Measurement of flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol and glycerol in rat heart and epididymal adipose tissue: Effects of insulin, adrenaline and alloxan-diabetes

1. Flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol, glyceride fatty acids and glycerol was calculated in the perfused rat heart and incubated epididymal adipose tissue from the incorporation of (14)C from [U-(14)C]-glucose (into glyceride glycerol, glyceride fatty acids and glycerol in the medium), and from measurements of the specific activity of l-glycerol 3-phosphate, and the effects of insulin, adrenaline and alloxan-diabetes were studied. Measurements were also made of the uptake of glucose and the outputs of lactate, pyruvate and glycerol. 2. New methods are described for the measurement of radioactivity in small amounts of metabolites (glycerol, glucose 6-phosphate and fructose 6-phosphate and l-glycerol 3-phosphate) in which use has been made of alterations in charge induced by enzymic conversions to effect resolution by ion-exchange chromatography. 3. In hearts the specific activity of l-glycerol 3-phosphate was less than that of glucose in the medium but similar to that of lactate released during perfusion. Because repeated measurements of the specific activity of l-glycerol 3-phosphate was impracticable, the specific activity of lactate has been used as an indirect measurement of glycerol phosphate specific activity. 4. In fat pads, specific activity of lactate was the same as that of glucose in the medium and thus the specific activity of l-glycerol 3-phosphate was taken to be the same as that of medium glucose. 5. In hearts from alloxan-diabetic rats, despite decreased glucose uptake and l-glycerol 3-phosphate concentration, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased about threefold. 6. In fat pads, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased by insulin (twofold), by adrenaline in the presence of insulin (fivefold) and by diabetes in pads incubated with insulin (1.5-fold). These increases could not be correlated either with increases in glucose uptake, which was unchanged by adrenaline and decreased in diabetes, or with the concentration of l-glycerol 3-phosphate, which was decreased by adrenaline and unchanged in diabetes. 7. These results are discussed in relation to the control of glyceride synthesis in heart and adipose tissue and to the regulation of glyceride fatty acid oxidation in the perfused rat heart.     

Effect of experimental diabetes on glycolytic intermediates and regulation of phosphofructokinase in rat lens

The pattern of glycolytic intermediates in the lens of alloxan-diabetic rats was indicative of regulation at phosphofructokinase. The changes in metabolites influencing phosphofructokinase activity in the diabetic, relative to the normal, rat lens were: glucose 6-phosphate, 182%; fructose 6-phosphate, 107%; fructose diphosphate, 57%. There was also a marked decrease in phosphoenolpyruvate, pyruvate, lactate and ATP but no significant change in other triose phosphates or cyclic AMP. The resuts are considered in relation to the early changes in [Ca²⁺] known to occur in lens in diabetes and to the coordinating effect of fructose diphosphate on flux through the glycolytic route.     

Measurement of concentrations of metabolites in adipose tissue and effects of insulin, alloxan-diabetes and adrenaline

1. Methods are described for the extraction and assay of ATP, ADP, AMP, glucose 6-phosphate, l-glycerol 3-phosphate and citrate in rat epididymal adipose tissue incubated in vitro for 1hr. At this time of incubation rates of glucose uptake and outputs of glycerol, free fatty acids, lactate and pyruvate were shown to be constant. 2. In fat pads incubated in medium containing glucose (3mg./ml.) and albumin (20mg./ml.) the concentrations (in mmumoles/g. wet wt.) were: ATP, 70; ADP, 36; AMP, 9.0; glucose 6-phosphate, 3.0; l-glycerol 3-phosphate, 3.3; citrate, 8.1. 3. The volume of intracellular water calculated from ([(3)H]water space-[(14)C]sorbitol space), ([(14)C]urea space-inulin space) and (weight loss on drying-[(14)C]sorbitol space) was 1.4ml./100g. wet wt. of tissue. The intracellular volume was not changed by insulin, alloxan-diabetes or adrenaline. 4. When compared in terms of mumoles/ml. of intracellular water the concentration of ATP in adipose tissue was less than in heart and diaphragm muscles. The concentrations of ADP and AMP were greater both in absolute terms and relative to ATP. Insulin, alloxan-diabetes and adrenaline had no significant effects on the concentrations of the adenine nucleotides in adipose tissue. 5. The concentration of glucose 6-phosphate was increased by insulin and lowered by alloxan-diabetes and adrenaline. The concentration of l-glycerol 3-phosphate was increased by insulin, unchanged by alloxan-diabetes and lowered by adrenaline. The concentration of citrate was increased by adrenaline and alloxan-diabetes and unchanged by insulin. 6. The effect of glucose concentration in the medium on rates of glucose uptake in adipose tissue from normal rats and alloxan-diabetic rats was investigated. The K(u) of glucose uptake was 29-44mg./100ml. and the V(max.) was 0.77mg./g. wet wt. of tissue/hr. Insulin increased the V(max.) and alloxan-diabetes diminished it, but neither agent significantly altered the K(u). 7. The significance of these results in relation to control of metabolism of adipose tissue is discussed.     

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.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

Effect of experimental hypothyroidism on the control of 6-phosphofructo-1-kinase activity in rat jejunal mucosa.

Changes in the activity of 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) from the epithelial cells of rat small intestine during experimental hypothyroidism were studied. Hypothyroidism resulted in significant decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine, free thyroxine and insulin. These changes were associated with a significant increase in the plasma concentration of thyrotropin. The total activity and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)) of jejunal PFK of hypothyroid rats were significantly diminished as compared to control rats. PFK of hypothyroid rats was more sensitive to inhibition by ATP. The mucosal enzyme of both control and hypothyroid state was sensitive to stimulation by AMP and fructose 2,6-bisphosphate. It is concluded that during hypothyroidism the rate of glycolytic pathway in the small intestine is reduced as a result of a fall in glucose uptake, and the subsequent kinetic changes of PFK are primarily to maintain the concentrations of fructose 6-phosphate (and glucose 6-phosphate) during the reduced glycolytic flux. These changes in PFK activity may be caused by changes in plasma insulin concentrations, glucose utilization and fructose 2,6-bisphosphate concentrations.     

Control of glycolysis and gluconeogenesis in rat kidney cortex slices

1. Glucose uptake or glucose formation has been studied in kidney cortex slices to investigate metabolic control of phosphofructokinase and fructose-diphosphatase activities. 2. Glucose uptake is increased and glucose formation is decreased by anoxia, cyanide or an uncoupling agent. Under these conditions the intracellular concentrations of glucose 6-phosphate and ATP decreased whereas that of fructose diphosphate either increased or remained constant, and the concentrations of AMP and ADP increased. 3. Glucose uptake was decreased, and glucose formation from glycerol or dihydroxyacetone was increased, by the presence of ketone bodies or fatty acids, or after starvation of the donor animal. Under these conditions, the concentrations of glucose 6-phosphate and citrate were increased, whereas those of fructose diphosphate and the adenine nucleotides were unchanged (see also Newsholme & Underwood, 1966). 4. It is concluded that anoxia and cell poisons increase glucose uptake and decrease gluconeogenesis by stimulating phosphofructokinase and inhibiting fructose diphosphatase, whereas ketone bodies, fatty acids or starvation increase gluconeogenesis and decrease glucose uptake through the citrate inhibition of phosphofructokinase.     

Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate.

Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.     

Regulation of adult and fetal myocardial phosphofructokinase. Relief of cooperativity and competition between fructose 2,6-bisphosphate, ATP, and citrate

To clarify the physiological role of fructose 2,6-bisphosphate in the perinatal switching of myocardial fuels from carbohydrate to fatty acids, the kinetic effects of fructose 2,6-bisphosphate on phosphofructokinase purified from fetal and adult rat hearts were compared. For both enzymes at physiological pH and ATP concentrations, 1 microM fructose 2,6-bisphosphate induced a greater than 10-fold reduction in S0.5 for fructose 6-phosphate and it completely eliminated subunit cooperativity. Fructose 2,6-bisphosphate may thereby reduce the influence of changes in fructose 6-phosphate concentration on phosphofructokinase activity. Based on double-reciprocal plots and ATP inhibition studies, adult heart phosphofructokinase activity is more sensitive to physiological changes in ATP and citrate concentrations than to changes in fructose 2,6-bisphosphate concentrations. Fetal heart phosphofructokinase is less sensitive to ATP concentration above 5 mM and equally sensitive to citrate inhibition. The fetal enzyme has up to a 15-fold lower affinity for fructose 2,6-bisphosphate, rendering it more sensitive to changes in fructose 2,6-bisphosphate concentration than adult heart phosphofructokinase. Together, these factors allow greater phosphofructokinase activity in fetal heart while retaining sensitive metabolic control. In both fetal and adult heart, fructose 2,6-bisphosphate is primarily permissive: it abolishes subunit cooperativity and in its presence phosphofructokinase activity is extraordinarily sensitive to both the energy balance of the cell as reflected in ATP concentration and the availability of other fuels as reflected in cytosolic citrate concentration.     

Interaction of D-fructose and fructose 1-phosphate with yeast phosphofructokinase and its influence on glycolytic oscillations.

Fermentation of D-fructose- and D-glucose induced glycolytic oscillations of different period lengths in Saccharomyces carlsbergensis. Recent studies suggested, that D-fructose or one of its metabolites interacted with phosphofructokinase (ATP:D-fructo-6-phosphate 1-phosphofructokinase, EC 2.7.1.11), the core of the glycolytic 'oscillator'. In order to explore the kinetics of interaction, the influence of D-fructose and fructose 1-phosphate on purified yeast phosphofructokinase was studied. D-fructose concentrations up to 0.3 mM stimulated the enzyme, while a further increase led to competitive inhibition. The Hill coefficient for fructose 6-phosphate decreased from 2.8 to 1.0. Fructose 1-phosphate acted in a similar way, up to 1 mM activation and inhibition competitive to fructose 6-phosphate at higher concentration (2.0--3.5 mM) with the same effect on the Hill coefficient. The inhibition patterns obtained with D-fructose or fructose 1-phosphate suggest a sequential random reaction mechanism of yeast phosphofructokinase with fructose 6-phosphate and MgATP2-. The mode of interaction of phosphofructokinase with D-fructose and fructose 1-phosphate is discussed. The influence of both effectors resulted in altered enzyme kinetics, which may cause the different period lengths of glycolytic oscillations.     

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.     

The control of rat-heart phosphofructokinase by citrate and other regulators

1. The effects of ATP, inorganic phosphate and citrate on the relationship between fructose 6-phosphate concentration and initial velocity of reaction has been investigated with a partially purified preparation of rat-heart phosphofructokinase. 2. At low concentrations of ATP (<80mum) rate curves for fructose 6-phosphate approximated to Michaelis-Menten kinetics. At higher ATP concentrations rate curves were sigmoid, the K(m) for fructose 6-phosphate increased and the reaction appeared to be first-order with respect to fructose 6-phosphate at concentrations above its K(m) and of a higher order at concentrations below its K(m). Inorganic phosphate lowered the K(m) for fructose 6-phosphate and the concentration at which the apparent kinetic order decreased. 3. At 40mum-ATP, citrate was an activator at low concentration (<100mum) and an inhibitor at higher concentrations. At 0.5mm-ATP, citrate was inhibitory at all concentrations tested. 4. A new method for phosphofructokinase assay using [U-(14)C]fructose 6-phosphate is described which allows measurements to be made of the velocity of the forward reaction at known concentrations of the products of the reaction. With this method confirmatory evidence has been obtained that concentrations of ATP, AMP, phosphate and citrate may regulate phosphofructokinase in the perfused rat heart.     

Regulation of anaerobic glycolysis in Ehrlich ascites tumour cells.

1) In intact Ehrlich ascites tumour cells the anaerobic glycolytic flux rate and pattern of intermediates have been investigated at different pH values of the extracellular medium. 2) As predicted from the dependence of the lactic acid dehydrogenase equilibrium on pH a strong negative correlation between log ([lactate]/[pyruvate]) and pH has been found. 3) The steady state fluxes of glycolysis at pH 8.0 and 7.4 are rather equal, despite significant differences in the intracellular concentrations of glycolytic intermediates. At pH 8.0 the concentrations of ATP, glucose 6-phosphate, and fructose 6-phosphate are lower, and the concentrations of ADP, AMP, fructose 1,6-bisphosphate, triose phosphates, phosphoglycerates, and phosphoenolpyruvate are higher than at pH 7.4. 4) From the analysis of the pH dependent changes of metabolites it follows that different mechanisms are responsible for maintaining equal actual activities of hexokinase, phosphofructokinase and pyruvate kinase at pH 7.4 and 8.0. 5) From an application of the linear theory of enzymatic chains and a calculation of the control strength of the regulatory important enzymes results that hexokinase is evidently rate-limiting for glycolysis, and phosphofructokinase is also significantly influencing the glycolytic flux. Pyruvate kinase and glyceraldehyde phosphate dehydrogenase, on the other hand, do not significantly affect the rate of the overall glycolytic flux in ascites.     

Hormonal regulation of fructose 2,6-bisphosphate levels in epididymal adipose tissue of rat

The participation of fructose 2,6-bisphosphate on glycolysis stimulated by insulin and adrenaline in incubated white adipose tissue of rat was investigated. Adrenaline addition to incubated fat-pads strongly decreased the intracellular levels of fructose 2,6-bisphosphate. When the tissue was preincubated with glucose, the presence of insulin in the incubation medium increased fructose 2,6-bisphosphate levels 2-fold. These variations were related to changes in the substrates, ATP and fructose 6-phosphate. It therefore appears that fructose 2,6-bisphosphate may be involved in the control of insulin-induced glycolysis, but it does not seem to play a role in the stimulation of glucolysis by adrenaline.     

The effect of Mg2+ upon 6-phosphofructokinase activity in Ehrlich ascites tumor cells in vivo

The effect of Mg2+ addition to intact Ehrlich ascites tumor cells (EATC) has been investigated. A decrease of glucose 6-phosphate (G6P) content and an increase of fructose 1,6-diphosphate (FDP) content are detected in glucose utilizing EATC incubated with increasing Mg2+ concentrations (from 0 to 5.0 mM). The strong enhancement of FDP/G6P ratio is taken as evidence for in vivo stimulation of phosphofructokinase 1 (PFK) (ATP:D-fructose-6-phosphate 1-phosphotransferase; EC 2.7.1.11). A similar effect can be observed when glucose is replaced by fructose as the glycolytic substrate. Stimulation of PFK is paralleled by substantial depletion of ATP. Cytochalasin B prevents the observed phenomena. Cell total Mg increases by about 15% when EATC are incubated with 5 mM Mg2+. The overall data show that extracellular Mg2+ may modulate glycolytic flux in EATC in vivo. Implications and significance of these phenomena in the regulation of cancer cell metabolic features are discussed.     

Effects of insulin and work on fructose 2,6-bisphosphate content and phosphofructokinase activity in perfused rat hearts

The effects of insulin and increased cardiac work on glycolytic rate, metabolite content, and fructose 2,6-bisphosphate (Fru-2,6-P2) content were studied in isolated perfused rat hearts. Steady-state rates of glycolysis increased 5-fold with the addition of insulin to the perfusate or by increasing cardiac pressure-volume work and correlated well in most conditions with changes in substrate concentration (Fru-6-P) and with concentration of the activator, Fru-2,6-P2. There was no correlation with changes in other well known regulators including citrate, ATP, AMP, Pi, or cytosolic phosphorylation potential. Using phosphofructokinase purified from hearts perfused under identical conditions, allosteric kinetic experiments were performed using the metabolite and effector concentrations determined from in vivo experiments. Reaction rates for phosphofructokinase calculated in vitro agreed well with the glycolytic rates measured in vivo and correlated with changes in Fru-6-P but not with other effectors. However, higher Fru-2,6-P2 levels were more effective in maintaining phosphofructokinase activity at high ATP and citrate levels. Kinetic experiments did not indicate a covalent modification of phosphofructokinase. These data indicate that control of cardiac phosphofructokinase and glycolysis may be accomplished by changes in the availability of substrate, Fru-6-P, and activator, Fru-2,6-P2, rather than by citrate, adenine nucleotides, or cytosolic phosphorylation potential as previously suggested.     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.     

Role of fructose 2,6-bisphosphate in the stimulation of glycolysis by anoxia in isolated hepatocytes.

1. Incubation of hepatocytes from fed or starved rats with increasing glucose concentrations caused a stimulation of lactate production, which was further increased under anaerobic conditions. 2. When glycolysis was stimulated by anoxia, [fructose 2,6-bis-phosphate] was decreased, indicating that this ester could not be responsible for the onset of anaerobic glycolysis. In addition, the effect of glucose in increasing [fructose 2,6-bisphosphate] under aerobic conditions was greatly impaired in anoxic hepatocytes. [Fructose 2,6-bisphosphate] was also diminished in ischaemic liver, skeletal muscle and heart. 3. The following changes in metabolite concentration were observed in anaerobic hepatocytes: AMP, ADP, lactate and L-glycerol 3-phosphate were increased; ATP, citrate and pyruvate were decreased: phosphoenolpyruvate and hexose 6-phosphates were little affected. Concentrations of adenine nucleotides were, however, little changed by anoxia when hepatocytes from fed rats were incubated with 50 mM-glucose. 4. The activity of ATP:fructose 6-phosphate 2-phosphotransferase was not affected by anoxia but decreased by cyclic AMP. 5. The role of fructose 2,6-bisphosphate in the regulation of glycolysis is discussed.     

Ribose 1,5-bisphosphate regulates rat kidney cortex phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) is a major enzyme of the glycolytic pathway, catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. In this study, we demonstrated the effect of ribose 1,5-bisphosphate on phosphofructokinase purified from rat kidney cortex. Ribose 1,5-bisphosphate relieved the phosphofructokinase from ATP inhibition and increased the affinity for fructose 6-phosphate at nanomolar concentrations. These activating effects of ribose 1,5-bisphosphate were enhanced in the presence of AMP. Ribose 1,5-bisphosphate reduced the inhibition of the phosphofructokinase induced by citrate. These results suggest that ribose 1,5-bisphosphate is an activator of rat kidney cortex phosphofructokinase and synergistically regulates the enzyme activity with AMP.