Hexose metabolism in pancreatic islets. Participation of Ca2(+)-sensitive 2-ketoglutarate dehydrogenase in the regulation of mitochondrial function

A rise in extracellular D-glucose concentration results in a preferential and Ca2(+)-dependent stimulation of mitochondrial oxidative events in pancreatic islet cells. The possible participation of Ca2(+)-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 Ca2+ 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 Ca2(+)-dependency, the presence of Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 stimulation by D-glucose of [2-3H]glycerol detritiation

1. In pancreatic islets, a rise in glucose concentration is known to increase the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization. The opposite situation was found to prevail in parotid cells. 2. In rat pancreatic islets, D-glucose caused a concentration-related stimulation of 3H2O production from [2-3H]glycerol, but failed to affect 3H2O production from [1(3)-3H]glycerol or 14CO2 production from [U-14C]glycerol. At the low concentration used in most of these experiments (i.e. 1.0 mM), glycerol failed to affect D-[U-14C]glucose oxidation. 3. These findings suggest that the preferential stimulation by D-glucose of mitochondrial oxidative events in pancreatic islets represents an unusual situation in secretory cells and involves an accelerated circulation in the glycerol phosphate shuttle.     

Comparison between D-[3-3H]- and D-[5-3H]glucose and fructose utilization in pancreatic islets from control and hereditarily diabetic rats

The metabolism of D-glucose and/or D-fructose was investigated in pancreatic islets from control rats and hereditarily diabetic GK rats. In the case of both D-glucose and D-fructose metabolism, a preferential alteration of oxidative events was observed in islets from GK rats. The generation of 3HOH from D-[5-3H]glucose (or D-[5-3H]fructose) exceeded that from D-[3-3H]glucose (or D-[3-3H]fructose) in both control and GK rats. This difference, which is possibly attributable to a partial escape from glycolysis of tritiated dihydroxyacetone phosphate, was accentuated whenever the rate of glycolysis was decreased, e.g., in the absence of extracellular Ca(2+) or presence of exogenous D-glyceraldehyde. D-Mannoheptulose, which inhibited D-glucose metabolism, exerted only limited effects upon D-fructose metabolism. In the presence of both hexoses, the paired ratio between D-[U-14C]fructose oxidation and D-[3-3H]fructose or D-[5-3H]fructose utilization was considerably increased, this being probably attributable, in part at least, to a preferential stimulation by the aldohexose of mitochondrial oxidative events. Moreover, this coincided with the fact that D-mannoheptulose now severely inhibited the catabolism of D-[5-3H]fructose and D-[U-14C]fructose. The latter situation is consistent with both the knowledge that D-glucose augments D-fructose phosphorylation by glucokinase and the findings that D-mannoheptulose, which fails to affect D-fructose phosphorylation by fructokinase, inhibits the phosphorylation of D-fructose by glucokinase.     

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

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. Inorganic and organic calcium antagonists, as well as Ca2+ 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. 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. 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. 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.     

Preferential stimulation by glucose of its oxidation relative to glycolysis in purified insulin-producing cells.

A rise in extracellular D-glucose concentration increases to a greater relative extent the conversion of both D-[5-3H]glucose to 3HOH and D-[6-14C]glucose to 14CO2 in rat purified insulin-producing cells than previously observed in pancreatic islets. In the pure B-cells, the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization increases, in a sigmoidal manner, as a function of the hexose concentration. The preferential stimulation by D-glucose of mitochondrial oxidative events is proposed to represent an unusual but essential feature of the metabolic and, hence, functional response of these fuel-sensor cells.     

Crabtree effect in tumoral pancreatic islet cells

The relationship between glycolysis and respiration was examined in a model of pancreatic B-cell dysfunction, namely in tumoral insulin-producing cells of the RINm5F line. A rise in D-glucose concentration from 2.8 to 16.7 mM increased the utilization of D-[5-3H]glucose and production of [14C]lactate from D-[U-14C]glucose, whereas decreasing the oxidation of either D-[U-14C]glucose or D-[6-14C]glucose. Whereas 2.8 mM D-glucose augmented O2 uptake above basal value, a further rise in D-glucose concentration to 16.7 mM decreased respiration, which remained higher, however, than basal value. Whether at low or high concentration, D-glucose exerted a pronounced sparing action upon the oxidation of endogenous nutrients in cells prelabeled with either L-[U-14C]glutamine or [14C]palmitate and, nevertheless, augmented above basal value the rate of lipogenesis, ATP/ADP content, adenylate charge, and cytosolic NADH/NAD+ and NADPH/NADP+ ratios. The generation of ATP resulting from the catabolism of either exogenous D-glucose or endogenous nutrients was not affected by the rise in hexose concentration from 2.8 to 16.7 mM. Thus, in sharp contrast with the situation found in normal islet cells, a rise in D-glucose concentration, instead of stimulating mitochondrial oxidative events, caused, through a Crabtree effect, inhibition of hexose oxidation and O2 consumption in tumoral islet cells.     

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.     

Enzymatic,metabolic and secretory perturbations in pancreatic islets of thyroidectomized rats

In thyroidectomized rats, the activity of FAD-linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2-ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of³HOH from [2-³H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham-operated rats, at least in the presence of a high concentration of D-glucose. Nevertheless impaired oxidation of both D-[3,4-¹⁴C]glucose and D-[6-¹⁴C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D-glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and a decreased output of insulin by islets incubated at low (2·8 mM ), but not higher, concentrations of D-glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD-linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non-insulin-dependent diabetes.     

The stimulus-secretion coupling of amino acid-induced insulin release. Insulinotropic action of L-alanine

Available information on the fate and insulinotropic action of L-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na(+) co-transport in the insulinotropic action of L-alanine. In the present study conducted in islets prepared from normal adult rats, L-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or L-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of L-[U-14C]alanine was unaffected by D-glucose, but inhibited by L-leucine. Inversely, L-alanine decreased the oxidation of D-[U-14C]glucose, but failed to affect L-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na(+) co-transport in the secretory response to L-alanine cannot be ignored.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

Hexose metabolism in pancreatic islets: unequal oxidation of the two carbons of glucose-derived acetyl residues.

The fate of the C1 and C2 of glucose-derived acetyl residues was examined in rat pancreatic islets. The production of 14CO2 from D-[2-14C]glucose exceeded that from D-[6-14C]glucose, in the same manner as the oxidation of [1-14C]acetate exceeded that of [2-14C]acetate. The difference in 14CO2 output from D-[2-14C]glucose and D-[6-14C]glucose was matched by complementary differences in the generation of 14C-labeled acidic metabolites and amino acids. Even the production of 14C-labeled L-lactate was somewhat higher in the case of D-[6-14C]glucose than D-[2-14C]glucose. The ratio between D-[2-14C]glucose and D-[6-14C]glucose oxidation progressively decreased at increasing concentrations of the hexose (2.8, 7.0, and 16.7 mM), was higher after 30 than 120 min incubation, and was decreased in the presence of a nonmetabolized analogue of L-leucine. These findings are consistent with the view that the difference between D-[6-14C]glucose and D-[2-14C]glucose oxidation is mainly attributable to the inflow into the Krebs cycle of unlabeled metabolites generated from endogenous nutrients, this being compensated by the exit of partially labeled metabolites from the same cycle. The present results also indicate that the oxidation of glucose-derived acetyl residues relative to their generation in the reaction catalyzed by pyruvate dehydrogenase is higher than that estimated from the ratio between D-[6-14C]glucose and D-[3,4-14C]glucose conversion to 14CO2.     

Anomeric dissociation between glucokinase activity and glycolysis in pancreatic islets

In pancreatic islet homogenates incubated in the presence of a high glucose concentration (40 mM), the beta-anomer of D-glucose is phosphorylated at a higher rate than the alpha-anomer, whether in the absence or presence of exogenous glucose 6-phosphate. However, in intact islets also exposed to 40 mM D-glucose, the production of 3H2O from D-[5-3H] glucose, the oxidation of D-[U-14C] glucose and the glucose-induced increment in either lactate production or 45Ca net uptake, as well as the release of insulin from isolated perfused pancreases, are not higher with beta- than alpha-D-glucose. It is concluded that the rate of glucose utilization by islet cells is not regulated solely by the activity of hexokinase and/or glucokinase.     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

Metabolic Response to Nonglucidic Nutrient Secretagogues and Enzymatic Activities in Pancreatic Islets of Adult Rats after Neonatal Streptozotocin Administration

In islets from adult rats injected with streptozotocin during the neonatal period, both a nonmetabolized analog of L-leucine and 3-phenylpyruvate augmented ¹⁴CO₂ output from islets either prelabeled with L-[U-¹⁴C]glutamine or exposed to D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose in a manner qualitatively comparable to that found in islets from control rats. The islets of diabetic rats differed, however, from those of control rats by their unresponsiveness to both the L-leucine analog and a high concentration of D-glucose in terms of increasing ³HOH generation from [2-³H]glycerol, an impaired sparing action of the hexose upon ¹⁴CO₂ output from islets prelabeled with [U-¹⁴C]palmitate, and, most importantly, by a decreased rate of D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose oxidation when either incubated at a high concentration of the hexose (16.7 mM) or stimulated by nonglucidic nutrient secretagogues at a low concentration of D-glucose (2.8 mM). In islet homogenates, the activity of glyceraldehyde phosphate dehydrogenase, glutamate decarboxylase, and NADP-malate dehydrogenase was lower in diabetic than control islets. Such was not the case for glutamatealanine transaminase, glutamate-aspartate transaminase, or glutamate dehydrogenase. The neonatal injection of streptozotocin thus affected, in the adult rats, the activity of several islet enzymes. Nevertheless, the metabolic data suggest that an impaired circulation in the glycerol phosphate shuttle, as observed in response to stimulation of the islets by either a high concentration of D-glucose or nonglucidic nutrient secretagogues, represents an essential determinant of the preferential impairment of glucose-induced insulin release in this model of non-insulin-dependent diabetes.     

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: Enzymatic,metabolic and cationic determinants

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.     

Hexose metabolism in pancreatic islets. Effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets.

Anaplerotic reactions leading to the de novo synthesis of fatty acids, were recently proposed to participate in the coupling of metabolic to secretory events in the process of glucose-stimulated insulin release. In an attempt to validate such a proposal, the effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release was investigated in glucose-stimulated rat pancreatic islets. The inhibitor of ATP citrate-lyase, when tested in the 1.0-2.0 mM range, failed to affect glucose-stimulated insulin release, but also failed to inhibit the incorporation of 14C-labelled acetyl residues derived from L-[U-14C]leucine into islet lipids. A partial inhibition of fatty acid labelling by 3H2O was only observed in islets incubated for 120 min in the presence of 5.0 mM (-)-hydroxycitrate and absence of CaCl2. These findings suggest that (-)-hydroxycitrate is not, under the present experimental conditions, a useful tool to abolish fatty acid synthesis in intact pancreatic islets.     

Interrelationships (Between Glucose Metabolism, Energy State, and the Cytosolic Free Calcium Concentration in Cortical Synjaptosomes from the Guinea Pig

The stoichiometries of glycolysis and pyruvate oxidation were determined in cortical synaptosomes under varying rates of ATP consumption. Glycolysis was measured by using D-3-[3H]glucose as a marker and pyruvate oxidation by using D-3,4-[14C]glucose, which has to be metabolized to 1-[14C]pyruvate before being decarboxylated by the pyruvate dehydrogenase complex of intrasynaptosomal mitochondria. Cytosolic free Ca2+ concentration [( Ca2+]c) was determined in parallel and was manipulated by using EGTA in the incubation. The results show that in nonstimulated synaptosomes glycolysis and pyruvate oxidation are tightly coupled and stoichiometric. In the absence of Ca2+, when [Ca2+]c drops from 260 nM to 40 nM, glucose utilization increases, following the increase in energy demand, which has been shown to be due to elevated Na+ cycling. KCl depolarization, veratridine, and a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, all stimulate glycolysis and pyruvate oxidation stoichiometrically, independently of the presence of external Ca2+. A rise in [Ca2+]c, therefore, is not required to regulate mitochondrial pyruvate metabolism. It is concluded that synaptosomes exhibit a high degree of respiratory control, that they rely on glucose oxidation for their energetics, and that stimulation of energy production can be achieved independently of changes in [Ca2+]c.     

Hexose metabolism in pancreatic islets. Ca(2+)-dependent activation of the glycerol phosphate shuttle by nutrient secretagogues.

A method is proposed for the measurement of the flux through the glycerol phosphate shuttle in pancreatic islets. Such a flux is taken as the ratio between the production of 3HOH and the specific radioactivity of L-[2-3H]glycerophosphate in islets exposed to [2-3H]glycerol. D-Glucose and non-glucidic nutrient secretagogues, such as 2-ketoisocaproate and 2-aminobicyclo[2,2,1]heptane-2-carboxylate, stimulate, in a Ca(2+)-dependent manner, circulation in the glycerol phosphate shuttle. The shuttle flux is commensurate with the fraction of pyruvate generation which is not coupled with L-lactate production. These findings support the view that a rise in D-glucose concentration leads to activation of the FAD-linked mitochondrial glycerophosphate dehydrogenase through an increase in cytosolic Ca2+ concentration.     

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.     

Pancreatic fate of 14C-labelled hexoses

In order to assess the respective contribution of the exocrine and endocrine moieties of the pancreas to the overall net uptake of selected monosaccharides by the pancreatic gland, the apparent distribution space of L-[1-14C]glucose, 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was measured in pieces of pancreas obtained from either control rats or animals injected with streptozotocin. Although the time course for the uptake of 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was much slower in the pieces of pancreas than that previously documented in isolated pancreatic islets, no significant difference could, as a rule, be detected between the results obtained in pancreatic pieces of control and streptozotocin rats. A comparable situation prevailed in the pancreas of animals examined 3 min after the intravenous injection of 3-O-[14C-methyl]-D-glucose. D-Glucose inhibited the uptake of 3-O-[14C-methyl]-D-glucose and that of D-[U-14C]fructose. Likewise, 3-O-methyl-D-glucose inhibited the uptake of D-[U-14C]glucose. Cytochalasin B (20 microm) also inhibited the uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose, but not that of D-[U-14C]fructose. D-Mannoheptulose hexaacetate, but not the unesterified heptose, inhibited the metabolism of tritiated and 14C-labelled D-glucose, as well as the net uptake of D-[U-14C]glucose and D-[U-14C]mannose and, to a lesser extent, that of D-[U-14C]fructose. These findings indicate that despite marked differences between endocrine and exocrine pancreatic cells in terms of both the time course for the uptake of several hexoses and the inhibition of their phosphorylation by D-mannoheptulose, little or no preferential labelling of the endocrine moiety of the pancreas by the 14C-labelled hexoses is observed, at least when judged from their distribution space in pancreatic pieces or the whole pancreatic gland. Nevertheless, the findings made with D-mannoheptulose and its hexaacetate ester raise the view that this heptose could conceivably be used to achieve a sizeable preferential labelling of the endocrine pancreas under the present experimental conditions.