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.     

Rates of flux through the pentose cycle in perfused rat liver. A procedure for the calculation of rates of substrate flux from 14CO2 production from [1-14C]glucose

A method for the determination of substrate flux through the pentose cycle was developed employing [1-14C]glucose in experiments with perfused rat livers. The method consists first of a kinetic analysis which differentiates between the production of 14CO2 from [1-14C]glucose via the pentose cycle and via the citrate cycle and, second of a calculation of the specific radioactivity of the hexose monophosphate pool from measured rates of glycolysis and the specific radioactivity of lactate released into the perfusate. The method was validated by experiments comparing the results of tracer infusions with [1-14C]glucose, [6-14C]glucose and [3-14C]pyruvate. In livers from fed rats perfused with 10 mM glucose, the rate of substrate flux through the pentose cycle was around 0.2 mumol X min-1 X g-1; it was about 20% of the substrate flux via glycolysis. The kinetic data were inconsistent with the existence of an L-type pentose cycle in liver.     

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.     

Effect of growth rate on the glucose metabolism of yeast grown in continuous culture. Radiorespirometric studies

Specifically labelled¹⁴C-d-glucose was used to estimate the percentage participation of glycolysis and pentose phosphate cycle in the glucose catabolism ofCandida utilis andSaccharomyces cerevisiae. The two yeasts were cultivated at various growth rates (0.1 to 0.5 h^?1) in a chemostat on synthetic medium limited with glucose under aerobic conditions. The results show a considerable increase in the percentage participation of pentose phosphate cycle in the glucose catabolized bySaccharomyces cerevisiae with the increase in specific growth rate. However, inCandida utilis, the specific growth rate does not influence significantly the part of glucose catabolized via pentose phosphate cycle, but its absolute values are relatively higher than inSaccharomyces cerevisiae. A rough quantitative estimate indicates that a maximum of 60 to 72% of the assimilated glucose is catabolized through the pentose phosphate cycle while inSaccharomyces cerevisiae the percentage participation of the pentose phosphate cycle varies from 24 to 60% (maximum) and 9 to 34% (minimum).     

The significance of sedoheptulose 1,7-bisphosphate in the metabolism and regulation of the pentose pathway in liver

Rat liver cytosolic enzyme preparation catalyses the formation of sedoheptulose 1,7-P2 (60% of total heptulose-P formed) from hexose 6-P and triose 3-P (reverse mode of pentose pathway operation). Smaller amounts of sedoheptulose 1,7-P2 are also formed from ribose 5-P during the non-oxidative synthesis of hexose 6-P (forward pentose pathway operation). The apparent absence of erythrose 4-P in biological systems may be explained by its contribution to carbons 4,5,6 and 7 of sedoheptulose 1,7-P2 as well as its pronounced ability to exist in dimeric form. Apart from the aldolase catalyzed formation of sedoheptulose 1,7-P2, 6-phosphofructokinase also catalyses its formation from sedoheptulose 7-P and fructose 1,6-bisphosphatase catalyses its dephosphorylation. These three enzymes may contribute to the regulation of carbon flux through the near equilibrium reactions of the non-oxidative pentose phosphate pathway in vivo. The phosphotransferase enzyme of the L-type pentose pathway is also able to catalyse the interconversion of sedoheptulose mono and bisphosphates via D-glycero D-ido octulose-P.     

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.     

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.     

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.     

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.     

Metabolism of Specifically Labeled Glucose, Glucose 1-Phosphate, and Glucose 6-Phosphate via the Oxidative Pentose Phosphate Cycle in a Reconstituted Spinach Chloroplast System in Darkness and in the Light

Using isolated spinach (hybrid 424) chloroplasts deprived of their envelopes (reconstituted chloroplast system), the metabolism of glucose, glucose 1-phosphate, and glucose 6-phosphate via the oxidative pentose phosphate cycle was analyzed. The activity of oxidative pentose phosphate cycle was monitored by continuous sampling of the CO₂ released during the decarboxylation process of 6-phosphogluconate.     

Alternative Pathways of Glucose Utilization in Brain: Changes in the Pattern of Glucose Utilization and of the Response of the Pentose Phosphate Pathway to 5-Hydroxytryptamine During Aging

Abstract: The oxidation of differentially labelled glucose, pyruvate and glutamate in brain slices from rats aged 20 days to 26 months has been studied and the partition of the glucose used into the glycolytic-tricarboxylic acid cycle pathway, the pentose phosphate pathway and the glutamate-GABA shunt has been calculated. Over the time range 4 to 26 months, there is an approximately 20% decrease in the production of CO₂ via the glycolytic-tricarboxylic acid cycle route, as there is in the rate of glucose phosphorylation. The glutamate-GABA pathway falls by about 50% over this same time span. The broad activity of the pentose phosphate pathway falls rapidly and cannot be detected in the brains of rats aged 18 months or more, whereas the fully stimulated pathway, i.e. in the presence of the artificial electron acceptor phen-azine methosulphate, declines only marginally over this period, falling sharply only after 23 months. The pentose phosphate pathway is stimulated by the presence of 5-hy-droxytryptamine and this stimulation appears to increase with age.     

The NADPH-producing pathways (pentose phosphate and malic enzyme) are regulated by the NADPH consumption in rat mammary gland

We have studied the changes in the activity of the pentose phosphate cycle and the malic enzyme produced by the activation or inhibition of different NADPH-consuming pathways. Kynurenate, an acetyl-CoA-carboxylase inhibitor produced a decrease in the flux through the NADPH-producing pathways pentose phosphate cycle and malic enzyme. Acini (isolated from mammary gland) incubated in the presence of ter-butyl-hydroperoxide, a compound which is metabolized via a NADPH-consuming pathway, showed a substantial increase in the pentose phosphate cycle and the malic enzyme pathways.     

Importance of the pentose phosphate pathway for d-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis

d-Glucose catabolism of a phosphofructokinase-deficient yeast Rhodotorula gracilis has been studied. By using d-glucose specifically ¹⁴C-labelled at different positions and measuring the distribution of the label in various fractions of cell metabolism, the following results were found. 1. The pentose phosphate pathway, being the main pathway of d-glucose catabolism, simultaneously converts glucose molecules into pentose phosphates oxidatively by using two NADP-linked dehydrogenases and via the non-oxidative transketolase–transaldolase pathway. 2. From the correlation of the ¹⁴CO₂ liberation and the d-glucose consumption and from the fact that the pentose phosphate moiety in nucleic acids is almost equally labelled from d-[1-¹⁴C]- and d-[6-¹⁴C]-glucose, it is concluded that of the glucose utilized about 80% undergoes transformation via the non-oxidative pentose phosphate pathway. Only about 20% of glucose is directly decarboxylated to pentose phosphate. 3. For further degradation it is postulated that the pentose phosphates are split into C₂ fragments and glyceraldehyde 3-phosphates. 4. All three loci of oxidative decarboxylation appear to be effective in Rh. gracilis, the oxidative part of the pentose phosphate pathway, the decarboxylation of pyruvate in the later part of the glycolytic pathway as well as the oxidation in the tricarboxylic acid cycle. 5. d-Glucose molecules taken up are only partially oxidized to CO₂: about four-fifths of each glucose molecule metabolized is incorporated into cell constituents. 6. The quantitative interrelations of the fluxes of d-glucose subunits along the catabolic pathways have been estimated and are discussed.     

Pentose cycle and reducing equivalents in rat mammary-gland slices

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with (14)C and in positions 1, 2, 3 and 6, and with (3)H in all six positions. Glucose carbon atoms are incorporated into CO(2), fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of (3)H into fatty acids is greatest from [3-(3)H]glucose. (3)H from [5-(3)H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-(14)C]- and [6-(14)C]-glucose was less, and that from [2-(14)C]- and [3-(14)C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from (14)C yields in CO(2) and fatty acids, and from the degradation of galactose from [2-(14)C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-(14)C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10-20% is converted into lactose, 20-30% is metabolized via the pentose cycle and the rest is metabolized via the Embden-Meyerhof pathway. About 10-15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80-100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Comparative effects of insulin and proinsulin in vitro on pathways of glucose utilization and lipid synthesis in the lactating rat mammary gland

The effects of insulin and proinsulin have been measured on the rates of glucose oxidation via the pentose phosphate pathway and incorporation into lipid in slices of lactating rat mammary gland. Half-maximal stimulation of glucose oxidation was observed with 1-3 x 10(-8)M insulin while 1 x 10(-7)M proinsulin was required to achieve half-maximal stimulation. A similar, approximately 10-fold, difference in potency was observed in relation to lipid synthesis. The present results appear to indicate that the maximum stimulation of either glucose oxidation via the pentose phosphate pathway or lipid synthesis by proinsulin did not reach the same level as that found for insulin.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

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.     

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.     

The pentose phosphate cycle is regulated by NADPH/NADP ratio in rat liver

The changes in the activity of the pentose phosphate cycle produced by the activation or inhibition of different NADPH-consuming pathways have been studied. The inhibition of fatty acid synthesis by kynurenate produced to the same extent, inhibition of the pentose phosphate cycle activity and an increase (about twofold) in the NADPH/NADP ratio. The addition of ter-butyl-hydroperoxide or paraquat, which is metabolized via NADPH-consuming pathways, produced the activation of the pentose phosphate cycle and a decrease in the NADPH/NADP ratio (about threefold). The plot of the NADPH/NADP ratio versus the pentose phosphate cycle activity gave a straight line with a regression index of 0.999. The regulation of the pentose phosphate cycle mainly by the intracellular NADPH/NADP ratio is discussed.