Pancreatic Islet Metabolism Research Articles

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation

1. 1. d-Glucose (0.5–16.7 mM) preferentially stimulates aerobic glycolysis and d-[3,4- 14C] glucose oxidation, relative to d-[5- 3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. 2. Inorganic and organic calcium antagonists, as well as Ca 2+ deprivation, only cause a minor decrease in the ratio between d-[3,4- 14C]glucose oxidation and d-[5- 3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and d[3,4- 14C]glucose oxidation in islets exposed to 6.0 mM d-glucose. Nevertheless, BCH augments [1- 14C]pyruvate and [2- 14C]pyruvate oxidation. 4. 4. The glucose-induced increment in the paired ratio between d-[3,4- 14C]glucose oxidation and d-[5- 3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. 5. These findings suggest that the preferential effect of d-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca 2+-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.

Hexose metabolism in pancreatic islets: Enzyme-to-enzyme tunnelling of hexose 6-phosphates

The fate of unlabelled d-glucose and d-[2- 3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of d-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between d-[2- 3H]glucose 6-phosphate and d-[1- 3H] fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from d-[2- 3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from d-[2- 3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of d-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.

Hexose metabolism in pancreatic islets: Participation of Ca 2+-sensitive 2-ketoglutarate dehydrogenase in the regulation of mitochondrial function

A rise in extracellular d-glucose concentration results in a preferential and Ca 2+-dependent stimulation of mitochondrial oxidative events in pancreatic islet cells. The possible participation of Ca 2+-dependent mitochondrial dehydrogenases, especially 2-ketoglutarate dehydrogenase, in such an unusual metabolic situation was explored in intact islets, islet homogenates and isolated islet mitochondria. In intact islets exposed to a high concentration of d-glucose, the removal of extracellular Ca 2+ impaired d-[6- 14C]glucose oxidation whilst failing to affect the cytosolic or mitochondrial ATP ADP ratios. In islet homogenates, the activity of 2-ketoglutarate dehydrogenase displayed exquisite Ca 2+-dependency, the presence of Ca 2+ causing a 10-fold increase in affinity for 2-ketoglutarate. In intact islet mitochondria, the oxidation of 2-[1- 14C]ketoglutarate also increased as a function of extramitochondrial Ca 2+ availability. Moreover, prior stimulation of intact islets by d-glucose resulted in an increased capacity of mitochondria to oxidize 2-[1- 14C]ketoglutarate. The absence of extracellular Ca 2+ during the initial stimulation of intact islets impaired but did not entirely suppress such a memory phenomenon. It is proposed that the mitochondrial accumulation of Ca 2+ in nutrient-stimulated islets indeed accounts, in part at least, for the preferential stimulation of mitochondrial oxidative events in this fuel-sensor organ.

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 [γ- 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.

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. 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 V max 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 K m of glyceraldehyde phosphate dehydrogenase with respect to glyceraldehyde (4.8 m m) is similar to the concentration of glyceraldehyde that gives one-half maximal rates of insulin release from pancreatic islets, whereas the K m of triokinase with respect to glyceraldehyde is much lower (< 50 μ m). 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 P i 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.

Hexose metabolism in pancreatic islets. Feedback control of d-glucose oxidation by functional events

Hexose metabolism in pancreatic islets. Feedback control of d-glucose oxidation by functional events

Hexose metabolism in pancreatic islets. Feedback control of d-glucose oxidation by functional events

A rise in extracellular d-glucose concentration in pancreatic islet cells causes a greater relative increase in the oxidation of pyruvate and acetyl residues than in glycolysis. A possible explanation for such an unusual situation was sought in the present study. The preferential stimulation of mitochondrial oxidative events was found to display a sigmoidal dependency on hexose concentration, and an exponential time course during prolonged exposure of the islets to a high concentration of d-glucose. The preferential stimulation of mitochondrial oxidative events was abolished in islets incubated in the presence of cycloheximide and absence of Ca 2+, in which case the oxidation of d-[6- 14C]glucose was more severely inhibited than that of d-[3,4- 14C]glucose. Likewise, the inhibitor of protein biosynthesis and the absence of Ca 2+ affected the oxidation of l-[U- 14C]leucine preferentially, relative to that of l-[1- 14C]leucine, in islets exposed to a high, but not a low, concentration of the amino acid. These results demonstrate that in pancreatic islets it is possible to dissociate both glycolysis from mitochondrial oxidative events and the oxidation of acetyl residues from their generation rate. Moreover, the experimental data suggest that nutrient-responsive and ATP-requiring functional processes exert a feedback control on mitochondrial respiration in this fuel-sensor organ.

Hexose metabolism in pancreatic islets: Metabolic and secretory responses to d-fructose

d-Fructose (3.3 to 33.0 mmol/liter) caused a concentration-related increase in insulin output from rat islets exposed to d-glucose (3.3 to 7.0 mmol/liter), such an increase not being more marked in mouse islets. The fructose-induced increment in insulin release, relative to that evoked by d-glucose, was two times higher in islets exposed to d-glucose than in islets stimulated by d-mannose, 2-ketoisocaproate, or nonnutrient secretagogs. Likewise, the metabolism of d-fructose in islet cells was significantly different in the absence or presence of d-glucose. Thus, the ketose was largely channeled into the pentose phosphate pathway in glucose-deprived, but not so in glucose-stimulated, islets. In both glucose-deprived and glucose-stimulated islets, however, the magnitude of the secretory response to d-fructose was commensurate with the increase in ATP production attributable to its catabolism. These findings indicate that the metabolic fate of hexoses—and, hence, their insulinotropic capacity—is not ruled solely at the level of their phosphorylation.

Hexose metabolism in pancreatic islets: Regulation of mitochondrial hexokinase binding

A major fraction of hexokinase was found to be bound, presumably to mitochondria, in both normal and tumoral rat pancreatic islet cells examined after either mechanical disruption or digitonin treatment. Spermidine enhanced the binding and glucose 6-phosphate caused the release of hexokinase to and from islet mitochondria, in a manner comparable to that seen in parotid or brain homogenates. In hepatocytes, some hexokinase, but no glucokinase, was found in the bound form. In islet cells, however, the pattern of glucokinase binding was similar to that of hexokinase. It is speculated that the preferential location of both hexokinase and glucokinase on mitochondria may favor the maintenance of a high cytosolic ATP content in islet cells.

Hexose metabolism in pancreatic islets: Absence of glucose-6-phosphatase in rat islet cells

Homogenates of either rat or mouse pancreatic islets, pure rat B cells or insulin-producing cells of the RINm5F line catalyzed the hydrolysis of d-glucose-6-phosphate. Relative to protein content, the enzymic activity, which was mainly associated with particulate rather than soluble subcellular material, was much lower in endocrine pancreatic cells than in liver. The rat islet enzyme differed from liver glucose-6-phosphate by its lower affinity for d-glucose-6-phosphate, its lower pH optimum, its greater relative efficiency towards l-glycerol-3-phosphate as distinct from d-glucose-6-phosphate, its restricted lability during exposure to pH 5.0, its inability to act as a glucose-6-phosphate:glucose phosphotransferase, and its insensitivity to inhibition by D-glucose. It is concluded that rat islet cells are virtually devoid of true glucose-6-phosphatase activity.

Hexose metabolism in pancreatic islets: Compartmentation of hexokinase in islet cells

Hexokinase activity was found in both soluble (cytosolic) and particulate subcellular fractions prepared from rat pancreatic islet homogenates. The bound enzyme was associated with mitochondria rather than secretory granules. Relative to the total hexokinase activity, the amount of bound enzyme was higher in islet homogenates prepared at pH 6.0 (72 ± 7%) than in islets homogenized at pH 7.4 (38 ±1%). The affinity of hexokinase for equilibrated d-glucose was not different in the cytosolic and mitochondrial fractions. In both fractions, hexokinase displayed a greater affinity for α- than β- d-glucose, but a higher maximal velocity with the β- than α-anomer. Glucose 6-phosphate inhibited to a greater extent cytosolic than mitochondrial hexokinase. A high K m glucokinase-like enzymic activity was also present in both subcellular fractions. It is proposed that the ambiguity of hexokinase plays a propitious role in the glucose-sensing function of pancreatic islet cells.

Regulation of energy metabolism in pancreatic islets by glucose and tolbutamide.

The kinetics of insulin secretion and oxygen uptake in response to D-glucose and tolbutamide were compared in mouse pancreatic islets. In addition, the role of decreased ATP as a driving force for secretagogue-induced oxygen consumption was examined. D-glucose (10-30 mmol/l) triggered a biphasic insulin release which always coincided with a monophasic increase in islet oxygen uptake. In the presence of D-glucose (5-30 mmol/l), tolbutamide (3-500 mumol/l) consistently elicited an initial peak of insulin secretion which was followed by a continued decline. Tolbutamide-induced secretory profiles were accompanied by similar respiratory profiles. Oxygen consumption per ng of insulin released during the test phase was higher after elevation of the glucose concentration than after addition of tolbutamide. In conjunction with 5 or 10 mmol/l D-glucose, but not with 15 or 30 mmol/l D-glucose, tolbutamide (30-100 mumol/l) lowered islet ATP content significantly (p less than 0.02). Phosphocreatine was not found in isolated islets, although they contained substantial creatine kinase activity. It is concluded that the driving force for tolbutamide-induced oxygen uptake is a decrease in the phosphorylation potential caused by the work load imposed by stimulation of the secretion process. However, a major proportion of the respiratory response to glucose also results from enhancement of biosynthesis.

Branched-chain amino and keto acid metabolism in pancreatic islets

The metabolism of l-leucine in pancreatic islets is reviewed. Regulatory sites may involve (i) the transport and intracellular distribution of l-leucine, (ii) its transamination as modulated by the availability of 2-keto acids, (iii) the activity of branched-chain 2 keto acid dehydrogenase and (iv) the further catabolism of isovaleryl coenzyme A. Emphasis is also placed on the activation of glutamate dehydrogenase by l-leucine, its interference with the utilization of endogenous nutrients and the reciprocal effects of l-leucine and either l-glutamine or l-asparagine upon their respective metabolism. The relevance of l-leucine metabolism to its insulinotropic action is underlined.

Hexose metabolism in pancreatic islets. Galactose transport, phosphorylation and oxidation.

In rat pancreatic islets, the apparent space of distribution of galactose is not different from that of other hexoses. In homogenates of islets or tumoral insulin-producing cells, galactose is phosphorylated at a very low rate relative to either glucose phosphorylation in the same tissues or galactose phosphorylation by liver homogenates. In intact islets, galactose increases modestly the glucose 6-phosphate content and is oxidized at a much lower rate than glucose. Galactose slightly increases insulin output in the presence of a stimulatory concentration of glucose but fails to provoke insulin release in the absence of glucose, whether in islets removed from rats fed a normal or galactose-rich diet. The low rate of galactose oxidation and its poor insulinotropic capacity appear attributable to the weak activity of galactokinase in pancreatic islets.

Enflurane and Adenosine 3???,5???-Cyclic Monophosphate Metabolism in Pancreatic Islets

The effects of enflurane at a concentration of 4% by volume in the gas phase upon rates of insulin secretion were correlated with islet contents of adenosine 3',5'-cyclic monophosphate (cyclic AMP). At this concentration, enflurane was without effect upon the basal rate of insulin release in the presence of 2 mM glucose. By contrast, the anesthetic led to 84 and 86% inhibitions, respectively, of rates of insulin secretion stimulated by 20 mM glucose and 20 mM glucose plus 1 mM theophylline. Enflurane also led to small reductions in islet cyclic AMP content under all conditions of incubation, and a statistically significant (P less than 0.02) effect of the anesthetic in lowering cyclic AMP content was seen in islets exposed to 20 mM glucose plus 1 mM theophylline. Enflurane was without effect upon the activity of the low Km cyclic AMP phosphodiesterase of the islet under the conditions employed. By contrast, the anesthetic led to significant inhibitions of both basal and fluoride-stimulated islet adenylate cyclase activity (34%, P less than 0.01 and 23%, P less than 0.005, respectively). It is concluded that under the conditions employed, enflurane leads to inhibition of islet adenylate cyclase activity and to small resultant reductions in islet cyclic AMP content. These effects appear to be of insufficient magnitude to explain completely the observed degree of insulin secretory inhibition by the anesthetic.

Hexose metabolism in pancreatic islets: phosphoglycerate 2,3-mutase and enolase activities in rat islets

Rat islet homogenates display both phosphoglycerate 2,3-mutase and enolase activities. When phosphoglycerate 2,3-mutase is activated by 2,3-diphosphoglycerate, the reaction velocity becomes close to that of enolase. The islet content in 2,3-diphosphoglycerate is sufficiently high to allow virtually full activation of phosphoglycerate 2,3-mutase.

Hexose metabolism in pancreatic islets. Inhibition of hexokinase.

In islet homogenates, hexokinase-like activity (Km 0.05 mM; Vmax. 1.5 pmol/min per islet) accounts for the major fraction of glucose phosphorylation. Yet the rate of glycolysis in intact islets incubated at low glucose concentrations (e.g. 1.7 mM) sufficient to saturate hexokinase only represents a minor fraction of the glycolytic rate observed at higher glucose concentrations. This apparent discrepancy between enzymic and metabolic data may be attributable, in part at least, to inhibition of hexokinase in intact islets. Hexokinase, which is present in both islet and purified B-cell homogenates, is indeed inhibited by glucose 6-phosphate (Ki 0.13 mM) and glucose 1,6-bisphosphate (Ki approx. 0.2 mM), but not by fructose 2,6-bisphosphate. In intact islets, the steady-state content of glucose 6-phosphate (0.26-0.79 pmol/islet) and glucose 1,6-bisphosphate (5-48 fmol/islet) increases, in a biphasic manner, at increasing concentrations of extracellular glucose (up to 27.8 mM). From these measurements and the intracellular space of the islets, it was estimated that the rate of glucose phosphorylation as catalysed by hexokinase represents, in intact islets, no more than 12-24% of its value in islet homogenates.

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.

Phospholipid metabolism in pancreatic islets.

The secretion of insulin can be elicited by a wide spectrum of stimuli including nutrients, hormones and neurotransmitters as well as a large number of pharmacological agents such as tumor-promoters and sulphonylureas. The diversity of these secretagogues suggests that islets may be activated through a number of distinct biochemical mechanisms. The work discussed in this review suggests that certain of the above-mentioned secretagogues, especially nutrient and neurotransmitter stimuli, may induce insulin secretion by a mechanism involving enhanced metabolism of inositol-containing lipids. The way in which this process is coupled to secretion is not known, although several possibilities exist. The hydrolysis of phosphoinositides and release of inositol phosphates may result, respectively in altered calcium permeability of the plasma membrane and mobilization of calcium from intracellular sources. The accompanying production of diacylglycerol might also influence membrane permeability and fluidity and also lead to activation of protein kinase C. Diacylglycerol can be phosphorylated to form phosphatidic acid which may play a role as an endogenous ionophore. Finally, inositol lipid breakdown could lead, through diacylglycerol and/or phosphatidic intermediates, to the liberation of arachidonic acid and subsequent conversion to active metabolites of the cyclo-oxygenase and lipoxygenase pathways. Thus, enhanced phospholipid metabolism in islets could, theoretically, result in the generation of a range of intracellular signals which mediate or modulate insulin secretion during stimulation by certain types of secretagogues. Continued investigation is clearly neccessary in order to elucidate the mechanisms by which such secretagogues provoke increased phospholipid metabolism and to understand the role(s) of this process in the regulation of islet function.

Pancreatic islet metabolism and redox state during stimulation of insulin secretion with glucose and fructose.

The mechanism of potentiation of insulin secretion by fructose was investigated. Twenty mM fructose + 3 mM glucose stimulated insulin secretion in a biphasic manner similar to what is found during stimulation with 20 mM glucose, whereas 20 mM fructose alone did not affect secretion. Fructose utilization was measured as formation of tritiated water from 5-3H-fructose. At 27.8 mM fructose the utilization rate was 258 pmol/2 h/10 islets, which is less than the utilization rate of 2.8 mM glucose. 20 mM glucose increased the islet NADH/NAD+ and NADPH/NADP+ redox ratios as well as islet concentration of ATP and PEP. 20 mM fructose + 3 mM glucose did not affect the concentration of ATP and PEP or the NADH/NAD+ redox ratio. The NADPH/NADP+ ratio was significantly decreased (60%) after 2.5 min incubation with .20 mM fructose + 3 mM glucose. It is concluded that fructose potentiation of insulin secretion is not primarily dependent on fructose metabolism and that any conceivable effect on plasma membrane ion fluxes as caused by a reduction of plasma membrane disulfides, may be caused by mechanisms other than a mere increase in the pyridine nucleotide substrates for the transhydrogenation process.