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.     

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.     

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 Ca2+, 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 Ca2+ 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. 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.     

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.     

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.     

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.     

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.     

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.     

Inhibition of insulin receptor phosphorylation by indomethacin

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the -subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the -subunit of the solubilized insulin receptor by 6007o and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-¹⁴C]-methyl-D glucose transport by 50070 (P < 0.01), D-[6-¹⁴C]-glucose oxidation by 5007o (P < 0.01), D-[6-¹⁴C]-glucose conversion to lipid by 30010 (P < 0.01), and D-[1-¹⁴C]-glucose conversion to lipid by 6007o (P<0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-¹⁴C]-methyl-D-glucose transport by 75070 (P<0.01), D-[6-¹⁴C]-glucose oxidation by 20% (P<0.05), D-[1-¹⁴C]-glucose oxidation by 35070 (P<0.01), D-[6-¹⁴C] glucose conversion to lipid by 25010 (P<0.01), and D-[1-¹⁴C] glucose conversion to lipid by 4501o (P<0.01). In contrast, insulin binding to its receptor, basal D-[1-¹⁴C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions. This supports the hypothesis that insulin's metabolic effects are linked to activation of the insulin receptor protein kinase and indicates that there may be heterogeneity in the mechanisms of intracellular metabolic control by insulin.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium

Summary The discrimination between the isotopes of hydrogen in the reaction catalyzed by yeast phosphoglucoisomerase is examined by NMR, as well as by spectrofluorometric or radioisotopic methods. The monodirectional conversion of D-glucose 6-phosphate to D-fructose 6-phosphate displays a lower maximal velocity with D-[2-²H]glucose 6-phosphate than unlabelled D-glucose 6-phosphate, with little difference in the affinity of the enzyme for these two substrates. About 72% of the deuterium located on the C₂ of D-[1-¹³C,2-²H]glucose 6-phosphate is transferred intramolecularly to the C₁ of D-[1-¹³C,1-²H]fructose 6-phosphate. The velocity of the monodirectional conversion of D-[U-¹⁴C]glucose 6-phosphate (or D-[2-³H]glucose 6-phosphate) to D-fructose 6-phosphate is virtually identical in H₂O and D₂O, respectively, but is four times lower with the tritiated than ¹⁴C-labelled ester. In the monodirectional reaction, the intramolecular transfer from the C₂ of D-[2-³H]glucose 6-phosphate is higher in the presence of D₂O than H₂O. Whereas prolonged exposure of D-[1-¹³C]glucose 6-phosphate to D₂O, in the presence of phosphoglucoisomerase, leads to the formation of both D-[1-¹³C,2-²H]glucose 6-phosphate and D-[1-¹³C,1-²H]fructose 6-phosphate, no sizeable incorporation of deuterium from D₂O on the C₁ of D-[1-¹³C]fructose 1,6-bisphosphate is observed when the monodirectional conversion of D-[1-¹³C]glucose 6-phosphate occurs in the concomitant presence of phosphoglucoisomerase and phosphofructokinase. The latter finding contrasts with the incorporation of hydrogen from ¹H₂O or tritium from ³H₂O in the monodirectional conversion of D-[2-³H]glucose 6-phosphate and unlabelled D-glucose 6-phosphate, respectively, to their corresponding ketohexose esters.     

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.     

Transglutaminase activity in pancreatic islets

1. Pancreatic islet homogenates catalyze, in a Ca²⁺-dependent fashion, the incorporation of [2,5-³H]histamine, [1,4-¹⁴C]putrescine, [1,2-³H]agmatine, [¹⁴C]methylamine, L-[U-¹⁴C]lysine in N,N-dimethylcasein. 2. Using [2,5-³H]histamine as the amine donor, the K_m for Ca²⁺ and histamine amounts to 90μM and 0.7 mM, respectively. 3. The incorporation of [2,5-³H]histamine into N,N-dimethylcasein is inhibited by monodansylcadaverine, N-p-tosyl glycine, bacitracin and methylamine, the relative extent of inhibition depending on the respective concentrations of Ca²⁺, inhibitor and amine donor. 4. Bacitracin and methylamine, but not N-p-tosyl glycine, cause a dose-related inhibition of glucose-stimulated insulin release. 5. It is concluded that, in pancreatic islets, the Ca²⁺-responsive transglutaminase activity plays a critical role in the process of glucose-induced insulin release.     

Inhibition by L-valine and L-norleucine of 3-phenylpyruvate-induced insulin release

Insulin release induced by 3-phenylpyruvate in isolated rat pancreatic islets was inhibited by L-valine, L-norleucine or aminooxyacetate. The inhibitory effect of these three agents coincided with a lesser stimulation by 3-phenylpyruvate of 14CO2 output from islets prelabelled with L-[U-14C] glutamine. Conversely, 3-phenylpyruvate augmented the rate of conversion of L-valine to 2-ketoisovalerate and that of L-norleucine to 2-ketocaproate. However, 3-phenylpyruvate, which increased 2-ketoisovalerate oxidative decarboxylation, inhibited 14CO2 production by islets exposed to D, L-[1-14C] norleucine. These findings reveal that distinct nutrient secretagogues (e.g. 3-phenylpyruvate and L-norleucine), which are each able to stimulate insulin release, may act antagonistically upon the secretory process when used in combination. The present results also emphasize the relevance of both mitochondrial oxidation and intracellular transfer of reducing equivalents as determinants of the secretory response to such nutrients as 3-phenylpyruvate and norleucine.     

Ionic control of renal gluconeogenesis IV. Effect of extracellular phosphate concentration

Isolated rat renal tubules from glucose from pyruvate, malate, glycerol and α-ketoglutarate. The rate of glucose formation from all but glycerol is enhanced by an increase in Ca²⁺ concentration. Because changes in inorganic phosphate concentrations influence the uptake and retention of calcium by isolated cells, the effect of changes in phosphate concentration upon renal gluconeogenesis was examined. It was found that changing phosphate concentration altered the metabolism of isolated rat renal tubules in three ways which dependend upon the Ca²⁺ concentration. In the absence of Ca²⁺, increasing phosphate concentration from 0.07 to 1.2 mM led to a stimulation of the decarboxylation of [U-¹⁴C]malate, [1-14C]pyruvate, [2-¹⁴C]-pyruvate, α-keto[5-¹⁴C]glutarate and [1,3-¹⁴C₂]glycerol, and to an increase in ATP concentration but had no effect upon the rate of glucose formation from malate, pyruvate, α-ketoglutarate but a slight stimulation of glucose production from glycerol. A further increase in phosphate above 1.2 mM had no effect on any of these parameters. In the presence of either low (0.2 mM) or high (2.0 mM) Ca²⁺, changing phosphate concentration had no effect upon the decarboxylation of any of these substrates except glycerol whose decarboxylation was stimulated by increasing medium phosphate concentration. In the presence of calcium, increasing phosphate concentration led to an inhibition of glucose formation from malate, pyruvate and α-ketoglutarate but not from glycerol. Also in the presence of calcium both parathyroid hormone and cyclic AMP stimulated glucose formation, and under these conditions increasing phosphate concentration led to an inhibition of glucose formation. In tubules treated with parathyroid hormone an increase in phosphate concentration from 0.07 to 6.0 mM led to a significant increase in cyclic AMP concentration even though the rate of glucose formation decreased.Analysis of metabolite concentrations and rates of substrates decarboxylations, under a variety of conditions, revealed that P_i altered renal gluconeogenesis at a site different from those controlled by changes in Ca²⁺ concentration. The P_i-control site was tentatively identified as the glyceraldehyde phosphate dehydrogenase-glycerate kinase reaction sequence. However, the effect of changing P_i concentration upon parathyroid hormone-induced alterations in cyclic AMP concentration could not be explained by this action of P_i, and was probably due to an effect of P_i upon cellular calcium distribution. Thus, changes in P_i concentration appear to have two cellular effects, only one of which is related to a change in cellular calcium metabolism.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

D-glucose metabolism in normal dispersed islet cells and tumoral INS-1 cells

The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of ³HOH from D-[5-³H]glucose and ¹⁴C-labelled CO₂, acidic metabolites and amino acids from D-[U-¹⁴C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-¹⁴C]glucose oxidation and D-[5-³H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types.     

2-Oxocarboxylic acids and function of pancreatic islets in obese–hyperglycaemic mice. Insulin secretion in relation to 45Ca uptake and metabolism

The effects of aliphatic 2-oxocarboxylic acids, at concentrations of up to 40mm, on the function of pancreatic islets from ob/ob (obese–hyperglycaemic) mice were investigated. 1. 2-Oxopentanoate, dl-3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate and 2-oxohexanoate all induced insulin release by isolated incubated islets and a biphasic insulin-secretory pattern in perfused mouse pancreas. The last two substances were similar in potency to glucose. Pyruvate, 2-oxobutyrate, 3-methyl-2-oxobutyrate and 2-oxo-octanoate did not induce insulin release significantly. 2. 2-Oxocarboxylic acids with significant insulin-secretory potency also induced significant ⁴⁵Ca uptake by isolated incubated islets. 3. The rates of decarboxylation of [1-¹⁴C]pyruvate, 3-methyl-2-oxo[1-¹⁴C]butyrate and 4-methyl-2-oxo[1-¹⁴C]pentanoate were twice as high as the rates of oxidation of the corresponding U-¹⁴C-labelled compounds. However, whereas the rates of metabolism of labelled pyruvate and 3-methyl-2-oxobutyrate steadily increased over the concentration range 1–40mm, those of labelled 4-methyl-2-oxopentanoate and d-[U-¹⁴C]glucose levelled off at concentrations above 10mm. 4. Omission of ⁴⁰CaCl₂ from the incubation medium reduced the rate of oxidation of the insulin secretagogue [U-¹⁴C]4-methyl-2-oxopentanoate, but left that of the non-(insulin secretagogue) [U-¹⁴C]3-methyl-2-oxobutyrate unaffected. 5. Only glucose, and not pyruvate, 3-methyl-2-oxobutyrate and 4-methyl-2-oxopentanoate, significantly inhibited oxidation of endogenous fatty acids. 6. It is suggested that stimulus–secretion coupling and the resulting exocytosis of insulin in pancreatic β-cells may modulate both fuel oxidation and ⁴⁵Ca uptake.