Possible regulatory interactions between compartmentalized glycolytic systems during initiation of glycolylsis in ascites tumor cells

Changes were measured in the rates of respiration and in the levels of glycolytic intermediates during the first 5 min after addition of 1.6 mM glucose to a suspension (5%, v/v) of respiring Ehrlich ascites carcinoma cells incubated in an isotonic 50 mM tris(hydroxymethyl)methylglycine buffer (pH 7.4) at 38 °C. The rates of accumulation of lactate and glycolytic intermediates were used to calculate the in vitro velocities of glycolytic enzymes.The initial velocities of hexokinase (EC 2.7.1.1), fructose-6-phosphate kinase (EC 2.7.1.11) and lactate dehydrogenase (EC 1.1.1.27) in μmoles glucose equivalents/ ml cells per min were 14, 11 and 4, respectively. The velocities of the two kinases fell sharply to less than 5 between 5 and 10 s, while the velocity of the dehydrogenase declined gradually over the first minute. The initial burst of activity in the kinases, which lasted for about 8 s, was associated with a rapid accumulation of phosphate ester and a negative net ATP generation by glycolysis. The accumulation of phosphate ester is almost exactly matched by the generation of ATP by the “tail end” of glycolysis (triose-P to lactate) in this period. After this time (10–25 s) the rate of oxidative phosphorylation calculated as six times the rate of O₂ consumption, is nearly identical to the combined rate of ATP utilization by hexokinase and fructose-6-phosphate kinase. As observed previously, oxamate (42 mM) blocked lactate dehydrogenase but did not depress the rate of phosphate ester accumulation.These various observations and correlations can be interpreted in terms of a dual glycolytic system. The accumulation of phosphate ester during the first 8 s is attributed to the operation of a partial glycolytic system, System B, which includes only the first three or four enzymes of glycolysis, and which draws upon an ATP pool (Pool I) previously employed in assorted cytoplasmic phosphorylations. The ADP generated by System B is rephosphorylated by and regulates the rate of a complete glycolytic system A, which converts glucose to lactate with little intermediate accumulation. The tail end of System A generates a new pool of ATP (Pool II) and controls the rate of glucose input through its head end, which is supplied by ATP being produced by oxidative phosphorylation. This scheme of interlocking controls is transient and alters after 8 s, when System B slows to a stop.     

Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis

The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K+ channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K+ channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (with the mitochondrial inhibitor FCCP present), mitochondrial oxidative phosphorylation, or the creatine kinase system. In the presence of an exogenous ATP-consuming system, however, glycolytic substrates (with FCCP present) were superior to substrates for either oxidative phosphorylation or the creatine kinase system at suppressing ATP-sensitive K+ channels. All three groups of substrates were equally effective at preventing cell shortening. In 6 of 38 excised inside-out membrane patches, ATP-sensitive K+ channels activated by removing ATP from the bath were suppressed by a complete set of substrates for the ATP-producing steps of glycolysis but not by individual glycolytic substrates, which is consistent with the presence of key glycolytic enzymes located near the channels in these patches. Under whole-cell voltage-clamp conditions, inclusion of 15 mM ATP in the patch electrode solution dialyzing the interior of the cell did not prevent activation of the ATP-sensitive K+ current under control conditions or during exposure to complete metabolic inhibition. In isolated arterially perfused rabbit interventricular septa, selective inhibition of glycolysis caused an immediate increase in 42K+ efflux rate, which was prevented by 100 microM glyburide, a known blocker of ATP-sensitive K+ channels. These observations suggest that key glycolytic enzymes are associated with cardiac. ATP-sensitive K+ channels and under conditions in which intracellular competition for ATP is high (e.g., in beating heart) that act as a preferential source of ATP for these channels.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

The effect of potassium depletion on the initial kinetics of glycolysis in ascites tumor cells

Ehrlich ascites carcinoma cells depleted of K⁺ and provided with 5.5 mM K⁺ in isosmotic 50 mM tris(hydroxymethyl)methylglycine buffer at pH 7.4 and 38 °C take up K⁺ from the medium at a rate of 6 μmoles/ml intracellular fluid per min. Depleted cells exposed to K⁺ for 2 min prior to glucose addition exhibit a higher initial rate of glycolysis, a lower glycose-6-P accumulation, and a higher fructose-1,6-P₂ accumulation than depleted cells incubated in a K⁺-free medium. Both the K⁺ transport and the effect of K⁺ on glycolysis are blocked by 2 mM oubain.Calculation of thein vitro velocities of glycolytic enzymes from the rates of accumulation of lactate and glycolytic intermediates shows that the presence of K⁺ accelerates the velocities of fructose-6-phosphate kinase and lactate dehydrogenase about 2-fold and the velocity of hexokinase about 1.5-fold during the first 15 s. In either the presence or absence of K⁺, the hexokinase velocity is highest immediately after glucose addition and declines sharply with time; this decline is greater than would be predicted by product inhibition by the accumulated glucose-6-P. The maximal stimulation of fructose-6-phosphate kinase attibutable to the increasing intarcellular K⁺ concentration is only 1.25-fold. These observations indicate that the initial acceleration in glycolysis is not simply mediated through a direct K⁺ activation of fructose-6-phosphate kinase.The calculated theoretical rate of ATP generation by glycolysis shows that glycolysis is an ATP-utilizing system for the first 5–10 s both in the presence and in the absence of K⁺. Hence, the initial stimulation of glycolysis by K⁺ is not a consequence of an increased rate of ATP hydrolysis associated with K⁺ transport, although this mechanism may be responsible for the stimulation of steady-state glycolysis.The initial rate of phosphate ester (hexose and triose phosphates) accumulation corresponds to be rate of ATP generation by the “tail-end” of glycolysis, or twice the rate of lactate accumulation, in either the absence or presence of K⁺, but both the rate and the maximal level of ester accumulated are higher in the presence of K⁺. This implies that the oxidatively generated pool of ATP which is diverted from endogenous reactions to hexokinase and fructose-6-phosphate kinase on the introduction of glucose is larger in the presence of K⁺.Valinomycin (0.27 μM) under certain conditions can produce effects on the glycolysis of non-depleted cells which superficially resemble the effects of K⁺ on depleted cells. However, unlike K⁺, valinomycin stimulates the initial rate of glycolytic ATP generation, and abolishes the initial correspondence between the ATP generation by the “tail-end” of glycolysis and phosphate ester accumulation. These observations are interpreted to mean that valinomycin introduces an ATPase activity effective on glycolytically generated ATP.Comparison of the theoretical ATP generation in the presence and absence of K⁺ indicates that approximately one ATP is hydrolyzed for each K⁺ transported.     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Influence of Temperature on Glucose Metabolism of Round Spermatids of Rats

Glucose utilization by spermatids was found to be 17.37±0.37 nmoles/hr/10⁶ cells at 34°C and 28.94±1.12nmoles/hr/10⁶ cells at 40°C. A good parallelism was observed between the increased rate of glucose utilization and lactate production at 40°C. There was no significant change in the levels of glycolytic intermediates in the cells, except for marked accumulations of fructose-1, 6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in the presence of glucose (1 mM). Glucose oxidation in the citrate cycle by spermatids was higher at 40°C than at 34°C, but was never greater than 2% of the overall rate of glucose utilization. In addition, glucose did not prevent decrease of ATP at either 34 or 40°C. The effects of temperature on the activities of 11 glycolytic enzymes were examined. The activities of aldolase and phosphoglyceromutase were similar between 30 and 34°C, but increased markedly at 40°C. The higher temperature increased the V_max values, without affecting the Kms. The activities of other glycolytic enzymes were similar at the different temperatures. These findings indicate that the increased overall rate of glucose utilization in glycolysis at higher temperature is due to increased Vmax values of aldolase and phosphoglyceromutase.     

Mechanism of activation of glucocerebrosidase by co-beta-glucosidase (glucosidase activator protein)

Experiments with respiration deficient (rho-), ADP/ATP transport deficient (op1) and double (op1 rho-) mutants, with glycolytic and tricarboxylic acid cycle substrates showed that the substrate-induced acidification of yeast suspensions is closely associated with glycolysis. The glucose/proton stoichiometry is 2.5 : 1 to 4 : 1 depending on glucose concentration. The kinetics of the process are complex, the acidification curve having a very fast initial component and two slower exponential components. The first component suggests an initial proton efflux from endogenous sources, triggered by exogenous substrates. The acidification process exhibits two Km values at about 1 and 15 mM D-glucose, indicating two distinct saturable pathways of proton extrusion. The total extent of acidification and thus the final pHout reaches a saturation value with increasing glucose concentration and suspension density. Both the total extent and the rate of acidification are subject to control by extracellular pH which reflects the tendency of the cells to build a fixed [H+]out/[H+]in ratio. When the control is lifted, both quantities are considerably increased. A crucial role in the substrate-induced acidification is thus played by active membrane processes and their control mechanisms.     

Study on ATP-generating system and related hexokinase activity in mitochondria isolated from undifferentiated or differentiated HT29 adenocarcinoma cells

The functional properties of mitochondria bound hexokinase are compared in two subpopulations of the HT29 human colon cancer cell-line: (1) the HT29 Glc+ cells, cultured in the presence of glucose, which are poorly differentiated and highly glycolytic and (2) the HT29 Glc- cells, adapted to grow in a glucose-free medium, which are 'enterocyte-like' differentiated and less glycolytic when given glucose (Zweibaum et al. (1985) J. Cell Physiol. 122, 21-28). The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase are found to be twice as high in Glc+ cells when compared to Glc- cells. Besides, the respiration rate is decreased in Glc+ cells compared to Glc- cells. These results correlate with the higher glycolytic rate in Glc+ cells. In many tissues, it has been shown that the binding of hexokinase to the mitochondrial outer membrane allows a preferential utilization of the ATP generated by oxidative phosphorylation which, in turn, is activated by immediate restitution of ADP. In highly glycolytic cancer cells, although a large fraction of hexokinase is bound to the mitochondria, the existence of such a channeling of nucleotides is still poorly documented. The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from both Glc+ and Glc- cells either with exogenous ATP or with ATP generated by mitochondria supplied with ADP and succinate (endogenous ATP). Diadenosine pentaphosphate (Ado2P5), oligomycin and carboxyatractyloside (CAT) were used in combination or separately as metabolic inhibitors of adenylate kinase, ATP synthase and ATP/ADP translocator, respectively. Exogenous ATP appears to be 6.5-times more efficient than endogenous ATP in supporting hexokinase activity in the mitochondria from Glc+ cells and only 1.8-times cells. The rate of oxidative phosphorylation being higher in mitochondria from Glc- cells, hexokinase activity is higher in this model when ATP is generated by respiration. Furthermore, in Glc+ mitochondria, the adenylate kinase reaction appears to be an important source of endogenous ATP for bound hexokinase, while, in Glc- mitochondria, hexokinase activity is almost totally dependent on the ATP generated by oxidative phosphorylation. This result might be explained by our previous finding that mitochondria from Glc+ cells lack contact sites between outer and inner membrane, whereas numerous contacts were observed in mitochondria from Glc- cells (Denis-Pouxviel et al. (1987) Biochim. Biophys. Acta 902, 335-348).     

Endogenous regulation of 2-deoxyglucose uptake in C6 glioma cells correlates with cytoskeleton-mediated changes of surface morphology

The cellular basis of the membrane-limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time-dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2-deoxyglucose (2-DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F-actin content. Because the changes of 2-DG uptake rates occurred independent of the intracellular 2-DG concentration, the bulk of this 2-DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2-DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton-mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.     

A stage in glycolysis controls the metabolic adjustments of vertebrate rod photoreceptors upon illumination

The factors affecting the metabolic adjustments of toad rod photoreceptors were studied by monitoring the oxygen utilization of excised retinas and by measuring rod outer segment ATP and GTP concentrations. Respiratory adjustments upon illumination were observed when glucose or fructose was provided in the perfusate, but not when a glycolytic inhibitor was added to the perfusate containing glucose and pyruvate, or when a substrate beyond glycolysis or from a later stage of glycolysis was substituted for glucose. The amplitudes of the respiratory adjustments to illumination were dependent on the concentration of glucose in the perfusate. The ATP and GTP concentration changes were dependent on respiratory adjustments, including glycolytic effects, and on the levels of illumination. The data suggest a control point within glycolysis for light-induced adjustments of respiration, possibly at phosphofructokinase.     

The role of glucose,pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous l-lactate (3–6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.     

Energy metabolism of cultured TM4 cells and the action of gossypol

The energy metabolism of cultured TM4 cells, a cell line originally derived from mouse testicular cells, has been studied in relation to the action of gossypol. In the absence of externally added substrates, TM4 cells consumed oxygen at 37 +/- 5 nmoles O2 X mg protein-1 X h-1. Pyruvate stimulated oxygen consumption in a dose-dependent fashion up to 23%. Addition of glucose to the cells suspended in substrate-free medium inhibited oxygen consumption. At 5.5 mM glucose, the inhibition of oxygen consumption was 45 +/- 9%. The rate of aerobic lactate production from endogenous substrates was less than 7 nmoles lactate X mg protein-1 X h-1, even in the presence of optimal concentrations of the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone. The rate of aerobic lactate production was 920 +/- 197 nmoles X mg protein-1 X h-1 at external glucose concentrations of 2 mM or greater. The formation of aerobic glycolytic adenosine triphosphate (ATP) in 5 mM glucose comprised about 80% of the total ATP production. Gossypol stimulated both aerobic lactate production and oxygen consumption of the transformed testicular cells in a dose-dependent manner. The effect of gossypol on glucose transport, aerobic lactate production, and oxygen consumption is consistent with the hypothesis that gossypol modifies energy metabolism in these cells mainly by partially uncoupling mitochondrial oxidative phosphorylation. The possible impairment of cell and tissue function under gossypol treatment would depend on the metabolic properties of each specific differentiated cell.     

Substrate oxidation and ATP supply in AS-30D hepatoma cells

The oxidation of several metabolites in AS-30D tumor cells was determined. Glucose and glycogen consumption and lactic acid production showed high rates, indicating a high glycolytic activity. The utilization of ketone bodies, oxidation of endogenous glutamate, and oxidative phosphorylation were also very active: tumor cells showed a high respiration rate (100 ng atoms oxygen (min x 10(7) cells)(-1)), which was 90% oligomycin-sensitive. AS-30D tumor cells underwent significant intracellular volume changes, which preserved high concentrations of several metabolites. A high O(2) concentration, but a low glucose concentration were found in the cell-free ascites liquid. Glutamine was the oxidizable substrate found at the highest concentration in the ascites liquid. We estimated that cellular ATP was mainly provided by oxidative phosphorylation. These data indicated that AS-30D hepatoma cells had a predominantly oxidative and not a glycolytic type of metabolism. The NADH-ubiquinol oxido reductase and the enzyme block for ATP utilization were the sites that exerted most of the control of oxidative phosphorylation (flux control coefficient = 0.3-0.42).     

Regulatory properties of human erythrocyte hexokinase during cell ageing

Human red blood cell hexokinase exists in multiple molecular forms with different isoelectric points but similar kinetic and regulatory properties. All three major isoenzymes (HK Ia, Ib, and Ic) are inhibited competitively with respect to Mg.ATP by glucose 6-phosphate (Ki = 15 microM), glucose 1,6-diphosphate (Ki - 22 microM), 2,3-diphosphoglycerate (Ki = 4 mM), ATP (Ki = 1.5 mM), and reduced glutathione (Ki = 3 mM). All these compounds are present in the human erythrocyte at concentrations able to modify the hexokinase reaction velocity. However, the oxygenation state of hemoglobin significantly modifies their free concentrations and the formation of the Mg complexes. The calculated rate of glucose phosphorylation, in the presence of the mentioned compounds, is practically identical to the measured rate of glucose utilization by intact erythrocytes (1.43 +/- 0.15 mumol h-1 ml red blood cells-1). Hexokinase in young red blood cells is fivefold higher when compared with the old ones, but the concentration of many inhibitors of the enzyme is also cell age-dependent. Glucose 6-phosphate, glucose 1,6-diphosphate, 2,3-diphosphoglycerate, ATP, and Mg all decay during cell ageing but at different rates. The free concentrations and the hemoglobin and Mg complexes of both ATP and 2,3-diphosphoglycerate with hemoglobin in the oxy and deoxy forms have been calculated. This information was utilized in the calculation of glucose phosphorylation rate during cell ageing. The results obtained agree with the measured glycolytic rates and suggest that the decay of hexokinase during cell ageing could play a critical role in the process of cell senescence and destruction.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines,insect and primary liver cells

Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodoptera frugiperda) cell line using radioactive substrates. A high degree of similarity in many aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always close to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucose- or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/or productivity. © 1996 Wiley-Liss, Inc.     

Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions.

Anaerobic and aerobic chemostat cultures of Saccharomyces cerevisiae were performed at a constant dilution rate of 0.10 h(-1). The glucose concentration was kept constant, whereas the nitrogen concentration was gradually decreasing; i.e., the conditions were changed from glucose and energy limitation to nitrogen limitation and energy excess. This experimental setup enabled the glycolytic rate to be separated from the growth rate. There was an extensive uncoupling between anabolic energy requirements and catabolic energy production when the energy source was present in excess both aerobically and anaerobically. To increase the catabolic activity even further, experiments were carried out in the presence of 5 mM acetic acid or benzoic acid. However, there was almost no effect with acetate addition, whereas both respiratory (aerobically) and fermentative activities were elevated in the presence of benzoic acid. There was a strong negative correlation between glycolytic flux and intracellular ATP content; i.e., the higher the ATP content, the lower the rate of glycolysis. No correlation could be found with the other nucleotides tested (ADP, GTP, and UTP) or with the ATP/ADP ratio. Furthermore, a higher rate of glycolysis was not accompanied by an increasing level of glycolytic enzymes. On the contrary, the glycolytic enzymes decreased with increasing flux. The most pronounced reduction was obtained for HXK2 and ENO1. There was also a correlation between the extent of carbohydrate accumulation and glycolytic flux. A high accumulation was obtained at low glycolytic rates under glucose limitation, whereas nitrogen limitation during conditions of excess carbon and energy resulted in more or less complete depletion of intracellular storage carbohydrates irrespective of anaerobic or aerobic conditions. However, there was one difference in that glycogen dominated anaerobically whereas under aerobic conditions, trehalose was the major carbohydrate accumulated. Possible mechanisms which may explain the strong correlation between glycolytic flux, storage carbohydrate accumulation, and ATP concentrations are discussed.     

The tricarboxylic acid cycle and glycolysis in relation to ion transport by the ciliary body

1. The respiration and aerobic glycolysis of pig ciliary processes in oxygenated phosphate and bicarbonate buffers have been investigated. 2. Significant amounts of lactic acid are produced only in the presence of added glucose, but this does not change the endogenous respiration rate. 3. Succinate and citrate increase the oxygen uptake considerably, but pyruvate has almost no effect; oxaloacetate and fumarate stimulate slightly in the presence of glucose. Aspartate and fumarate together stimulate pyruvate utilization and are oxidized as fast as citrate. 4. Ouabain inhibits the oxidation of glucose and other substrates by limiting the ADP supply from the sodium transport system. Cyanide and azide inhibit respiration and stimulate glycolysis. 5. The transport mechanism depends largely on ATP from oxidative phosphorylation and regulates the rate of respiration and glycolysis by controlling ADP production from the Na(+)-K(+)-activated adenosine triphosphatase.