The role of the pentose phosphate shunt in glucose-induced insulin release: In vitro studies with 6-aminonicotinamide, methylene blue, NAD, NADH, NADP, NADPH and nicotinamide on isolated pancreatic rat islets

The possible role of the pentose phosphate shunt in insulin release was investigated in vitro with collagenase isolated pancreatic islets of rats. Parameters measured were insulin released into the medium and measured by an immunoassay and formation of ¹⁴CO₂ from glucose labeled either in the C-1 or C-6 position. The in vitro effect of the following substances was studied:

1. 1. 6-Aminonicotinamide, an antimetabolite in the synthesis of pyridine nucleotides. In islets of animals pretreated with 6-amino nicotinamide 6 h previously and in the presence of 3 mg/ml glucose in the incubation medium, 6-aminonicotinamide markedly reduced oxidation of [1-¹⁴C]glucose but did not affect that of glucose labeled in C-6. Concomitantly there was a marked decrease in insulin release. This action of 6-aminonicotinamide did not take place when it was added only to the incubation medium. Pretreatment with 6-aminonicotinamide did not change the insulin concentration of the islets, making it unlikely that it interfered with insulin synthesis. The effect of 6-aminonicotinamide is consistent with partial inhibition of the pentose shunt.

2. 2. Methylene blue: this agent was selected because it is known from studies with red blood cells that it will oxidize NADPH and thus stimulate activity of the pentose shunt. In concentrations of 0.5 and 2 μg/ml, methylene blue markedly stimulated oxidation of [1-¹⁴C]glucose but not that of C-6. Simultaneously there was a dose related decrease of insulin released.

3. 3. Pyridine nucleotides: in the absence of glucose only NADPH exhibited a significant effect of insulin release. If glucose (3 mg/ml) was present 1 or 10 mM of NAD⁺ or NADH exhibited a significant effect, NADP⁺ or NADPH were less effective. If the pentose shunt was blocked by pretreatment with 6-aminonicotinamide, all 4 pyridine nucleotides stimulated insulin release. Similarly there was an increase in oxidation of [1-¹⁴C]glucose, consitent with restimulation of the pentose shunt.

4. 4. Nicotinamide by itself exhibited a small effect; however, it was much less than the one produced by equimolar concentrations of the pyridine nucleotides.

Conclusion: Restricted availability of NADPH either less production or by fast removal leads to a decrease in glucose-induced insulin release. Pyridine nucleotides will restimulate 6-aminonicotinamide blockade insulin release and glucose oxidation by the pentose shunt. Recently it has been proposed by others that the polyol pathway may play a key role in insulin release, our data are consistent with such a hypothesis. Furthermore they do support a major role of the pentose shunt in insulin release.     

Alternative Pathways of Glucose Utilization in Brain; Changes in the Pattern of Glucose Utilization in Brain Resulting from Treatment of Rats with 6-Aminonicotinamide

The effect of 6-aminonicotinamide (6AN) treatment on the activities of alternative pathways of glucose metabolism in 20-day-old rat brain was evaluated by measurements of yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, or 6 and uniformly labeled glucose, and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. At the highest dose of 6AN used (35 mg/kg body weight) there was a significant decrease in the 14CO2 yields via the pentose phosphate pathway, the glycolytic route, tricarboxylic acid (TCA) cycle, and via the glutamate-gamma-aminobutyric acid pathway. Giving a graded series of doses (20-35 mg 6AN/kg body weight) revealed a hierarchy of responses in which the pentose phosphate pathway, lactate, glyceride-glycerol, and fatty acid formation were most sensitive, followed, in sequence, by the pyruvate dehydrogenase reaction, the glutamate-gamma-aminobutyrate route and, finally, the TCA cycle. The nature of the blocks in the various pathways was examined by the use of metabolite profiles.     

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.     

Synergistic activation of 2-deoxy-D-glucose uptake in rat and murine peritoneal macrophages by human macrophage colony-stimulating factor-stimulated coupling between transport and hexokinase activity and phorbol-dependent stimulation of pentose phosphate-shunt activity.

1. Transport and accumulation of 2-deoxy-D-glucose (2dGlc) in rat and murine peritoneal macrophages were investigated by using C-1-3H-labelled and C-2,6-3H-labelled 2dGlc. 2. There was active accumulation of both C-1- and C-2,6-labelled 2dGlc by quiescent rat and murine macrophages via a phloretin-inhibitable transport system. 3. The rate of uptake and accumulation of 2dGlc (C-1 label) was increased by exposure to human macrophage colony-stimulating factor (mCSF-1) (1000 units/ml) in both murine and rat macrophages. This indicates that mCSF-1 enhances coupling between hexokinase activity and glucose transport at the endofacial surface of the transporter. 4. Phorbol 12-myristate 13-acetate ('phorbol') at 40 nM stimulated 2dGlc in rat macrophages entirely by increasing the C-2,6 label uptake. This indicates that phorbol stimulates 2dGlc uptake mainly by increasing the activity of the pentose phosphate pathway. 5. Simultaneous exposure to phorbol and mCSF-1 stimulates 2dGlc uptake to a greater extent than found with either phorbol or mCSF-1 alone. This result is explained by a simultaneous enhancement of pentose phosphate-pathway activity and of hexokinase activity acting at the endofacial surface of the cell membrane. The dual activation of these serial processes coupled to the loss of the reaction products of the pentose phosphate-shunt pathway from the cells in the form of reactive oxygen intermediates, protons and CO2 could explain the synergistic action of phorbol and mCSF-1 in activation of sugar transport in macrophages.     

Direct Measurement of in Vivo Flux Differences between Electrophoretic Variants of G6pd from Drosophila Melanogaster

Demonstrating that naturally occurring enzyme polymorphisms significantly impact metabolic pathway flux is a fundamental step in examining the possible adaptive significance of such polymorphisms. In earlier studies of the glucose-6-phosphate dehydrogenase (G6PD) polymorphism in Drosophila melanogaster, we used two different methods, exploiting both genotype-dependent interactions with the 6Pgd locus, and conventional steady-state kinetics to examine activity differences between the two common allozymes. In this report we use 1-14C- and 6-14C-labeled glucose to estimate directly genotype-dependent flux differences through the pentose shunt. Our results show that G6pdA genotype possesses statistically lower pentose shunt flux than G6pdB at 25 degrees. We estimate this to be about a 32% reduction, which is consistent with the two former studies. These results reflect a significant responsiveness of pentose shunt flux to activity variation at the G6PD-catalyzed step, and predict that the G6PD allozymes generate a polymorphism for pentose shunt flux.     

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

Formation of a pentose phosphate cycle metabolite, erythrose-4-phosphate, from initial compounds of glycolysis by transketolase from the rat liver

Using ion-exchange chromatography of sucrose phosphates on Dowex-1, it was demonstrated that the highly purified rat liver transketolase (specific activity 1.7 mumol/min.mg protein) is capable of catalyzing the synthesis of erythrose-4-phosphate, a metabolite of the pentose phosphate pathway non-oxidizing step, from the initial participants of glycolysis, i. e., glucose-6-phosphate and fructose-6-phosphate. As can be evidenced from the reaction course, the second product of this synthesis is octulose-8-phosphate. The reaction was assayed by accumulation of erythrose-4-phosphate. The soluble fraction from rat liver catalyzes under identical conditions the synthesis of heptulose-7-phosphate (but not erythrose-4-phosphate), which points to the utilization of the erythrose-4-phosphate formed in the course of the transketolase reaction by transaldolase which is also present in the soluble fraction. The role of the transketolase reaction reversal from the synthesis of pentose phosphate derivatives to glycolytic products is discussed. The transketolase reaction provides for the relationship between glycolysis and the anaerobic step of the pentose phosphate pathway which share common metabolites, i. e. glucose-6-phosphate and fructose-6-phosphate.     

A New Type of Hexose Monophosphate Shunt in Chlorella sorokiniana: CO(2) Release from Differentially Labeled Glucose

Using differentially labeled glucose as a substrate to probe the operation of the hexose monophosphate shunt (pentose cycle) in Chlorella sorokiniana, we found that the labeling patterns for the release of ¹⁴CO₂ over the first 5 minutes are compatible with the operation of the recently described L-type pentose shunt. Experimentally, this L-type differs from the F-type or `textbook' variety in that no radioactivity is obtained from C-2 labeled glucose, and the small amount derived from C-6 labeled glucose is due to a second pass of the glucose molecule (derived from the pentose cycle) through the pentose cycle.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

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.     

The pentose cycle (hexose monophosphate shunt). Rigorous evaluation of limits to the flux from glucose using 14CO2 data, with applications to peripheral ganglia of chicken embryos

The difference between the 14CO2 outputs from [1-14C]glucose and [6-14C]glucose has frequently been used as a measure of activity in the hexose monophosphate shunt without considering the exact significance of this difference. Assuming only 1) that all C-1 of glucose is released to CO2 on entry to the shunt and 2) that the shunt provides the only mechanism for increasing C-1 of glucose over C-6 of glucose in CO2, it is very simply shown that the flux from glucose to the shunt is not less than the difference between the 14CO2 outputs at any time after adding labeled glucose nor more than the steady-state output of 14CO2 from [1-14C]glucose. Moreover, absence of a 14CO2 difference does not prove that the shunt is absent or inactive. The value for the minimum flux rate can be maximized by following the time course of the C-1 - C-6 difference in 14CO2 during the transient phase before isotopic equilibration is complete, but useful values can be obtained when the time course is not available. The above relationships are applicable to gluconeogenic as well as non-gluconeogenic tissues. Applications of these relationships to peripheral ganglia from chicken embryos, in which the 14CO2 difference passes through a maximum during incubation, show that 27-37% of the glucose taken up enters the pentose cycle in sympathetic ganglia from 10-day-old embryos, while 17-36% enters the cycle in 15-day-old dorsal root ganglia.     

Early and late changes in the metabolic pattern of the working myocardial fibres and Purkinje fibres of the human heart under ischaemic and inflammatory conditions: An enzyme histochemical study

Summary In this communication, the results of an enzyme histochemical study on the working myocardial fibres and Purkinje fibres of the atrioventricular conducting system of the human heart under ischaemic and inflammatory conditions are presented. The material was selected from patients showing changes which could be classified in three major groups: (1) early changes due to acute ischaemia either in the myocardial fibres or in the conducting system or in both; (2) chronic ischaemic changes due to cardiovascular insufficiency, such as old infarction, or coronary arteriosclerosis or both; and (3) inflammatory conditions such as myocarditis.The activity and location of about 20 enzymes that play a role in the aerobic and anaerobic pathways of energy metabolism were examined. The activity and location of some hydrolytic enzymes and the glycogen and lipid content were also studied.The most important findings were an obvious depletion of the glycogen reserves under acute ischaemic changes in both types of fibre. This was associated with a transient or permanent reduction in activity of many enzymes that play a role in aerobic and anaerobic metabolism. Further, there was an instantaneous and persistent increase in the activity of the NADPH-regenerating enzymes of the pentose phosphate pathway and of glyceraldehyde-3-phosphate dehydrogenase, the rate-limiting enzyme of glycolysis under ischaemic conditions. Chronic ischaemic changes were characterized by a gradual long-term increase in the activity of many anaerobic glycolytic enzymes. Moreover, there was an absence of activity of acetylcholine esterase immediately after the onset of infarction in the fibres of the conducting system. Lastly, a slight increase in lipid content was found in the hypertrophic chronic ischaemic fibres and in old infarcted areas. Cardiac fibres in inflamed areas showed a marked increased activity of the pentose phosphate shunt enzymes and a less pronounced increased activity of most anaerobic and hydrolytic enzymes. In contrast to the cardiac fibres in infarcted areas, the fibres in inflamed areas did not reveal a decrease or absence of activity of aerobic enzymes such as succinate dehydrogenase.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Use of [2-14C]glucose and [5-14C]glucose for evaluating the mechanism and quantitative significance of the ''liver-cell'' pentose cycle.

1. Expressions are derived for the steady-state measurement of the quantitative contribution of the liver-type pentose phosphate cycle to glucose metabolism by tissues. One method requires the metabolism of [5-14C]glucose followed by the isolation and degradation of glucose 6-phosphate. The second procedure involves the metabolism of [2-14C]glucose and the isolation and degradation of a triose phosphate derivative, usually lactate or glycerol. 2. Measurements of 14C in C-2 and C-5 of glucose 6-phosphate are required and the values of the C-2/C-5 ratios can be used to calculate the quantitative contribution of the L-type pentose cycle in all tissues. 3. The measurement of 14C in C-1, C-2 and C-3 of triose phosphate derivatives can be used to calculate the quantitative contribution of the L-type pentose cycle relative to glycolysis. 4. The effect of transaldolase and transketolase exchange reactions, reactions of gluconeogenesis and non-oxidative formation of pentose 5-phosphate, isotopic equilibration of triose phosphate pools and isotopic equilibration of fructose 6-phosphate and glucose 6-phosphate, which could interfere with a clear interpretation of the data using [2-14C]- and [5-14C]glucose are discussed.     

Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc.     

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.     

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate.

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and 'futile' cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.     

Therapeutic Potential of Riboflavin,Niacin and Ascorbic Acid on Carbohydrate Metabolizing Enzymes in Secondary Endometrial Carcinoma Bearing Rats

Curative potential of riboflavin, niacin and ascorbic acid against tamoxifen mediated endometrial carcinoma was established by studies on carbohydrate metabolizing enzymes. The enzymes investigated were glycolytic enzymes namely, hexokinase; aldolase; phosphoglucoisomerase and the gluconeogenic enzymes namely, glucose-6-phosphatase and fructose-1, 6-biphosphatase in endometrial carcinoma bearing rats. A significant increase in glycolytic enzymes and a subsequent decrease in gluconeogenic enzymes were observed in plasma, liver and kidney of endometrial carcinoma animals. The administration of riboflavin (45 mg/kg bw/day), niacin (100 mg/kg bw/day) and ascorbic acid (200 mg/kg bw/day) along with tamoxifen (45 mg/kg bw/day) caused a significant decrease in the activity of glycolytic enzymes and a significant increase in the activities of gluconeogenic enzymes to near normal levels in experimental animals. Our results suggest that riboflavin, niacin and ascorbic acid have potential combination therapy against tamoxifen mediated secondary endometrial carcinoma in experimental rats. However, there were no deleterious side effects observed in combinants alone treated animals.     

The activity of the pentose phosphate pathway in isolated liver cells

Isolated liver cells have been used to assess the relative contribution of the pentose phosphate pathway to glucose metabolism. The incorporation of carbon from specifically labelled glucose into ¹⁴CO₂ by isolated cells gave values (μg.atoms/g.cells/hr) of: C-1, 7.9; C-6, 1.3; C-U, 3.4. The corresponding figures for liver slices were: C-1, 2.3; C-6, 1.6; C-U, 3.0. The most striking difference was the 3.5-fold increase in the oxidation of C-1 of glucose. Isolated cells retain more than 50% of ATP and have a content of intermediates of the glycolytic pathway closely similar to freeze-clamped liver. The relative importance of the pentose phosphate pathway in isolated liver cells, approximately 16% of glucose catabolised, is consistent with the enzyme profile of liver and the reductive synthetic reactions of the tissue.