Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

Carbohydrate metabolism of the perfused rat liver

1. The rates of gluconeogenesis from most substrates tested in the perfused livers of well-fed rats were about half of those obtained in the livers of starved rats. There was no difference for glycerol. 2. A diet low in carbohydrate increased the rates of gluconeogenesis from some substrates but not from all. In general the effects of a low-carbohydrate diet on rat liver are less marked than those on rat kidney cortex. 3. Glycogen was deposited in the livers of starved rats when the perfusion medium contained about 10mm-glucose. The shedding of glucose from the glycogen stores by the well-fed liver was greatly diminished by 10mm-glucose and stopped by 13.3mm-glucose. Livers of well-fed rats that were depleted of their glycogen stores by treatment with phlorrhizin and glucagon synthesized glycogen from glucose. 4. When two gluconeogenic substrates were added to the perfusion medium additive effects occurred only when glycerol was one of the substrates. Lactate and glycerol gave more than additive effects owing to an increased rate of glucose formation from glycerol. 5. Pyruvate also accelerated the conversion of glycerol into glucose, and the accelerating effect of lactate can be attributed to a rapid formation of pyruvate from lactate. 6. Butyrate and oleate at 2mm, which alone are not gluconeogenic, increased the rate of gluconeogenesis from lactate. 7. The acceleration of gluconeogenesis from lactate by glucagon was also found when gluconeogenesis from lactate was stimulated by butyrate and oleate. This finding is not compatible with the view that the primary action of glucagon in promoting gluconeogenesis is an acceleration of lipolysis. 8. The rate of gluconeogenesis from pyruvate at 10mm was only 70% of that at 5mm. This ;inhibition' was abolished by oleate or glucagon.     

Induction and suppression of the key enzymes of glycolysis and gluconeogenesis in isolated perfused rat liver in response to glucose, fructose and lactate

1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.     

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.     

The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro

1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-(14)C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-(14)C]glucose, except that incorporation into [(14)C]lactate was decreased, and significant amounts of [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-(14)C]glucose 6-phosphate into all metabolites except [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-(14)C]glucose other than to increase markedly the incorporation into, and amount of, [(14)C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12mumol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-P(i) or 26mm-HCO(3) (-) was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.     

The rate of gluconeogenesis from various precursors in the perfused rat liver

1. The rates of gluconeogenesis from many precursors have been measured in the perfused rat liver and, for comparison, in rat liver slices. All livers were from rats starved for 48hr. Under optimum conditions the rates in perfused liver were three to five times those found under optimum conditions in slices. 2. Rapid gluconeogenesis (rates of above 0·5μmole/g./min.) were found with lactate, pyruvate, alanine, serine, proline, fructose, dihydroxyacetone, sorbitol, xylitol. Unexpectedly other amino acids, notably glutamate and aspartate, and the intermediates of the tricarboxylic acid cycle (with the exception of oxaloacetate), reacted very slowly and were not readily removed from the perfusion medium, presumably because of permeability barriers which prevent the passage of highly charged negative ions. Glutamine and asparagine formed glucose more readily than the corresponding amino acids. 3. Glucagon increased the rate of gluconeogenesis from lactate and pyruvate but not from any other precursor tested. This occurred when the liver was virtually completely depleted of glycogen. Two sites of action of glucagon must therefore be postulated: one concerned with mobilization of liver glycogen, the other with the promotion of gluconeogenesis. Sliced liver did not respond to glucagon. 4. Pyruvate and oxaloacetate formed substantial quantities of lactate on perfusion, which indicates that the reducing power provided in the cytoplasm was in excess of the needs of gluconeogenesis. 5. Values for the content of intermediary metabolites of gluconeogenesis in the perfused liver are reported. The values for most intermediates rose on addition of lactate. 6. The rates of gluconeogenesis from lactate and pyruvate were not affected by wide variations of the lactate/pyruvate ratio in the perfusion medium.     

The effect of glycerol and dihydroxyacetone on hepatic adenine nucleotides

1. The changes in the metabolite content in the isolated perfused rat liver and in the perfusion medium were measured after loading the liver with glycerol or dihydroxyacetone. 2. Glycerol was rapidly taken up by livers from fed and starved rats; glucose, lactate and pyruvate were discharged into the medium. The [lactate]/[pyruvate] ratio in the medium rose from 10 to 30 and in the tissue from 9.6 to 36.6. 3. The most striking effects of glycerol loading were: (i) the accumulation in the liver of alpha-glycerophosphate, which increased from 0.13 to 8.45mumol/g at 40min; (ii) the decrease in the concentration of adenine nucleotides to 70% of the control value at 40min. 4. The P(i) content of the tissue also fell, from 4.25 to 2.31mumol/g at 10min, but the sum of the phosphates measured rose from the normal value of 13.8 to 18.8mumol/g at 40min, because of an uptake of P(i) from the medium. 5. Omission of phosphate from the standard perfusion medium increased the depletion of adenine nucleotides on glycerol loading. 6. Dihydroxyacetone was more rapidly metabolized than glycerol. Again glucose, lactate and pyruvate were the main products. The [lactate]/[pyruvate] ratio remained below 10. 7. Dihydroxyacetone caused an increase of the fructose 1-phosphate content from 0.23 to 0.39mumol/g at 10min. The adenine nucleotide content of the tissue was not significantly decreased by dihydroxyacetone loading. 8. The rate of removal of both glycerol and dihydroxyacetone was about 60% greater in the livers from fed than in those from starved animals. 9. The results extend previous findings by Burch et al. (1970), who administered glycerol and dihydroxyacetone intraperitoneally.     

Influence of ethanol on the liver metabolism of fed and starved rats

1. The influence of ethanol on the metabolism of livers from fed and starved rats has been studied in liver-perfusion experiments. Results have been obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in livers from starved rats than in livers from fed rats. Ethanol had no effect on the oxygen consumption of either type of liver. After the addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely, the change being faster in livers from starved rats. 3. With livers from fed rats glucose was released from the liver into the perfusion medium. This release was slightly greater when ethanol was present. With livers from starved rats no release of glucose was observed, and when ethanol was added a marked uptake of glucose from the medium was found. A simultaneous release of glycolytic end products, lactate and pyruvate, into the medium occurred. 4. Acetate was the main metabolite accumulating in the perfusion medium when ethanol was oxidized. With livers from starved rats a slightly increased formation of ketone bodies was found when ethanol was present. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 10 to 87 with livers from fed rats and from 20 to 171 with livers from starved rats when the livers were perfused with ethanol in the medium. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 0·8 to 7·6 with livers from fed rats and from 1·0 to 9·5 with livers from starved rats when ethanol was added to the medium. 6. The effects of ethanol are discussed and related to changes in the redox state of the liver that produce new conditions for some metabolic pathways.     

Gluconeogenesis from fructose predominates in periportal regions of the liver lobule

Gluconeogenesis from fructose was studied in periportal and pericentral regions of the liver lobule in perfused livers from fasted, phenobarbital-treated rats. When fructose was infused in increasing concentrations from 0.25 to 4 mM, corresponding stepwise increases in glucose formation by the perfused liver were observed as expected. Rates of glucose and lactate production from 4 mM fructose were around 100 and 75 mumol/g/h, respectively. Rates of fructose uptake were around 190 mumol/g/h when 4 mM fructose was infused. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, decreased glucose formation from fructose maximally by 20% suggesting that a fraction of the lactate formed from fructose is used for glucose synthesis. A good correlation (r = 0.92) between extra oxygen consumed and glucose produced from fructose was observed. At low fructose concentrations (less than 0.5 mM), the extra oxygen uptake was much greater than could be accounted for by glucose synthesis possibly reflecting fructose 1-phosphate accumulation. Furthermore, fructose diminished ATP/ADP ratios from about 4.0 to 2.0 in periportal and pericentral regions of the liver lobule indicating that the initial phosphorylation of fructose via fructokinase occurs in both regions of the liver lobule. Basal rates of oxygen uptake measured with miniature oxygen electrodes were 2- to 3-fold higher in periportal than in pericentral regions of the liver lobule during perfusions in the anterograde direction. Infusion of fructose increased oxygen uptake by 65 mumol/g/h in periportal areas but had no effect in pericentral regions of the liver lobule indicating higher local rates of gluconeogenesis in hepatocytes located around the portal vein. When perfusion was in the retrograde direction, however, glucose was synthesized nearly exclusively from fructose in upstream, pericentral regions. Thus, gluconeogenesis from fructose is confined to oxygen-rich upstream regions of the liver lobule in the perfused liver.     

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.     

Effects of ischaemia on content of metabolites in rat liver and kidney in vivo

1. The time-course of changes in content of intermediates of glycolysis in rat liver and kidney cortex after severance of blood supply was investigated. 2. The decline in content of ATP was more rapid in kidney (1.7-0.5mumol/g in 30s) than in liver (2.7-1.6mumol/g in 60s). In both tissues AMP and P(i) accumulated. 3. Net formation of lactate was 1.7mumol/g during the second minute of ischaemia in liver from well-fed rats, 1.1mumol/g in liver from 48h-starved rats, and about 1.0mumol/g during the first 30s of ischaemia in kidney. Net formation of alpha-glycerophosphate was rapid, especially in liver. 4. In kidney the concentration of beta-hydroxybutyrate rose, but that of alpha-oxoglutarate and acetoacetate decreased. 5. In both organs the concentrations of fructose diphosphate and triose phosphates increased during ischaemia and those of other phosphorylated C(3) intermediates decreased. 6. The concentration of the hexose 6-phosphates rose rapidly during the first minute of ischaemia in liver, but decreased during renal ischaemia. 7. In kidney the content of glutamine fell after 2min of ischaemia, and that of ammonia and glutamate rose. 8. The redox states of the cytoplasmic and mitochondrial NAD couple in kidney cortex were similar to those in liver. 9. The regulatory role of glycogen phosphorylase, pyruvate kinase and phosphofructokinase is discussed in relation to the observed changes in the concentrations of the glycolytic intermediates.     

Effect of verapamil on glycogenolysis and gluconeogenesis in the perfused rat liver

In perfused livers from fed rats, rates of glucose production (glycogenolysis) were 133 +/- 12 mumol/g/hr. Infusion of 2 microM verapamil into these livers decreased the rates of glucose production significantly to 97 +/- 15 mumol/g/hr within 10 min. Conversely, rates of production of lactate plus pyruvate (glycolysis) of 64 +/- 6 mumol/g/hr were not significantly altered by verapamil (60 +/- 3 mumol/g/hr). When 50 microM verapamil was infused, however, rates of both glycogenolysis and glycolysis were diminished to 56 +/- 11 and 43 +/- 5 mumol/g/hr, respectively. In perfused livers from fasted rats, infusion of 20 mM fructose increased the rates of production of glucose (gluconeogenesis) significantly from 11 +/- 7 to 121 +/- 17 mumol/g/hr. These rates reached 138 +/- 7 mumol/g/hr upon the simultaneous infusion of verapamil (2 microM). In these livers, fructose also increased rates of production of lactate from 6 +/- 2 to 132 +/- 11 mumol/g/hr, which were further increased to 143 +/- 8 mumol/g/hr when 2 microM verapamil was infused. The results show that calcium-dependent processes involved in hepatic carbohydrate metabolism respond differently to the calcium channel blocker verapamil. Low concentrations of verapamil inhibited glycogenolysis significantly while having no effect on either glycolysis or gluconeogenesis. These data suggest that these two processes have different sensitivities to changes in intracellular calcium concentrations and/or different sources of regulatory calcium.     

Glucose infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

After a pulse of [3-14C]pyruvate, 24 hr starved rats were infused through the portal vein with two different doses of glucose (7.8 or 20.8 mg/min) or the medium, and blood was collected from the inferior cava vein at the level of the suprahepatic veins. The highest dose of glucose enhanced the appearance of [14C]glucose in blood from the 2nd to the 20th min after tracer delivery. It also enhanced production of [14C]glycogen and concentration of glycogen in the liver after 5 and 20 min. At 20 min of glucose infusion the appearance of [14C]glyceride glycerol in liver as well as liver lactate concentration and lactate/pyruvate ratio were increased. The low dose of glucose used enhanced liver values of [14C]glycogen, [14C]glycogen specific activity and glycogen concentration. Our results support the hypothesis that in the starved rat glucose is converted into C3 units prior to being deposited as liver glycogen and based on the liver zonation model (Jungermann et al., 1983) it is proposed that glucose stimulated gluconeogenesis by shifting the liver to the cytosolic redox state as a secondary consequence of increased glycolytic activity.     

Effects of fructose concentration on carbohydrate metabolism, heat production and substrate cycling in isolated rat hepatocytes

1. Hepatocytes from starved rats were incubated with 5mm-glucose, labelled uniformly with (14)C and specifically with (3)H at positions 1, 2, 3 or 6, and with fructose at concentrations of 2.5, 7.5 or 25mm. 2. In the absence of other substrates only 1% of the radioactivity initially present in [U-(14)C]glucose appeared in the metabolic products, CO(2), lactate, pyruvate, amino acids and glycogen. 3. Fructose at 2.5mm caused a 30% increase in the glucose concentration and a 4-fold increase in the apparent oxidation of [U-(14)C]-glucose. 4. The formation of (3)H(2)O from [1-(3)H]-, [2-(3)H]-, [3-(3)H]- or [6-(3)H]-glucose was 2.4, 4.3, 2.15 or 1.6% respectively in the control incubations and 4.1, 10.4, 7.7 or 5.1% with 2.5mm-fructose. 5. Fructose at 7.5 and 25mm decreased the (3)H(2)O yields to less than the control values, but had no apparent effect on the amount of [U-(14)C]glucose metabolized. 6. In the incubations with 5mm-glucose and 25mm-fructose there were significant decreases in heat production, O(2) consumption and in the ratio of O(2) uptake to heat output. 7. Fructose at 2.5mm caused a 64% increase in heat output, but only a 43% increase in O(2) uptake. 8. The radioisotopic and calorimetric data demonstrate that physiological concentrations of fructose greatly increase metabolism in hepatocytes from starved rats. These data also indicate increased cycling at glucose/glucose 6-phosphate and at fructose 6-phosphate/fructose 1,6-bisphosphate in the presence of 2.5mm-fructose, although the rates of cycling were actually decreased relative to the amount of glucose catabolized. 9. At concentrations of 2.5, 7.5 and 25mm, fructose depressed hepatocyte ATP concentrations by 20, 65 and 80% respectively. Although fructose at 7.5 and 25mm increased glucose and lactate release, O(2) consumption, production of heat and formation of(3)H(2)O from [1-(3)H]-, [2-(3)H]-, [3-(3)H]- or [6-(3)H]-glucose were lowered to values equal to, or less than, controls. These effects probably reflect a severe derangement of hepatic metabolism due to excess phosphorylation of fructose when present at high concentrations.     

Studies on gluconeogenesis and ketogenesis in isolated hepatocytes from alloxan diabetic rats.

Gluconeogenesis and ketogenesis were studied in isolated hepatocytes obtained from normal and alloxan diabetic rats. Insulin treatment maintained near-normal blood glucose levels and caused an increase in glycogen deposition. The third day after insulin withdrawal the rats displayed a diabetic syndrome marked by progressive hyperglycemia and glycogen depletion. Net glucose production in liver cells isolated from alloxan diabetic rats progressively increased with time up to 72 hr after the last in vivo insulin injection. Maximal glucose production was observed at 72 hr with 10 mM alanine, lactate, pyruvate, or fructose. Glucose production decreased at 96 hr. The same pattern was observed with the incorporation of labeled bicarbonate into glucose. Ketogenesis in liver cells and hepatic lipid content also peaked at 72 hr.     

Influence of some mononucleotides and their corresponding nucleosides on the metabolism of carbohydrates in the isolated rat diaphragm muscle.

1. The influence of ATP on glucose metabolism was studied in the isolated rat diaphragm; it was shown that ATP increases the oxidation of glucose and the aerobic conversion of glucose into lactate, whereas it decreases glycogen synthesis. There was no influence of ATP on the anaerobic formation of lactate from glucose. 2. A maximum effect of ATP on the oxidation of glucose (about 160% increase) was obtained in the presence of 10mm-ATP; in the presence of 2mm-ATP the effect was about 65%, and was approximately constant from 10 to 90min. incubation period. 3. In a phosphate-free tris-buffered medium the oxidation of glucose was considerably decreased, but the percentage stimulation by ATP was about the same as in a phosphate-buffered medium. 4. ATP was shown to increase the oxidation of fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 1,6-diphosphate and, to a much smaller extent, pyruvate. 5. ADP stimulated the oxidation of glucose to the same extent as ATP at a concentration of 2mm and the effect with AMP was only slightly less; IMP and adenosine had only a small stimulatory effect at this concentration, whereas inosine had no effect.     

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.     

Fructose effect to suppress hepatic glycogen degradation

The effect of fructose on glycogen degradation was examined by measuring the flux of 14C from prelabeled glycogen in perfused rat livers. During 2-h refeeding of 24-h-fasted rats, newly synthesized hepatic glycogen was labeled by intraperitoneal injection of [U-14C] galactose (0.1 mg and 0.02 microCi/g of body weight). The livers of refed rats were then perfused in a nonrecirculating fashion for an initial 30 min with glucose alone (10 mM) for the following 60 min with glucose (10 mM) without (n = 5) or with fructose (1, 2, or 10 mM; n = 5 for each). When livers were exposed to fructose, release of label into the perfusate immediately declined and remained markedly suppressed through the end of perfusion (p less than 0.05). The suppression was dose-dependent; at steady state (50-70 min), label release was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose, respectively (p less than 0.0001). Suppression was not accompanied by significant changes in the activities of glycogen synthase or phosphorylase assessed in vitro. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (Fru-1-P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.60 mumol/g of liver with 1, 2, and 10 mM fructose, respectively; p less than 0.0001). Maximum inhibition of label release was 82%; the Fru-1-P concentration for half inhibition was 0.57 mumol/g of liver, well within the concentration of Fru-1-P attained during refeeding. We conclude that fructose enhances net glycogen accumulation in liver by suppressing glycogenolysis and that the suppression is presumably caused by allosteric inhibition of phosphorylase by Fru-1-P.