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.     

Autocrine regulation of glucose metabolism in pancreatic islets

We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured 14CO2 and 3H2O production from d-[U-14C]glucose and d-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced 14CO2 and 3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased 14CO2 and 3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in 14CO2 and 3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.     

Acetylcholine stimulates glucose metabolism by pancreatic islets

Acetylcholine stimulates insulin secretion in the presence of physiological concentrations of glucose. Stimulation of insulin secretion by acetylcholine is accompanied by an increase in glucose usage by isolated rat islets. Acetylcholine increased glucose usage by 38%, 28%, and 12% at 3.5 mM, 5.5 mM, and 10 mM glucose, respectively, compared to glucose usage by isolated islets incubated with glucose alone. Data showing increased glucose usage in islets treated with acetylcholine converge with data from an earlier report (J. Biol. Chem. 254 3921-3929 [1979]) showing a crossover point for glycolytic metabolites at phosphofructokinase to indicate that activation of glycolysis by acetylcholine results from increased phosphofructokinase activity and coordinate activation of hexokinase in intact islets.     

The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets

1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.     

Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium

The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2-acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP-diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.     

Hexose metabolism in pancreatic islets

Summary In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-¹⁴C]glucose than utilization of D-[5-³H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-¹⁴C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-¹⁴C]glucose oxidation, was impaired in the absence of extracellular Ca²⁺, and failed to be affected by NH₄ ⁺. Although the ratio between D-[6-¹⁴C]glucose oxidation and, D-[5-³H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca²⁺, as attributable not solely to stimulation of Ca²⁺ inflow into the islet cells but also to an increase in ATP availability.     

UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

Uncoupling Protein 2 (UCP2) is expressed in the pancreatic β-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.     

Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.     

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.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Long-term effects of glucose on insulin release and glucose oxidation by mouse pancreatic islets maintained in tissue culture

Rates of glucose oxidation and insulin release in response to a wide range of glucose concentrations were studied in short-term experiments in isolated mouse pancreatic islets maintained in tissue culture for 6 days at either a physiological glucose concentration (6.7mm) or at a high glucose concentration (28mm). The curves relating glucose oxidation or insulin release to the extracellular glucose concentration obtained with islets cultured in 6.7mm-glucose displayed a sigmoid shape similar to that observed for freshly isolated non-cultured islets. By contrast islets that had been cultured in 28mm-glucose showed a linear relationship between the rate of glucose oxidation and the extracellular glucose concentration up to about 8mm-glucose. The maximal oxidative rate was twice that of the non-cultured islets and the glucose concentration associated with the half-maximal rate considerably decreased. In islets cultured at 28mm-glucose there was only a small increase in the insulin release in response to glucose, probably due to a depletion of stored insulin in those B cells that had been cultured in a high-glucose medium. It is concluded that exposure of B cells for 6 days to a glucose concentration comparable with that found in diabetic individuals causes adaptive metabolic alterations rather than degeneration of these cells.     

Aromatic-L-amino-acid decarboxylase activity in mouse pancreatic islets

Aromatic-L-amino-acid decarboxylase activity has been measured in intact or homogenised pancreatic islets of ob/ob mice (Ume? ob/ob). The method used involves the trapping and measuring of the 14CO2 released from L-[1-14C]dihydroxyphenylalanine (L-dopa). Islets showed a decarboxylase activity which was dependent on pyridoxal phosphate and inhibitable by 0.1 mM benserazide or 0.1 mM alpha-monofluoromethyldopa. Maximum activity in intact islets was about 330 mmol/kg dry islet per h with an apparent Km of 3.3 mM. Islet homogenates had a Vmax of about 120 mmol/kg per h with a Km of 0.3 mM. L-5-Hydroxytryptophan, m-tyrosine and o-tyrosine interfered with the decarboxylation of L-dopa in a way that suggested a high activity also towards those substrates. L-Phenylalanine, L-tyrosine and D-glucose had no effect. At 0.05 mM L-dopa islet homogenates showed a much higher activity than homogenates of liver, kidney, or spleen. Islet uptake of L-[3H]dopa was well in excess of the decarboxylation rate and thus probably not rate-limiting. It is concluded that mouse pancreatic islets have a high activity of aromatic-L-amino-acid decarboxylase. This is in accordance with previous suggestions of a stimulatory effect of this enzyme on insulin secretion.     

Regulation of RNA metabolism in relation to insulin production and oxidative metabolism in mouse pancreatic islets in vitro

This study was undertaken to investigate the long-term effects of different substrates, in particular glucose, on the regulation of islet RNA metabolism and the relationship of this regulation to the metabolism and insulin production of the islet B-cell. For this purpose collagenase-isolated mouse islets were used either in the fresh state or after culture for 2 or 5 days in RPMI 1640 plus 10% calf serum supplemented with various test compounds. Islets cultured with 16.7 mM glucose contained more RNA than those cultured with 3.3 mM glucose. Culture of islets in glucose at low concentrations inhibited glucose-stimulated RNA synthesis and this inhibitory effect was reversed by prolonged exposure to high glucose concentrations. Culture with 10 mM leucine and 3.3 mM glucose or with 10 mM 2-ketoisocaproate and 3.3 mM glucose increased the total RNA content of islets as compared to that of islets cultured with 3.3 mM glucose alone. Islets cultured with 5 mM theophylline maintained a high RNA content in the presence of 3.3 mM glucose. Theophylline also increased the islet RNA content when added together with 16.7 mM glucose, as compared to 16.7 mM glucose alone. Theophylline probably exerted this effect by decreasing the rate of RNA degradation. Changes in islet RNA metabolism showed a close correlation to changes in islet total protein biosynthesis, whereas islet (pro)insulin biosynthesis and insulin release exhibited different glucose-dependency patterns. The response of islet oxygen uptake to glucose was similar to that of islet RNA and protein biosynthesis. It is concluded that the RNA content of the pancreatic islets is controlled at the levels of both synthesis and degradation. Glucose stimulates the RNA synthesis and inhibits its degradation. Moreover, the results suggest that regulation of RNA synthesis may be mediated through islet metabolic fluxes and the cAMP system.     

Evidence for the presence of glucose cycling in pancreatic islets of the ob/ob mouse

Pancreatic islets from ob/ob mice incubated with 3H2O and 5.5 mM glucose formed 3H-labeled glucose, 74 picoatoms incorporated/islet/h. Sixty-three percent of the 3H was bound to carbon 2 of the glucose. The amount of glucose-6-P dephosphorylated to glucose, determined from this incorporation, was 48 pmol/islet/h. Glucose utilization, measured by the formation of 3H2O from [5-3H]glucose, was 72 pmol/islet/h. The amount of glucose dephosphorylated was then about 40% of that phosphorylated. Thus, glucose-6-P is dephosphorylated to glucose to a significant extent by intact islets in vitro and presumably by the beta cells of the islets. The extent of this glucose cycling, i.e. glucose----glucose-6-P----glucose, may play a role in determining the extent of glucose-induced insulin secretion.     

Stimulation of phospholipid methylation by glucose in pancreatic islets

A two fold stimulation in the incorporation of [3H-methyl] groups from [3H-methyl] methionine into phospholipids was seen in intact pancreatic islets within six minutes of exposure to a glucose concentration that stimulates insulin release. Nonstimulatory sugars, L-glucose and D-galactose, as well as dibutyryl cAMP, did not affect phospholipid methylation in islet cells. A calcium channel blocker, verapamil, inhibited methylation. These studies suggest that the signal for glucose-induced insulin release could involve phospholipid methylation.     

Effects of glucose, glucose metabolites and calcium ions on adenylate cyclase activity in homogenates of mouse pancreatic islets.

The effects of glucose, a series of glucose metabolites, nicotinamide nucleotides, Ca2+ and p-chloromercuribenzenesulphonate on adenylate cyclase activity in homogenates of mouse pancreatic islets were studied. The basal activity of the adenylate cyclase was approx. 6 pmol of cyclic AMP formed/30 min per microng of DNA at 30 degrees C. The enzyme activity was stimulated by some 150% by fluoride. Starvation of the animals for 48h had no effect on either the basal or the fluoride-stimulated activity. The adenylate cyclase activity was increased by 40-50% when 17 mM-glucose, 10 micronM-phosphoenolpyruvate or 10 micronM-pyruvate was added to the assay medium. The effect of glucose was unchanged in the presence of 17 mM-mannoheptulose, and mannoheptulose alone had no effect. The other glycolytic intermediates, and the coenzymes NAD+, NADH and NADPH, at concentrations up to 1 mM were without any detectable effect on the rate of formation of cyclic AMP. The insulin secretagogue p-chloromercuribenzenesulphonate inhibited the adenylate cyclase markedly even at a concentration of 10 micronM. Calculated concentrations of free Ca2+ of 10 micronM and 0.1 mM inhibited adenylate cyclase by 29 and 71% respectively. It is concluded that both glucose itself and phosphoenolpyruvate and/or pyruvate are true activating ligands for islet and adenylate cyclase and that inhibition of the cyclase by Ca2+ may be of physiological significance.