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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.

ABSTRACT: 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.

SUBMITTER: Gumaa KA

PROVIDER: S-EPMC1185244 | biostudies-other | 1969 Dec

REPOSITORIES: biostudies-other

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Project description:Liver content of pentose-cycle intermediates and the activity of the three major cytoplasmic NADPH-producing enzymes and pentose-cycle enzymes were measured in three dietary states: 48 h-starved rats, rats fed on a standard diet ad libitum, and rats meal-fed with a low-fat high-carbohydrate diet. Measured tissue contents of pentose-cycle intermediates in starved liver were: 6-phosphogluconate, 4.7 +/- 0.5 nmol/g; ribulose 5-P, 3.7 +/- 0.5 nmol/g; xylulose 5-P, 4.3 +/- 0.4 nmol/g; sedoheptulose 7-P, 25.5 +/- 1.3 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 30.6 +/- 0.7 nmol/g. These values were in good agreement with values calculated from fructose 6-P and free glyceraldehyde 3-P, assuming the major transketolase, transaldolase, ribulose-5-P 3-epimerase and ribose-5-P isomerase reactions were all in near-equilibrium. Similar results were found in animals fed ad libitum. These relationships were not valid in animals fed on a low-fat high-carbohydrate diet, with tissue contents of metabolites in some cases being more than an order of magnitude higher than the calculated values. Measured tissue contents of pentose-cycle intermediates in these animals were: 6-phosphogluconate, 124.2 +/- 13.9 nmol/g; ribulose 5-P, 44.8 +/- 7.1 nmol/g; xylulose 5-P, 77.2 +/- 9.4 nmol/g; sedoheptulose 7-P, 129.9 +/- 10.1 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 157.0 +/- 11.3 nmol/g. In all animals, regardless of dietary state, tissue content of erythrose 4-P was less than 2 nmol/ml. Liver activities of glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase were increased from 3.5 +/- 0.9 mumol/g and 7.3 +/- 0.5 mumol/min per g in starved animals to 13.2 +/- 1.1 and 10.5 +/- 0.7 mumol/min per g in low-fat high-carbohydrate-fed animals. Despite these changes, the activities of transaldolase (3.4 +/- 0.3 mumol/min per g), transketolase (7.8 +/- 0.2 mumol/min per g) and ribulose-5-P 3-epimerase (7.5 +/- 0.4 mumol/min per g) were not increased in meal-fed animals above those observed in starved animals (3.4 +/- 0.2, 7.1 +/- 0.3 and 8.6 +/- 0.4 mumol/min per g respectively). The increase in the activity of oxidative pentose-cycle enzymes in the absence of any change in the non-oxidative pentose cycle appeared to contribute to the observed disequilibrium in the pentose cycle in animals meal fed on a low-fat high-carbohydrate diet.

Project description:Background:Previous literatures have implied that the liver fat deposition plays a crucial role in the development and progression of insulin resistance. In the present study, we aimed to investigate the association of liver fat content (LFC) with glucose metabolism status in the population of newly diagnosed type 2 diabetes mellitus (nT2DM), prediabetes mellitus (PDM) and normal controls (NC), and assessing if the LFC could as an indicator for the prediction of T2DM. Methods:A total of 242 subjects (including 141 nT2DM patients, 48 PDM subjects and 53 NC) were enrolled. The levels of LFC were quantified by using the proton magnetic resonance spectroscopy ([1H]-MRS) technique. Clinical and laboratory parameters of study subjects were collected by medical records and biochemical detection. One-way ANOVA or nonparametric test (Kruskal-Wallis) was applied for intergroup comparisons; intergroup comparison was performed in using of Bonferroni multiple-significance-test correction. Results:There were significantly increased LFC levels in nT2DM (14.72%?±?6.37%) than in PDM (9.62%?±?4.41%) and that of NC groups (5.11%?±?3.66%) (all p?<?0.001). The prevalence of nonalcoholic fatty liver disease (NAFLD) was also found to be increased in nT2DM (91.48%) than in PDM (85.41%) and that of NC (32.07%) groups. Correlation analysis revealed that the increase of LFC positively associated with fast plasma glucose (FPG), 2 h plasma glucose (PG), Delta G30 and homeostatic model assessment of insulin resistance (HOMA-IR), negatively associated with Delta Ins30, Delta C30, Ins30/G30 AUC, CP30/G30 AUC, Ins AUC/G AUC, CP AUC/G AUC, homeostatic model assessment for ?-cell function index (HOMA-?) and matsuda insulin sensitivity index (Matsuda ISI). Multilinear regression analysis showed that LFC, body mass index (BMI) and diastolic blood pressure (DBP) contributed for the prediction of HOMA-IR, and total cholesterol (TC), age, waist circumference (WC) and low-density lipoprotein cholesterol (LDL-C) were the significant contributors for HOMA-?. Conclusions:Our study revealed an increased LFC level and prevalence of NAFLD in nT2DM than in PDM and that of NC groups, the increase of LFC was closely associated with insulin resistance and impaired glucose metabolism status, may be regarded as potential indicator contributing to the development and progression of T2DM.

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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.

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