Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells

Drosophila melanogaster has become a prominent and convenient model for analysis of insulin action. However, to date very little is known regarding the effect of insulin on glucose uptake and metabolism in Drosophila. Here we show that, in contrast to effects seen in mammals, insulin did not alter [(3)H]2-deoxyglucose uptake and in fact decreased glycogen synthesis ( approximately 30%) in embryonic Drosophila Kc cells. Insulin significantly increased ( approximately 1.5-fold) the production of (14)CO(2) from D-[1-(14)C]glucose while the production of (14)CO(2) from D-[6-(14)C]glucose was not altered. Thus, insulin-stimulated glucose oxidation did not occur via increasing Krebs cycle activity but rather by stimulating the pentose phosphate pathway. Indeed, inhibition of the oxidative pentose phosphate pathway by 6-aminonicotinamide abolished the effect of insulin on (14)CO(2) from D-[U-(14)C]glucose. A corresponding increase in lactate production but no change in incorporation of D-[U-(14)C]glucose into total lipids was observed in response to insulin. Glucose metabolism via the pentose phosphate pathway may provide an important source of 5'-phosphate for DNA synthesis and cell replication. This novel observation correlates well with the fact that control of growth and development is the major role of insulin-like peptides in Drosophila. Thus, although intracellular signaling is well conserved, the metabolic effects of insulin are dramatically different between Drosophila and mammals.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Correlation between the rate of protein and nucleic acid synthesis and the intensity of various glucose metabolic pathways in rat liver cells

A sharp and strong suppression of protein synthesis by cycloheximide in liver cells of starving rats is paralleled with activation of RNA synthesis and glucose-6-phosphate dehydrogenase production. Subsequent reconstitution and stimulation of protein synthesis (6-12 hrs after cycloheximide injection) result in activation of hexokinase. Upon stimulation of DNA synthesis (48-60 hrs after cycloheximide injection) the activity of both enzymes is very low. Since glucose-6-phosphate dehydrogenase appears to be the limiting step of glucose decay via the pentose phosphate pathway, and hexokinase is the limiting step of glycolysis, it was assumed that RNA synthesis predominantly occurs via the pentose phosphate pathway, while that of proteins via glycolysis.     

Effect of alloxan-diabetes and treatment with anti-insulin serum on pathways of glucose metabolism in lactating rat mammary gland

1. The overall metabolic changes in lactating mammary gland in alloxan-diabetic and anti-insulin-serum-treated rats were assessed by measurement of the incorporation of (14)C from specifically labelled glucose, pyruvate and acetate into carbon dioxide and lipid, together with measurements of enzymes concerned with the pentose phosphate pathway and with citrate metabolism. 2. Alloxan-diabetes depressed the rate of formation of (14)CO(2) from [1-(14)C]glucose and [2-(14)C]glucose to approx. 10% of the control rate; this was partially reversed by addition of insulin in vitro. The quotient Oxidation of [1-(14)C]glucose/Oxidation of [6-(14)C]glucose fell from a value of 17.6 in the control group to 3.9 in the diabetic group and was restored to 14.3 in the presence of insulin in vitro. In keeping with these results it was shown that glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were significantly decreased in alloxan-diabetic rats. 3. Alloxan-diabetes depressed the decarboxylation and the oxidation of labelled pyruvate, but not the oxidation of labelled acetate. 4. The synthesis of lipid from specifically labelled glucose was greatly decreased, that from [2-(14)C]pyruvate was almost unchanged and that from [1-(14)C]acetate alone was increased in alloxandiabetic rats. However, the stimulation of lipid synthesis from acetate by glucose was small in the alloxan-diabetic rats compared with the controls. Insulin in vitro partially reversed all these effects. Both citrate-cleavage enzyme and acetate thiokinase activities were decreased in alloxan-diabetic rats. 5. Treatment of rats with anti-insulin serum depressed the formation of (14)CO(2) from [1-(14)C]glucose and [2-(14)C]glucose, but increased that from [6-(14)C]glucose. This was completely restored by the presence of insulin in vitro. The quotient Oxidation of [1-(14)C]glucose/Oxidation of [6-(14)C]glucose fell from a value of 17.6 in the control group to 3.8 in the anti-insulin-serum-treated group. There were no changes in the activity of glucose 6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but the hexokinase distribution changed and the content of the soluble fraction increased significantly. 6. The synthesis of lipid from specifically labelled glucose was depressed in anti-insulin-serum-treated rats; this effect was completely reversed by addition of insulin in vitro to the tissue slices.     

Oxytocin and glucose oxidation in the rat uterus

The effects of oxytocin on the biochemical pathways of glucose oxidation were investigated in the rat uterus. In the presence of oxytocin, glucose oxidation in uterine segments obtained from Sprague-Dawley rats at diestrus increased 1.5–2.0-fold above the basal rate. A half-maximal response was observed at about 3 nM oxytocin; the maximum response was equal to or greater than the response to 1.7 nM insulin. In stripped myometrial segments (denuded of the endometrial component), oxytocin stimulated glucose oxidation at estrus only; whereas in intact uterine segments, the stimulation of oxidation was observed at both estrus and diestrus. In contrast, stimulation of oxidation by carbachol in stripped myometrial segments was independent of the estrous state of the tissue. The ratio of [1-¹⁴C]glucose to [6-¹⁴C]glucose oxidation was measured to estimate the relative involvement of the pentose phosphate and the tricarboxylic acid pathways of metabolism. In myometrial tissue, stimulation of glucose oxidation by oxytocin appeared to proceed through the tricarboxylic acid cycle. In intact uterine segments, at diestrus, glucose oxidation involved largely the pentose phosphate pathway (suggesting increased glucose metabolism in endometrial tissue), whereas at estrus, in the intact tissue segments, oxytocin increased glucose oxidation largely via the tricarboxylic acid cycle, and appeared to do so predominantly in the myometrial tissue. Carbachol-stimulated glucose oxidation appeared to proceed mainly via the tricarboxylic cycle in the myometrial tissue, irrespective of the stage of the estrous cycle. In the uterus of the Brattleboro rat (either intact uterine segments or stripped myometrial strips), oxytocin stimulated glucose oxidation only at estrus, predominantly through the tricarboxylic acid cycle. These findings suggest that oxytocin, in addition to its known effect on the contractility of uterine and myoepithelial smooth muscle, may regulate glucose metabolism in both the myometrial and endometrial components of uterine tissue.     

The pentose cycle and insulin release in mouse pancreatic islets

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.     

Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: Effects of nerve growth factor and 6-aminonicotinamide

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of¹⁴CO₂ generated from 1-[¹⁴CO₂]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[¹⁴CO₂]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.Special Issue dedicated to Dr. O. H. Lowry.     

Short-term control of the pentose phosphate cycle by insulin could be modulated by the NADPH/NADP ratio in rat adipocytes and hepatocytes

The short-term activation of the pentose phosphate cycle by insulin in rat adipocytes and hepatocytes has been studied. This NADPH-producing pathway is regulated by the activation or inhibition of different NADPH-consuming pathways. The stimulation of the fatty acid synthesis by insulin produced an increase in the flux through the pentose phosphate cycle. Kynurenate produced a decrease in the fatty acid synthesis and, consequently a diminution in the flux through the pentose phosphate cycle. Incubation of adipocytes and hepatocytes in presence of kynurenate (10 mM and 3 mM respectively) and insulin (5 nM), prevents both insulin activation on fatty acid synthesis and pentose phosphate cycle. These results suggest that insulin activates the pentose phosphate cycle through the activation of fatty acid synthesis.     

Amino acids and glucose utilization by different metabolic pathways in ascites-tumour cells

The utilization of amino acids and glucose by ascites tumour cells has been studied in order to elucidate which are their relative roles as energy substrates or building blocks for biosynthetic purposes, as well as the quantitative contribution of the different metabolic pathways involved. 1. Glucose is utilized at a rate of 1.1 mumol x min-1 x g cells-1. 93% is transformed into lactate, 0.7% used by the pentose phosphate pathway, 1.5% by the tricarboxylic acid cycle and 2% is for lipid synthesis. 2. ATP production is derived: 78% from glucose conversion into lactate, 1% from glucose oxidation and 19% from glutamine oxidation. 3. Glucose starvation, in the presence of all amino acids, leads to a 70% decrease in the rate of protein synthesis, due to the drop in ATP levels. 4. Pentose phosphate pathway flux increases by 75% when glycolysing cells are incubated in the presence of all amino acids. 5. Pyruvate is decarboxylated at a rate of 66 nmol x min-1 x g cells-1, 45-80% of it is incorporated into lipids instead of being oxidized, depending on the incubation conditions. 6. Non-essential amino acids (aspartate and glutamate) are oxidized at a low rate. Glutamine is oxidized at a rate 20-times and 35-times that of glucose and glutamate respectively. Glutamine can not replace glucose as the main energy source. 7. Leucine utilization, 28 nmol x min-1 x g cells-1, is very high compared with normal cells, due to the high rate of lipid and protein synthesis. Its oxidation is similar to that of non-tumoural cells. 8. Sterols account for 80% of the lipids synthesized either from leucine or glucose.     

Vanadate activates pentose phosphate pathway and glycolysis, and raises fructose 2,6-bisphosphate concentration in slices of lactating rat mammary gland.

In mammary gland slices from lactating rats, vanadate increased the rate of glucose oxidation via the pentose phosphate pathway by 36% and raised the glucose flux via glycolysis by 47%. Furthermore, vanadate increased the fructose 2,6-bisphosphate (Fru-2,6-P2) level by 33%. The effect of vanadate on glucose oxidation was compared to the effect of insulin. The present data indicate that 0.5mM vanadate has an effect on glucose utilization similar to that of insulin but does not reach the same level.     

Pathways of Carbohydrate Metabolism in Microcyclus Species

Radiorespirometric and enzymatic studies were conducted to determine primary and secondary pathways of carbohydrate catabolism in Microcyclus aquaticus and M. flavus. M. aquaticus catabolizes both glucose and gluconate mainly via the Entner-Doudoroff and pentose phosphate pathways with some concurrent participation of the Embden-Meyerhof pathway. M. flavus, however, oxidizes glucose mainly via the Embden-Meyerhof pathway and gluconate via the Entner-Doudoroff pathway with some simultaneous operation of the pentose phosphate pathway. Both of the organisms showed evidence of the tricarboxylic acid cycle as a secondary pathway for the oxidation of carbohydrates.     

The effect of high glucose and high insulin concentrations on pentose phosphate shunt enzymes and malic enzyme in cultured human endothelial cells

The influence of glucose and insulin on pentose phosphate shunt enzymes and malic enzyme activity in cultured human endothelial cells has been investigated. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme were present in endothelial cells. Enzyme activities were not altered either by 20 mM glucose or 10(-8) M insulin after 3, 6 and 12 hour incubations respectively. Neither increased glucose nor increased insulin alter the activity of the pentose phosphate shunt. As a consequence fatty acid and cholesterol synthesis in the endothelial cell is unlikely to be altered in the presence of increased glucose or increased insulin.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate

By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.     

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.     

The utilization of glucose for the synthesis of milk components in the fed and starved lactating goat in vivo.

1. [U-14C]Glucose and [3-3H]glucose were infused into fed and starved lactating goats in order to study glucose metabolism in the mammary gland. 2. Glucose carbon was oxidized and metabolizet to milk lactose, citrate and triacylglycerol in the lactating goat udder. 3. Recycling of glucose carbon in the lactating animal accounted for 10-20% of the total glucose turnover in the whole animal. Recycling of glucose 6-phosphate in the udder accounted for about 25% of the glucose 6-phosphate metabolized. 4. Flux of glucose 6-phosphate through the pentose phosphate pathway was sufficient to account for 34% of the NADPH required for fatty acid synthesis in the gland in the fed animal. 5. Net metabolism of glucose 6-phosphate via the pentose phosphate pathway accounted for 17.8 and 1.2% of the glucose phosphorylated by the mammary gland in the fed and starved animal respectively. Metabolism of glucose 6-phosphate via the pentose phosphate pathway was sufficient to account for all the CO2 produced from glucose in the fed animal, but only 17% of the CO2 produced from glucose in the starved animal.     

Glucose oxidation in white adipose tissue from BIO 14.6 dystrophic hamsters

In studies of glucose oxidation in white retroperitoneal adipose tissue of BIO 14.6 dystrophic and F1B normal hamsters aged 55-67 and 368-379 days, no difference was found in the basal state of radiolabelled 14CO2 production using either D-[6-14C]glucose or D-[1-14C]glucose. When C6-labelled glucose was used, insulin induced a slightly greater increase in glucose oxidation in dystrophic adipose tissue at both ages. When C1-labelled glucose was used, insulin enhanced glucose oxidation in dystrophic tissue more than twice normal in tissues from young animals and five times normal in tissues from the old ones. The increase in oxidation with D-[1-14C]glucose likely represents enhanced activity of the pentose phosphate pathway, which has also been observed in certain tissues of other animals with inherited skeletal-muscle degeneration. The change can probably be classified as being compensatory, an attempt by tissues to maintain functional integrity.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.     

Changes in pathways of pentose phosphate formation in relation to phosphoribosyl pyrophosphate synthesis in the developing rat kidney. Effects of glucose concentration and electron acceptors

Phosphoribosyl pyrophosphate (PPRibP), required in nucleotide synthesis, increases 2-fold in rat kidney from 1 day post partum to adult stage; there is no accompanying increase in PPRibP synthetase activity measured in vitro. Ribose 5-phosphate is a key factor in the regulation of PPRibP synthesis. The activity and regulation of 3 routes of ribose 5-phosphate formation have been measured in renal growth: (i) the flux through the oxidative pentose phosphate pathway was high in the neonatal period but increased only +50% thereafter; (ii) the non-oxidative pentose phosphate pathway, including transketolase, increased by +145%; (iii) the rate-limiting enzymes of the glucuronate-xylulose route increased +200% from 1 day to the adult stage. The importance of systems reoxidizing NADPH was shown by: (i) the stimulation of renal PPRibP formation from glucose by phenazine methosulphate; (ii) the early involvement of the oxidative pentose phosphate pathway at the stage where NADPH is used for biosynthetic routes; (iii) the increasing involvement of the glucuronate-xylulose route, which acts as a transhydrogenase producing NADP+ in addition to pentose phosphate formation and (iv) the correlation between renal PPRibP content and the activity of aldose reductase, which, by utilization of NADPH, stimulates ribose 5-phosphate formation via the oxidative pentose phosphate pathway. Evidence is adduced that the contribution of the 3 routes of ribose 5-phosphate formation in the kidney varies at different stages of development.     

Metabolism of radiolabeled glucose by mouse oocytes and oocyte-cumulus cell complexes.

This study was carried out to examine the metabolism of [1-14C]-, [6-14C]-, and [5-3H]glucose by oocyte-cumulus cell complexes (OCC) and denuded oocytes (DO) and to test the hypothesis that metabolism of glucose through the pentose phosphate pathway is associated with meiotic induction. OCC or DO were cultured in hanging drops suspended from the cap of a microfuge tube, with NaOH serving as a trap to collect released 3H2O or 14CO2. Preliminary experiments established that this culture system supports both spontaneous and ligand-induced meiotic maturation. An initial time course experiment (1.5-6 h) showed that hypoxanthine-treated OCC from eCG-primed animals metabolized glucose principally via glycolysis, with an increase to 2.7-fold in response to FSH. Though more [1-14C]glucose was oxidized than [6-14C]glucose, its metabolism was about two orders of magnitude less than that of [5-3H]glucose. Also, FSH significantly increased oxidation of [1-14C]glucose but not [6-14C]glucose, indicating a preferential activation of the pentose phosphate pathway. Pyrroline carboxylate, an activator of the pentose phosphate pathway, increased the activity of this pathway to over 2-fold but failed to affect glucose oxidation through the tricarboxylic acid cycle. Glycolytic metabolism was increased by 25%. The addition of pyruvate to pyruvate-free medium resulted in significant reduction in the metabolism of all three glucose analogues. In OCC retrieved from hCG-injected, primed mice and cultured under hormone-free conditions, metabolic responses were similar to those in FSH-treated complexes cultured in hypoxanthine. DO metabolized glucose, but at a much reduced rate when compared to OCC. Pyruvate reduced the consumption of all three glucose analogues by DO. Pyrroline carboxylate reduced [5-3H]glucose metabolism by DO but had little effect on [1-14C]- and [6-14C]glucose oxidation. These data demonstrate metabolism of glucose by both DO and OCC, but reveal that cumulus cells are more active than the oocyte in this regard. In addition, induction of maturation by FSH, hCG, or pyrroline carboxylate was accompanied by a significant increase in the oxidation of [1-14C]glucose but not [6-14C]glucose by OCC, supporting a proposed role for the pentose phosphate pathway in meiotic induction.     

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.