Metabolism of radiolabelled energy-yielding substrates by rat Sertoli cells

The rates of metabolism in vitro of 3H- or 14C-labelled glucose, pyruvate, glutamine and leucine by Sertoli cells from immature rats were estimated. The overall rate of glucose utilization exceeded by far the rates of oxidation of pyruvate (derived from glucose) via the citric acid cycle and glucose metabolism via the oxidative branch of the pentose phosphate pathway. This pattern of glucose metabolism was not markedly altered after stimulation of glucose metabolism by FSH. The rate of oxidation of exogenous pyruvate indicated that the energy yield from glucose metabolism by Sertoli cells could be dependent on the extracellular concentrations of pyruvate and lactate. There is no evidence that a high rate of aerobic glycolysis is of vital importance for Sertoli cells. In medium containing glucose and all amino acids, 14C-labelled glutamine and leucine were converted to 14CO2 at considerable rates. It was calculated that the oxidation of glutamine and leucine in addition to glucose and fatty acids can yield much of the required energy of Sertoli cells.     

The pentose phosphate pathway in rabbit liver. Studies on the metabolic sequence and quantitative role of the pentose phosphate cycle by using a system in situ

1. The reactions of the pentose phosphate cycle were investigated by the intraportal infusion of specifically labelled [(14)C]glucose or [(14)C]ribose into the liver of the anaesthetized rabbit. The sugars were confined in the liver by haemostasis and metabolism was allowed to proceed for periods up to 5min. Metabolism was assessed by measuring the rate of change of the specific radioactivity of CO(2), the carbon atoms of glucose 6-phosphate, fructose 6-phosphate and tissue glucose. 2. The quotient oxidation of [1-(14)C]glucose/oxidation of [6-(14)C]glucose as measured by the incorporation into respiratory CO(2) was greater than 1.0 during most of the time-course and increased to a maximum of 3.1 but was found to decrease markedly upon application of a glucose load. 3. The estimate of the pentose phosphate cycle from C-1/C-2 ratios generally increased during the time-course, whereas the estimate of the pentose phosphate cycle from C-3/C-2 ratios varied depending on whether the ratios were measured in glucose or hexose 6-phosphates. 4. The distribution of (14)C in hexose 6-phosphate after the metabolism of [1-(14)C]ribose showed that 65-95% of the label was in C-1 and was concluded to have been the result of a rapidly acting transketolase exchange reaction. 5. Transaldolase exchange reactions catalysed extensive transfer of (14)C from [2-(14)C]glucose into C-5 of the hexose 6-phosphates during the entire time-course. The high concentration of label in C-4, C-5 and C-6 of the hexose 6-phosphates was not seen in tissue glucose in spite of an unchanging rate of glucose production during the time-course. 6. It is concluded that the reaction sequences catalysed by the pentose phosphate pathway enzymes do not constitute a formal metabolic cycle in intact liver, neither do they allow the definition of a fixed stoicheiometry for the dissimilation of glucose.     

The response of carbohydrate metabolism in potato tubers to low temperature

This work investigates the possible causes of cold-induced sweetening in potato by examining the impact of low temperature on carbohydrate metabolism in mature tubers. Metabolism in tuber discs was monitored by determining the redistribution of radiolabel following incubation in [U-(14)C]glucose. Estimates of flux based on the specific activity of hexose phosphates established that while incubation at 4 degrees C resulted in an immediate restriction in pathways of carbohydrate oxidation relative to activity at 25 degrees C, there was no corresponding increase in flux to soluble sugars. In contrast, prior storage at low temperature stimulated flux to sugars at both 4 and 25 degrees C. Comparison of (14)CO(2) release from specifically labeled glucose and gluconate fed to tuber discs at 4 and 25 degrees C indicated that flux through glycolysis was preferentially restricted relative to the oxidative pentose phosphate pathway at low temperature, irrespective of prior storage temperature. However, the degree of randomization of label between positions C1 and C6 in the fructosyl moiety of sucrose following metabolism of [1-(13)C]glucose established that there was no preferential inhibition of the recycling of triose phosphates to hexose phosphates at low temperature. These results indicate that sugar accumulation in tubers during storage in the cold is not a direct consequence of a constraint in carbohydrate oxidation, despite preferential restriction of glycolysis at low temperature. It is concluded that the cold lability of enzymes catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is not a major factor in cold-induced sweetening in plants and that this widely held hypothesis should be abandoned.     

Glucolysis in Pseudomonas putida: Physiological Role of Alternative Routes from the Analysis of Defective Mutants

A number of mutants in which glucolysis is impaired have been isolated from Pseudomonas putida. The study of their behavior shows that this organism possesses a single glucolytic pathway with physiological significance. The first step of the pathway consists in the oxidation of glucose into gluconate. Two proteins with glucose dehydrogenase activity appear to exist in P. putida but the reasons for this duplicity are not clear. The process continues with the formation of 2-ketogluconate which is in turn converted into gluconate-6-phosphate. This is proved by the fact that mutants unable to form gluconate-6-phosphate from 2-ketogluconate show extremely slow growth on glucose or gluconate (generation times are increased more than 100 times). Other possible routes for the conversion of glucose into gluconate-6-phosphate, the glucose-6-phosphate pathway, or the direct phosphorylation of the gluconate formed by glucose oxidation are only minor shunts in P. putida. The Entner-Doudoroff enzymes, which catalyze the conversion of gluconate-6-phosphate into pyruvate and triosephosphate, appear to be essential to grow on glucose and also on gluconate and 2-ketogluconate. A significative role of the pentose route in the catabolism of these substrates is not apparent from this study. In contrast, P. putida strains showing no activity of the Entner-Doudoroff enzymes grow readily on fructose, although there is evidence that this hexose is at least partially catabolized via gluconate-6-phosphate.     

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.     

Epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate stimulate lactate production and the pentose phosphate pathway in freshly isolated rat hepatocytes

Epidermal growth factor (EGF) and tetradecanoylphorbol acetate (TPA) rapidly stimulated the production of lactate by hepatocytes isolated from fed rats. Our results indicate that enzymes of both glycolysis and the pentose phosphate pathway are involved in these actions. EGF stimulated CO2 release from the 1-position of glucose, and caused a small but significant increase in pyruvate kinase activity. In addition, EGF caused a rise in fructose 1,6-bisphosphate and fructose 2,6-bisphosphate concentrations, indicating activation of phosphofructokinase. TPA did not alter the concentrations of these sugar phosphates, but did cause an increased lactate production and CO2 production from the 1-position of glucose similar to EGF. Furthermore, the EGF stimulation of lactate formation was independent of the presence of medium Ca2+. Phenylephrine stimulation of this process, in parallel incubations, was entirely dependent upon the presence of Ca2+ in the medium. We conclude that EGF stimulates glycolysis and the pentose phosphate pathway in isolated hepatocytes from fed rats. The duplication of these actions by TPA suggests that protein kinase C is a mediator of EGF action in hepatocytes.     

Gluconate Catabolism in Rhizobium japonicum

Gluconate catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled gluconate gave the following results for growing cells, with values expressed as per cent (14)CO(2) evolution: C-1 = 93%, C-2 = 57%, C-3 = 30%, C-4 = 70%, C-6 = 39%. The preferential release of (14)CO(2) from C-1 and C-4 indicate that gluconate is degraded primarily by the Entner-Doudoroff pathway but the inequalities between C-1 and C-4 and between C-3 and C-6 indicate that another pathway(s) also participates. The presence of gluconokinase and a system for converting 6-phosphogluconate to pyruvate also indicate a role for the Entner-Doudoroff pathway. The extraordinarily high yield of (14)CO(2) from C-1 labeled gluconate suggests that the other participating pathway is a C-1 decarboxylative pathway. The key enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, could not be demonstrated. Specifically labeled 2-ketogluconate and 2,5-diketogluconate were oxidized by gluconate grown cells and gave ratios of C-1 to C-6 of 2.73 and 2.61, respectively. These compare with a ratio of 2.39 obtained with specifically labeled gluconate. Gluconate dehydrogenase, the first enzyme in the ketogluconate pathway found in acetic acid bacteria, was found. Oxidation of specifically labeled pyruvate, acetate, succinate, and glutamate by gluconate-grown cells yielded the preferential rates of (14)CO(2) evolution expected from the operation of the tricarboxylic acid cycle. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the primary pathways of gluconate oxidation in R. japonicum. An ancillary pathway for the initial breakdown of gluconate would appear to be the ketogluconate pathway which enters the tricarboxylic acid cycle at alpha-ketoglutarate.     

Gluconate catabolism in cowpea rhizobia: evidence for a ketogluconate pathway

Enzymatic and radiorespirometric analysis of several strains of cowpea rhizobia revealed the presence of key enzymes of the Entner-Doudoroff (ED) pathway with the operation of the hexose cycle for the dissimilation of gluconate. These bacteria lack the oxidative pentose phosphate (PP) pathway when grown on gluconate. Gluconate-grown cells possessed an operational tricarboxylic acid (TAC) cycle. Enzymes of an ancillary pathway, the ketogluconate (KG) pathway for gluconate catabolism were detected. The presence of this pathway was confirmed by techniques of thin-layer chromatography and radiorespirometry.Abbreviations ED Entner Doudoroff - PP pentose phosphate - EMP Embden-Meyerhof-Parnas - KG ketogluconate - TCA tricarboxylic acid - DKG diketogluconate - PFK phosphofructokinase     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

Biosynthesis of the cell wall polysaccharides, mannan and glucan, by Candida spec. H as indication of different pathways of glucose breakdown

The catabolic and anabolic D-glucose transformation of the yeast Candida spec. H has been studied. By using 1-14C-D-glucose and 6-14C-D-glucose, measuring the 14CO2 liberation and the label of glucose and mannose isolated from glucan and mannan, the following results have been obtained.1. Beginning with 100 micromoles glucose . ml-1 in the batch growth medium, at first on an average 64% of the glucose having been catabolized to CO2 are directly decarboxylated to pentose phosphate by pentose phosphate pathway (PPW). Later on at an exogen concentration of 70 micromoles.ml-1 73% of glucose on an average having been catabolised to CO2 undergoes transformation via glycolyse and tricarbonacid cycle (G-TCC). 2. Only after getting this glucose concentration the maximal hexose incorporation rate into glucan and mannan can be obtained. 3. 20--40% of the hexose channeled into the polysaccharid-biosynthesis have been prepared by resynthesis from pentose phosphate via PPW. 4. The results are discussed in connection with the observed crabtree effect.     

Pathways of carbohydrate metabolism in peripheral nervous tissue. I. The contribution of alternative routes of glucose utilization in peripheral nerve and brain

The activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis have been measured in rat sciatic nerve and brain. Parallel studies have been made of the utilization of 14 C-labelled glucose and pyruvate in these two tissues. Comparison of the enzyme profiles and flux through alternative routes was based on activity relative to the rate of glucose phosphorylation as measured by the rate of formation of 3H2O from [2-3H]glucose. The contributions of the pentose phosphate pathway and lipogenesis to glucose utilization were substantially higher in sciatic nerve than brain. The relatively high activity of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) suggested a special role for these enzymes in sciatic nerve.     

The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The K(m) values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and P(i) found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [(14)C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the (14)CO(2) yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.     

Blood sugar formation from dietary carbohydrate is facilitated by the pentose phosphate pathway in an insect Manduca sexta Linnaeus

Dietary carbohydrate, the principal energy source for insects, also determines the level of the blood sugar trehalose. This disaccharide, a byproduct of glycolysis, occurs at highly variable concentrations that play a key role in regulating feeding behavior and growth. Little is known of how developing insects partition the metabolism of dietary carbohydrate to meet the needs for blood trehalose, ribose sugars and NADPH, as well as energy production. This study examined the effects of varying dietary sucrose levels between 3.4 and 34 g/l in an artificial diet on growth rate, depot fat content and blood sugar formation from (13)C-enriched glucose in Manduca sexta. (2-(13)C)Glucose or (1,2-(13)C(2))glucose were administered to larvae by injection and after 6 h blood was analyzed by nuclear magnetic resonance spectroscopy. [2-(13)C]Trehalose was the principal product of [2-(13)C]glucose, but trehalose was also (13)C-enriched at C1 and C3, demonstrating activity of the pentose phosphate pathway. The trehalose C1/C2 (13)C-enrichment ratio, a measure of the substrate cycled through the pentose pathway, significantly increased with increasing dietary sugar, and reached a mean of 0.22 at the highest level. Blood trehalose concentration increased from approximately 38 mM at the lowest dietary carbohydrate level to 75 mM at the highest. Moreover, blood trehalose, growth rate and depot fat all increased in precisely the same way in relation to the level of pentose cycling. Based on the multiplet (13)C-NMR signal structure of trehalose synthesized from [1,2-(13)C(2)]glucose by insects maintained on a high carbohydrate diet, it was established that the formation of trehalose from glucose phosphate derived directly from the administered substrate, with no involvement of the pentose pathway, was greater than that from glucose phosphate metabolized through the pentose pathway prior to trehalose synthesis. On the other hand, glucose phosphate first metabolized through the pentose pathway contributed more to pyruvate formation than did glucose phosphate formed from the labeled substrate metabolized directly to pyruvate via glycolysis; this finding based on the multiplet (13)C-NMR signal structure in alanine derived from pyruvate. The results suggest that as dietary carbohydrate increases blood sugar synthesis from glucose phosphate derived directly from dietary sugar is facilitated by the pentose pathway which provides an increasing amount of substrate to pyruvate formation.     

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.     

New reaction sequences for the non-oxidative pentose phosphate pathway.

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.     

Further evidence for the classical pentose phosphate cycle in the liver

Isolated rat hepatocytes were incubated with [3-(14)C]xylitol or d-[3-(14)C]xylulose plus xylitol or glucose at substrate concentrations. The glucose formed was isolated and degraded to give the relative specific radioactivities in each carbon atom. C-4 of glucose had the highest specific radioactivity, followed by C-3, with half to one-fifth that of C-4. Only about 1% of the total radioactivity was in C-1. The data are compared with the predictions of the classical pentose phosphate cycle [Horecker, Gibbs, Klenow & Smyrniotis (1954) J. Biol. Chem.207, 393-403], and the proposed new version of the pentose phosphate cycle in liver [Longenecker & Williams (1980) Biochem. J.188, 847-857], which they denoted as the ;L-type pentose cycle'. The Williams pathway predicts that the specific radioactivity of C-1 of glucose should be half that of C-4 (after correction for approximately equal labelling on C-3 and C-4 of hexose phosphate in the pathway involving fructose 1,6-bisphosphatase). The actual labelling in C-1 is 20-350-fold less than this. When the hepatocytes are incubated with phenazine methosulphate, to stimulate the oxidative branch of the pentose phosphate cycle, the predicted relationship between (C-2/C-3) and (C-1/C-3) ratios of specific radio-activities is nearly exactly in accord with the classical pentose phosphate cycle. Glucose and glucose 6-phosphate were isolated and degraded from an incubation of hepatocytes from starved/re-fed rats with [3-(14)C]xylitol. Although the patterns were of the classical type, there was more randomization of (14)C into C-2 and C-1 in the glucose 6-phosphate isolated at the end of the incubation than in the glucose which was continuously produced.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the Yeast Saccharomyces bulderi

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.     

Metabolism of Lactate in the Rat Brain During the Early Neonatal Period

The metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2 and lipids was higher than those found for glucose and 3-hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase-catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase-catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3-hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3-hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (-)-Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate and N-acetylaspartate.     

Loss of [13C]glycerol carbon via the pentose cycle. Implications for gluconeogenesis measurement by mass isotoper distribution analysis

Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods.