Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B-cell function

28Mg2+ uptake by rat islets was measured during incubation with various stimulators or inhibitors of insulin release. D-Glucose induced a dose-dependent increase in 28Mg2+ uptake after 10 min or 120 min. The threshold concentration was around 6 mM and the maximum effect was observed with 15-20 mM glucose. After 120 min 28Mg2+ uptake was also stimulated by the metabolized sugars mannose, N-acetylglucosamine or glyceraldehyde, was unaffected by the non-metabolized or poorly metabolized L-glucose, galactose, 3-O-methylglucose, 2-deoxyglucose, fructose or mannoheptulose and was inhibited by glucosamine. The effect of glucose was markedly impaired by mannoheptulose, glucosamine, aminooxyacetate and NH4Cl, but was only partially decreased by D600 or diazoxide, which were ineffective in a glucose-free medium. Tolbutamide or KCl slightly increased 28Mg2+ uptake. Alanine, leucine alone or with glutamine, and ketoisocaproate also stimulated 28Mg2+ uptake, whereas arginine and lysine decreased it. These changes in 28Mg2+ uptake, brought about by various modifiers of the B-cell function, are thus similar but not identical to the changes in Ca2+ uptake, and are not the consequence of insulin release. The stimulatory effect of glucose requires glucose metabolism by islet cells, but is only partially due to depolarization of the B-cell membrane.     

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.     

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.     

Inosine-stimulated insulin release and metabolism of inosine in isolated mouse pancreatic islets.

Inosine is a potent primary stimulus of insulin secretion from isolated mouse islets. The inosine-induced insulin secretion was totally depressed during starvation, but was completely restored by the addition of 5 mM-caffeine to the medium and partially restored by the addition of 5 mM-glucose. Mannoheptulose (3 mg/ml) potentiated the effect of 10 mM-inosine in islets from fed mice. The mechanism of the stimulatory effect of inosine was further investigated, and it was demonstrated that pancreatic islets contain a nucleoside phosphorylase capable of converting inosine into hypoxanthine and ribose 1-phosphate. Inosine at 10 mM concentration increased the lactate production and the content of ATP, glucose 6-phosphate (fructose 1,6-diphosphate + triose phosphates) and cyclic AMP in islets from fed mice. In islets from starved mice inosine-induced lactate production was decreased and no change in the concentration of cyclic AMP could be demonstrated, whereas the concentration of ATP and glucose 6-phosphate rose. Inosine (10 mM) induced a higher concentration of (fructose 1,6-diphosphate + triose phosphates) in islets from starved mice than in islets from fed mice suggesting that in starvation the activities of glyceraldehyde 3-phosphate dehydrogenase or other enzymes below this step in glycolysis are decreased. Formation of glucose from inosine was negligible. Inosine had no direct effect on adenylate cyclase activity in islet homogenates. The observed changes in insulin secretion and islet metabolism mimic what is seen when glucose and glyceraldehyde stimulate insulin secretion, and as neither ribose nor hypoxanthine-stimulated insulin release, the results are interpreted as supporting the substrate-site hypothesis for glucose-induced insulin secretion according to which glucose has to be metabolized in the beta-cells before secretion is initiated.     

The effect of sugars on (pro)insulin biosynthesis

Rates of incorporation of [4,5-(3)H]leucine into insulin plus proinsulin, designated ;(pro)insulin', and total protein in rat pancreatic islets were measured. Glucose stimulates rates of total protein and (pro)insulin biosynthesis, but (pro)insulin biosynthesis is stimulated preferentially. Mannose and N-acetylglucosamine also stimulate (pro)insulin and total protein biosynthesis; inosine and dihydroxyacetone stimulate (pro)insulin biosynthesis specifically. Fructose does not stimulate (pro)insulin biosynthesis when tested alone, but does so in the presence of low concentrations of glucose, mannose or N-acetylglucosamine. Many glucose analogues do not stimulate (pro)insulin biosynthesis. Mannoheptulose inhibits synthesis of (pro)insulin and total protein stimulated by glucose or mannose but not by dihydroxyacetone, inosine or N-acetylglucosamine; phloretin (9mum) inhibits N-acetylglucosamine-stimulated (pro)insulin biosynthesis preferentially. The data are in agreement with the view that the same glucose-sensor mechanism may control both insulin release and biosynthesis, and ;substrate-site' model is suggested. The threshold for stimulation of biosynthesis of (pro)insulin and total protein is lower than that found for glucose-stimulated insulin release; moreover the biosynthetic response to an elevation of glucose concentration is slower than that found for insulin release. The physiological implication of these findings is discussed. Caffeine and isobutylmethylxanthine, at concentrations known to increase islet 3':5'-cyclic AMP and potentiate glucose-induced insulin release, were without effect on rates of glucose-stimulated (pro)insulin biosynthesis.     

Kinetics of utilization of organic substrates by Mycoplasma mycoides subsp. mycoides in a salts solution: a flow-microcalorimetric study

The metabolism of various organic substrates by suspensions of Mycoplasma mycoides subsp. mycoides in a salts solution was followed by microcalorimetry. Enthalpy changes associated with metabolism were in good agreement with theoretical values. Substrate utilization showed Michaelis kinetics, allowing saturation constants (Km) and maximum specific rates of substrate utilization (Vmax) to be determined. In cells grown on a complex medium containing glucose, Km values were: glucose, fructose, N-acetylglucosamine, glycerol and pyruvate, less than 5 microM; lactate, 20 microM; glucosamine, 130 microns, and mannose, 1 mM. Values of Vmax for glycerol, pyruvate and lactate were similar and approximately twice those for glucose, mannose, glucosamine and N-acetylglucosamine; Vmax for fructose was one-quarter of that for glucose. In cells grown on complex medium in which glucose was replaced by mannose, glucosamine or N-acetylglucosamine, Vmax and Km for the respective growth sugars and for glucose were not significantly affected. However, in cells grown in the presence of fructose, Vmax for fructose increased to the value observed for glucose. It is suggested that M. mycoides is adapted to, and is constitutive for, the utilization of a single sugar (glucose), and a single amino sugar (N-acetylglucosamine), but that in the presence of fructose a fructose-utilizing pathway is induced.     

Hexose metabolism in pancreatic islets. The phosphorylation of fructose

Fructose, like glucose, rapidly equilibrates across the plasma membrane of pancreatic islet cells, but is poorly metabolized and is a weak insulin secretagogue in rat pancreatic islets. A possible explanation for such a situation was sought by investigating the modality of fructose phosphorylation in islet homogenates. Several findings indicated that the phosphorylation of fructose is catalyzed by hexokinase, but not fructokinase. First, at variance with the situation found in liver homogenates, the phosphorylation of fructose in the islet homogenate was unaffected by K+ and inhibited by glucose, mannose, glucose 6-phosphate or glucose 1,6-bisphosphate. Second, the Km for fructose was much higher in islets than in liver. Third, in islet homogenates the Km and Vmax for fructose were much higher than those for glucose or mannose phosphorylation, at low aldohexose concentrations, in good agreement with the properties of purified hexokinase. In intact islets fructose augmented the islet content in glucose 6-phosphate sufficiently to cause marked inhibition of its own rate of phosphorylation. These findings may account, in part at least, for the low rate of fructose utilization by rat pancreatic islets.     

Does glyceraldehyde enter pancreatic islet metabolism via both the triokinase and the glyceraldehyde phosphate dehydrogenase reactions? A study of these enzymes in islets

Glyceraldehyde has been known to be an insulin secretagogue for more than 15 years. It has been (reasonably) assumed that glyceraldehyde enters the glycolytic pathway via its phosphorylation by ATP to form glyceraldehyde phosphate, a reaction catalyzed by the enzyme triokinase, and that subsequent metabolism is identical to that of glucose. glucose. However, up to now there have been no studies verifying the presence of triokinase in the pancreatic beta cell. We report here that (1) the activity of triokinase in pancreatic islets is very low, indicating that the activity is intrinsically low and/or the enzyme was rapidly inactivated during the preparation of tissue for assay; (2) the activity is much lower than glucose phosphorylating activity (hexokinase plus glucokinase) in islets, even though glyceraldehyde is a more efficient insulin secretagogue than glucose; (3) glyceraldehyde phosphate dehydrogenase from pancreatic islets can use glyceraldehyde as a substrate in place of glyceraldehyde phosphate (the Vmax of glyceraldehyde phosphate dehydrogenase from islets when glyceraldehyde is the substrate is 20-fold that of triokinase when glyceraldehyde is the substrate); and (4) the Km of glyceraldehyde phosphate dehydrogenase with respect to glyceraldehyde (4.8 mM) is similar to the concentration of glyceraldehyde that gives one-half maximal rates of insulin release from pancreatic islets, whereas the Km of triokinase with respect to glyceraldehyde is much lower (less than 50 microM). These data suggest that besides stimulating insulin release in islets via its entering metabolism by phosphorylation to glyceraldehyde phosphate in the triokinase reaction, glyceraldehyde could be phosphorylated by Pi in the glyceraldehyde phosphate dehydrogenase reaction to form glycerate 1-phosphate which is probably unmetabolizable in islets. The second reaction could drastically increase the NADH/NAD ratio in islets without providing substrates for hydrogen shuttles that reoxidize cytosolic NADH. Since an increased NAD(P)H/NAD(P) ratio is believed to be a key part of the signal for insulin release, such a mechanism would explain the potent insulinotropism of glyceraldehyde in short-term experiments. In addition, the formation of unmetabolizable acids may explain the toxic effects of long-term exposure of islets to glyceraldehyde and why glyceraldehyde causes the beta cell to become acidic, whereas glucose does not.     

Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Effect of glucose on ATP dephosphorylation in rat spermatids

Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.     

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.     

Factors affecting hexose phosphorylation in Acetobacter xylinum

Fructose was oxidized and converted to cellulose by cells of Acetobacter xylinum grown on fructose or succinate, but not by cells grown on glucose. In resting fructose-grown cells, glucose strongly suppressed fructose utilization. Extracts obtained from fructose- or succinate-grown cells catalyzed the adenosine triphosphate (ATP)-dependent formation of the 6-phosphate esters of glucose and fructose, whereas glucose-grown cell extracts phosphorylated glucose but not fructose. Fructokinase and glucokinase activities were separated and partially purified from cells grown on glucose, fructose, or succinate. Whereas fructokinase phosphorylated fructose only, glucokinase was active towards glucose and less active towards mannose and glucosamine. The optimal pH for the fructokinase was 7.4 and for the glucokinase was 8.5. The K(m) values for the fructokinase were: fructose, 6.2 mm; and ATP, 0.83 mm. The K(m) values for the glucokinase were: glucose, 0.22 mm; and ATP, 4.2 mm. Fructokinase was inhibited by glucose, glucosamine, mannose, and deoxyglucose in a manner competitive with respect to fructose, with K(i) values of 0.1, 0.14, 0.5, and 7.5 mm, respectively. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP) inhibited both kinases noncompetitively with respect to ATP. The K(i) values were: 1.8 mm (ADP) and 2.1 mm (AMP) for fructokinase, and 2.2 mm (ADP) and 9.6 mm (AMP) for glucokinase. Fructose metabolism in A. xylinum appears to be regulated by the synthesis and activity of fructokinase.     

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.     

Effect of interferon and double-stranded RNA on B-cell function in mouse islets of Langerhans.

The direct effects of alpha- and beta-interferons on isolated mouse pancreatic islets were investigated in vitro and found to be similar. After 7 h incubation with interferon concentrations above 350 units/ml, glucose-stimulated (pro)insulin biosynthesis was significantly inhibited, with only a slight inhibition of total protein biosynthesis. Inhibition could be abolished in the additional presence of an anti-interferon antibody. Interferon did not affect insulin release, total insulin content, or glucose oxidation of the islets. The stimulation of (pro)insulin biosynthesis by adenosine, D-glyceraldehyde, mannose, N-acetylglucosamine and leucine was also inhibited by interferon, with no effect on insulin release. At concentrations of dsRNA (double-stranded RNA) said to induce interferon (1-100 micrograms/ml), glucose-stimulated (pro)insulin biosynthesis was inhibited without significantly affecting insulin release. The dsRNA may itself inhibit stimulated (pro)insulin biosynthesis or may function indirectly by the induction of interferon.     

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).