Environmental modulation of the anomeric specificity of glucose metabolism in normal and tumoral cells

In rat pancreatic islets and erythrocytes, alpha-D-glucose (2.8-5.6 mM) is better metabolized than beta-D-glucose, as judged from the conversion of D-[5-3H]glucose to 3H2O, augmentation in lactic acid production (or output) or oxidation of D-[U-14C]glucose. In tumoral cells, however, whether of the insulin-producing or lymphocytic leukemia type, the anomeric preference for alpha-D-glucose utilization is no longer present when the cells are incubated at comparable glucose concentrations (2.8-4.0 mM). Nevertheless, the tumoral insulin-producing cells are able to display preference for either alpha-D-glucose (at very low glucose concentrations in the 0.14-0.82 mM range) or beta-D-glucose (in the presence of 16.7 mM glucose). These findings indicate that the anomeric specificity of glucose metabolism may differ in distinct cell types, and can be modulated by the ambient glucose concentration. ambient glucose concentration.     

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.     

Pentose cycle pathway in normal and tumoral islet cells

Relative to protein content, the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the rate of glucose metabolism by the pentose cycle pathway in tumoral insulin-producing cells were similar to or higher than those found in normal rat islets. Hence, the decreased secretory response of tumoral cells to glucose is apparently not attributable to any major anomaly in glucose handling by the hexose monophosphate pathway.     

Anomeric specificity of glucose metabolism in the pentose cycle

The production of 3H2O from alpha- and beta-D-[5-3H]glucose and that of 14CO2 from either alpha- and beta-D-[1-14C] or alpha- and beta-D-[6-14C]glucose were measured in rat pancreatic islets and tumoral insulin-producing cells incubated at 7 degrees C. The ratio in 14CO2 output from D-[1-14C]glucose/D-[6-14C]glucose, the fraction of glucose metabolism occurring through the pentose cycle, and the flow rate through such a cycle were always higher in the presence of beta- than alpha-D-glucose. This indicates that the anomeric specificity of glucose-6-phosphate dehydrogenase is operative in intact islet cells.     

Effect of cytochalasin B on glucose uptake, utilization, oxidation and insulinotropic action in tumoral insulin-producing cells

Cytochalasin B (17-3 microM) virtually abolished 3-O-methyl-D-[U-14C]glucose uptake and D-[5-3H]glucose utilization in tumoral insulin-producing cells of the RINm5F line. This coincided with a marked decrease in D-[U-14C]glucose oxidation and suppression of the stimulant action of D-glucose upon insulin release. Cytochalasin B, however, augmented basal insulin release by the tumoral cells. The RINm5F cells appeared much more sensitive than normal islet cells to cytochalasin B, as judged by the relative magnitude of inhibition in either hexose uptake or utilization. In both cell types, the inhibitory action of cytochalasin B upon glucose metabolism seemed to be competitive, being more marked at low than high glucose concentration. These results are interpreted in support of the view that a decreased efficiency of hexose transport across the plasma membrane represents an essential deficiency of the RINm5F cells.     

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

Summary 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.     

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.     

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.     

Anomeric preference of glucose phosphorylation and glycolysis in human erythrocytes

Lactate output from the alpha and beta anomers of glucose was measured in intact human erythrocytes at 37 degrees C; and glucose anomer phosphorylation, in human erythrocyte homogenates. The rates of both glucose metabolism (lactate output) and phosphorylation were higher in the presence of beta-D-glucose as distinct from alpha-D-glucose at three glucose concentrations used (2, 5, and 10 mM). Thus, the v beta/v alpha ratios of metabolism and phosphorylation of glucose at 2 mM were 1.24 and 1.22, respectively. The results indicate that the beta preference of hexokinase, a rate-limiting enzyme in glycolysis, is reflected in beta-preferential glycolysis.     

Fructose metabolism via the pentose cycle in tumoral islet cells

In tumoral islet cells (RINm5F line) the phosphorylation of D-fructose is catalyzed by hexokinase rather than fructokinase. Fructose 6-phosphate appears to be preferentially channelled into the pentose cycle, as suggested by a ratio of D-[1-14C]fructose/D-[U-14C]fructose oxidation close to 2.7, the failure to generate 14C-labelled lactate from D-[1-14C]fructose and a poor metabolic response to menadione. When the islet cells are exposed to both D-fructose and D-glucose, however, the metabolism of the former hexose is dramatically modified, fructose 6-phosphate being now formed at a lower rate and preferentially channelled into the glycolytic pathway. These findings illustrate the existence of regulatory steps in fructose catabolism located distally to its site of phosphorylation.     

32P, 86Rb+ and 45Ca2+ handling by tumoral insulin-secreting cells (RINm5F line)

In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Metabolic and secretory response of tumoral-insulin producing cells to D-fructose and D-galactose

At variance with normal islet cells, tumoral insulin-producing cells of the RINm5F line were found to display a positive secretory response not solely to D-glucose and D-mannose, but also to D-fructose and D-galactose. All hexoses increased the ATP/ADP ratio, exerted a sparing action upon the oxidation of endogenous nutrients in cells prelabelled with either L-[U-¹⁴C]glutamine or [U-¹⁴C]palmitate, increased the output of lactic acid and, as judged from data collected in the presence of D-[U-¹⁴C]hexoses, underwent oxidation in the RINm5F cells. The secretory response to these four hexoses appeared commensurate with the extent of their metabolic effects.     

Metabolism, protein content, and in vitro embryonic development of goat cumulus-oocyte complexes matured with physiological concentrations of glucose and L-lactate

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.     

The role of glucose,pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous l-lactate (3–6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.     

Evidence for an alternative and non-phosphorylating pathway for NADH reoxidation in a yeast strain resistant to glucose repression

A yeast strain (SP1) resistant to glucose repression modified simultaneously in the fermentative and in the oxidative pathways (loss of alcohol dehydrogenase I and over production of cytochrome a + a3, being insensitive to the glucose effect) developed a secondary mitochondrial hydrogen pathway. Oxidative phosphorylation was measured with exogenous NADH as substrate on mitochondria derived from repressed or derepressed cells. In this strain, antimycin A promotes a partial inhibition of NADH oxidation but a complete inhibition of phosphorylation. Amytal partially inhibits oxidation of NADH but not phosphorylation. KCN inhibits NADH oxidation in a biphasic way (first level 0.1 mM, second level 5 mM) but phosphorylation was fully inhibited by 0.1 mM KCN. This alternative but non-phosphorylating pathway is insensitive to salicyl hydroxamate. The external NADH dehydrogenase, like cytochrome c oxidase is partially insensitive to catabolite repression. These results provide evidence for the presence in strain SP1 of an alternative mitochondrial pathway, going from the external NADH dehydrogenase to an oxidase, different from the normal NADH dehydrogenase ubiquinone pathway.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Alpha- and beta-anomeric preference of glucose-induced insulin secretion at physiological and higher glucose concentrations, respectively

We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. alpha-D-Glucose was phosphorylated faster than beta-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization.     

Impaired uptake of D-glucose by tumoral insulin-producing cells

At variance with the situation found in normal pancreatic islets, no equilibration of extracellular and intracellular D-glucose concentrations occurs in tumoral insulin-producing cells of the RINm5F line. This unexpected behaviour may account, in part at least, for the abnormal kinetics of glucose utilization in the tumoral cells and their poor secretory response to this hexose.