OE, at least partly through PKA-mediated effects on the K(ATP) channel.     

Membrane ion transport in erythrocytes of salt hypertensive Dahl rats and their F2 hybrids: the importance of cholesterol.

The possible association of salt hypertension and altered lipid metabolism with abnormalities of particular systems transporting sodium and potassium has been studied in erythrocytes of Dahl rats and their F2 hybrids fed a high-salt diet since weaning. Our attention was paid to the Na(+)-K+ pump, Na(+)-K+ cotransport and especially to passive membrane permeability for Na+ and Rb+ (Na+ and Rb+ leak), because the Na+ leak was found to be dependent on the genotype, age and salt intake of Dahl rats, whereas the Rb+ leak was suggested to be a potential marker of salt sensitivity in Dahl and Sabra rats. Young male Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats kept on a low-salt (0.3% NaCl) or high-salt diet (8% NaCl) were used for the progenitor study. The subsequent genetic study was based on 135 young male SS/Jr x SR/Jr F2 hybrids fed a high-salt diet since weaning. Ouabain (5 mmol/l) and bumetanide (10 micromol/l) were used to distinguish the contribution of the Na(+)-K+ pump, Na(+)-K+ cotransport and passive membrane permeability to measured net Na+ fluxes and unidirectional Rb+ (K+) movements. Compared to normotensive SR/Jr animals, salt-loaded SS/Jr rats had higher blood pressure (BP), elevated erythrocyte Na+ content, and increased Na+ and Rb+ leaks together with enhanced Na+ and Rb+ transport mediated by the Na(+)-K+ pump and Na(+)-K+ cotransport system. Salt hypertensive Dahl rats were also characterized by elevated plasma levels of total cholesterol and triglycerides, which were positively associated with BP of F2 hybrids (r=0.27 and 0.24, p< 0.01). In F2 hybrids, mean arterial pressure correlated significantly with erythrocyte Na+ content (r=0.24, p<0.01) and ouabain-sensitive Na+ extrusion, but not with the passive membrane permeability for Na+ or Rb+ (r=-0.02 and 0.06, not significant). Both of the above-mentioned significant associations could partially be ascribed to the dependence of erythrocyte Na+ content and ouabain-sensitive Na+ extrusion on plasma cholesterol (r=0.18 and 0.21, p<0.05). Our results support the idea that abnormal lipid metabolism and/or altered Na+,K(+)-ATPase function play an important role in the pathogenesis of salt hypertension in salt-sensitive Dahl rats.     

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.     

Are ionic fluxes of pancreatic beta cells a target for gastric inhibitory polypeptide?

Gastric inhibitory polypeptide (GIP), an incretin candidate, is suggested to amplify the glucose-induced insulin secretion. To evaluate its mode of action we examined whether GIP affects 86Rb+ efflux, 45Ca2+ uptake or efflux, and intracellularly recorded electrical activity of mouse pancreatic islets. GIP (5 nM) neither inhibited 86Rb+ efflux at 3 mM glucose nor modulated 86Rb+ efflux that was inhibited by 5.6 mM glucose or stimulated by the calcium ionophore A23187. 45Ca2+ uptake was increased by GIP in the presence of 16.7 mM which was not observed at 3 or 11 mM glucose. GIP elevated 45Ca2+ efflux from islets, but did not modify 45Ca2+ efflux when a virtually Ca2+ free medium was used. Electrical activity of beta cells induced by 16.7 mM glucose was significantly increased by 5 nM GIP. It is concluded that the amplification of insulin release by GIP is based on the effect of GIP on Ca2+ uptake.     

Effect of extracellular phosphate on Ca2+ and K+ fluxes in pancreatic islets

The idea that a lowering in cytosolic Ca2+ concentration may cause a decrease in K+ conductance in the pancreatic B-cell was tested by investigating the effect of a high extracellular phosphate concentration on 45Ca and 86Rb efflux from prelabelled rat pancreatic islets. Whether in the absence or presence of glucose, 20 mM phosphate tended to decrease 45Ca efflux. This effect was not suppressed in the absence of extracellular Ca2+, at least in glucose-deprived islets, suggesting that it may reflect a fall in cytosolic Ca2+ concentration. The administration of phosphate failed, however, to decrease 86Rb efflux from the islets. In the presence of extracellular Ca2+, 20 mM phosphate also failed to stimulate insulin release from islets perifused at low glucose concentration and inhibited insulin release stimulated by a high glucose concentration. These data indicate that the sequestration of Ca2+ in intracellular organelles and concomitant decrease in cytosolic Ca2+ concentration, as presumably provoked by a rise in extracellular phosphate concentration, is not sufficient to simulate the effect of glucose on K+ conductance.     

Interleukin-6 affects insulin secretion and glucose metabolism of rat pancreatic islets in vitro

Recently it has been postulated that interleukin-1 (IL-1) locally released by infiltrating mononuclear cells may destroy the pancreatic B cells during the development of insulin-dependent diabetes mellitus. Since IL-1 is a potent inducer of interleukin-6 (IL-6) in various cells, it is conceivable that IL-6 is a second mediator of the IL-1 action. In the present study the effects of IL-6 alone or in combination with IL-1 were studied on pancreatic islet function in vitro after tissue culture and compared with the effects observed after exposure to IL-1 only. Rat pancreatic islets were cultured in medium RPMI 1640 + 10% calf serum with or without the addition of human recombinant IL-6 (500-5000 pg/ml) for 48 h. The medium insulin accumulation was increased by 40-50% after culture with 500-2000 pg/ml IL-6, but was similar to the controls at 5000 pg/ml. When islets were cultured for 18 h only, also 5000 pg/ml IL-6 stimulated the medium insulin accumulation. IL-6 did not affect the islet insulin content and the rates of islet (pro)insulin and total protein biosynthesis. It inconsistently decreased the islet DNA content. In short-term experiments after 48-h culture with IL-6, there was a dose-dependent inhibition of the glucose-stimulated insulin release. On the other hand, islets cultured with IL-6 (5000 pg/ml) exhibited an elevated glucose oxidation and oxygen uptake, but a lower ATP content at 16.7 mM glucose and an unaffected glucose utilization and glutamine oxidation compared to the controls. This raises the possibility that IL-6 had induced a condition with an increased energy expenditure, resulting in an enhanced mitochondrial metabolism of glucose. Islets cultured with human recombinant IL-1 beta (25 units/ml) showed a strong inhibition of the insulin accumulation in the culture medium and of glucose-stimulated insulin release and a marked decrease in the islet DNA and insulin content. A combination of IL-1 (25 U/ml) + IL-6 (1000 pg/ml) did not alter the inhibitory action of IL-1 alone. The present findings thus show that IL-6 induces a dissociation between insulin secretion and glucose oxidation in islets in vitro. This has not been observed in islets exposed to IL-1, which suggests that IL-6 does not solely mediate the inhibitory effects of IL-1 on islet function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of membrane permeability by external and internal ATP in 3T6 cells grown in serum-free medium.

Cultures of 3T6 cells were plated in serum-free medium and grown in the presence of insulin (1 microgram/ml) and epidermal growth factor (0.5 ng/ml). External ATP (250 microM) applied to such cultures caused a rapid efflux of acid-soluble pools labeled with [3H]uridine, 2-deoxy[3H]glucose, or 86Rb+ and allowed the entry of p-nitrophenylphosphate. This increase in passive membrane permeability depended on ATP concentration, pH, and time of ATP contact with the cells, and it was not produced by GTP, UTP, or Pi. In the presence of compounds that decrease intracellular ATP, low concentrations of external ATP (40 microM) caused a massive synergistic stimulation of efflux. The efflux of acid-soluble pools was stopped (sealing) by bringing the cultures of 3T6 cells to neutral pH in the presence of Ca2+ and Mg2+. Exposure of 3T6 cells grown in serum-free medium to [gamma-32P]ATP under the conditions of permeabilization led to the selective labeling of a membrane protein with a molecular weight of 44,000 as revealed by NaDodSO4 polyacrylamide gel electrophoresis and autoradiography. The results show that the control of membrane permeability by ATP is completely independent of serum-deprived proteins. Furthermore, the protein band (Mr, 44 x 10(3)) that shows selective labeling by [32P]ATP during permeabilization is not an adsorbed serum component.     

Glyburide and tolbutamide induce desensitization of insulin release in rat pancreatic islets by different mechanisms.

Insulin secretion was studied in rat pancreatic islets after 24-h exposure to various glyburide or tolbutamide concentrations. Glucose-induced insulin release was significantly (P < 0.05) reduced in islets cultured with 0.1 microM glyburide or 100 microM tolbutamide (2098 +/- 187, 832 +/- 93, and 989 +/- 88 pg/islet.h in control, glyburide-exposed, and tolbutamide-exposed islets, respectively). When glyburide-treated islets were stimulated with glyburide or tolbutamide, insulin release was also impaired compared to that in control islets (P < 0.05). In contrast, tolbutamide-exposed islets showed an impaired response to tolbutamide, but a normal response to glyburide. To investigate the mechanism of the sulfonylurea-induced impairment of insulin secretion, we measured insulin release and Rb+ efflux (a marker of the K+ channel activity) in a perifusion system and islet Ca2+ uptake under static conditions. Insulin release in response to 16.7 mM glucose increased in control islets from 9.4 +/- 1.1 to 131 +/- 19 pg/islet.min (first phase secretion peak). Simultaneously, the fractional 86Rb+ efflux declined from 0.015 +/- 0.002% to 0.006 +/- 0.001% (change in decrement, -63.5%). Glucose-induced insulin release in glyburide- and tolbutamide-treated islets was significantly reduced (first phase peak, 22.1 +/- 5 and 39.7 +/- 8 pg/islet.min, respectively; P < 0.05), and the fractional 86Rb+ efflux decrement was -21 +/- 6% for glyburide (P < 0.005 vs. control islets) and -65 +/- 4% (not different from control) for tolbutamide. When glyburide- or tolbutamide-exposed islets were stimulated with the corresponding sulfonylurea, insulin release was impaired compared to that in control islets (P < 0.05), but, again, 86Rb+ efflux was impaired (P < 0.05) only in glyburide-exposed islets. When 45Ca2+ uptake was studied, the increase in glucose concentration from 2.8 to 16.7 mM increased calcium uptake in control islets from 1.76 +/- 0.58 to 7.27 +/- 1.36 pmol/islet.2 min (n = 4). Preexposure to 0.1 microM glyburide did not change calcium uptake at a glucose concentration of 2.8 mM (1.44 +/- 0.45 pmol/islet.2 min) but significantly reduced calcium uptake stimulated by 16.7 mM glucose (3.21 +/- 0.35 pmol/islet.2 min; n = 4; P < 0.005 compared to control islets). In contrast, preexposure to 100 microM tolbutamide did not change either basal or glucose-stimulated calcium uptake (1.44 +/- 0.45 and 6.90 +/- 0.81 pmol/islet.2 min, respectively; n = 4). These data show that in vitro chronic exposure of pancreatic islets to the sulfonylureas glyburide and tolbutamide impairs their ability to respond to a subsequent glucose or sulfonylurea stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)