ATP depletion, a possible role in the pathogenesis of hyperuricemia in glycogen storage disease type I.

Other investigators have shown that fructose infusion in normal man and rats acutely depletes hepatic ATP and P(i) and increases the rate of uric acid formation by the degradation of preformed nucleotides. We postulated that a similar mechanism of ATP depletion might be present in patients with glucose-6-phosphatase deficiency (GSD-I) as a result of ATP consumption during glycogenolysis and resulting excess glycolysis. The postulate was tested by measurement of: (a) hepatic content of ATP, glycogen, phosphorylated sugars, and phosphorylase activities before and after increasing glycolysis by glucagon infusion and (b) plasma urate levels and urate excretion before and after therapy designed to maintain blood glucose levels above 70 mg/dl and thus prevent excess glycogenolysis and glycolysis. Glucagon infusion in seven patients with GSD-I caused a decrease in hepatic ATP from 2.25 +/- 0.09 to 0.73 +/- 0.06 mumol/g liver (P <0.01), within 5 min, persisting in one patient to 20 min (1.3 mumol/g). Three patients with GSD other than GSD-I (controls), and 10 normal rats, showed no change in ATP levels after glucagon infusion. Glucagon caused an increase in hepatic phosphorylase activity from 163 +/- 21 to 311 +/- 17 mumol/min per g protein (P <0.01), and a decrease in glycogen content from 8.96 +/- 0.51 to 6.68 +/- 0.38% weight (P <0.01). Hepatic content of phosphorylated hexoses measured in two patients, showed the following mean increases in response to glucagon; glucose-6-phosphate (from 0.25 to 0.98 mumol/g liver), fructose-6-phosphate (from 0.17 to 0.45 mumol/g liver), and fructose-1,6-diphosphate (from 0.09 to 1.28 mumol/g) within 5 min. These changes, except for glucose-6-phosphate, returned toward preinfusion levels within 20 min. Treatment consisted of continuous intragastric feedings of a high glucose dietary mixture. Such treatment increased blood glucose from a mean level of 62 (range 28-96) to 86 (range 71-143) mg/dl (P <0.02), decreased plasma glucagon from a mean of 190 (range 171-208) to 56 (range 30-70) pg/ml (P <0.01), but caused no significant change in insulin levels. Urate output measured in three patients showed an initial increase, coinciding with a decrease in plasma lactate and triglyceride levels, then decreased to normal within 3 days after treatment. Normalization of urate excretion was associated with normalization of serum uric acid. We suggest that the maintenance of blood glucose levels above 70 mg/dl is effective in reducing serum urate levels and that transient and recurrent depletion of hepatic ATP due to glycogenolysis is contributory in the genesis of hyperuricemia in untreated patients with GSD-I.

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.

Stimulation by glucagon of the incorporation of U-14C-labeled substrates into glucose by isolated hepatocytes from fed rats.

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.

The role of phosphorylation in the alpha-adrenergic-mediated inhibition of rat hepatic pyruvate kinase.

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40-50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2-3-fold increase in the phosphate content of the enzyme. These changes were blocked by the alpha-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the alpha-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by alpha-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.

Effect of diabetes, insulin, starvation, and refeeding on the level of rat hepatic fructose 2,6-bisphosphate.

The influence of alloxan diabetes and starvation for 72 h on the level of rat hepatic fructose 2,6-biphosphate was investigated. Both diabetes and starvation decreased the level to 10% of the value found in livers of normal, fed rats (10 nmol/g liver). The activity of the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, 6-phosphofructo 2-kinase, was also decreased in livers of diabetic rats. Insulin administration for 24 h to diabetic rats restored the level of fructose 2,6-bisphosphate to normal. Refeeding a high carbohydrate diet for 24 h to starved rats resulted in fructose, 2,6-biphosphate levels that were 2.5-fold higher than that in livers of fed rats. The level of fructose 2,6-bisphosphate in diabetes and starvation, and after refeeding correlates as well with the rate of glycolysis and gluconeogenesis in these states and thereby provides further support for its role in regulating hepatic carbohydrate metabolism.

Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics.

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP-dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P2ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P2ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single NH2-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P2ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P2 levels. 6PF-2-K/Fru-2,6-P2ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P2, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P2. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P2 substrate cycles. The Fru-2,6-P2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P2ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH2-terminal and does not have a cAMP-dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypertrophy of brown adipocytes in brown and white adipose tissues and reversal of diet-induced obesity in rats treated with a beta3-adrenoceptor agonist.

In a previous study, we demonstrated that chronic treatment with a new beta3-adrenoceptor agonist, CL 316,243 [disodium (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-ben zodioxazole-2,2-dicarboxylate], promoted thermogenesis, caused the appearance of multilocular adipocytes in white adipose tissue (WAT), and retarded development of obesity in young rats eating a high-fat diet (Himms-Hagen et al., Am J Physiol 266: R1371-R1382, 1994). Objectives of the present study were to find out whether CL 316,243 could reverse established diet-induced obesity in rats and to identify the multilocular adipocytes that appeared in WAT. Infusion of CL 316,243 (1 mg/kg/day) reduced abdominal fat, with a decrease in enlarged adipocyte size but no loss of white adipocytes. The resting metabolic rate increased by 40-45%, but food intake was not altered. Abundant densely stained multilocular brown adipocytes expressing uncoupling protein (UCP) appeared in retroperitoneal WAT, in which a marked increase in protein content occurred. UCP content of interscapular brown adipose tissue (BAT) was also increased markedly. We suggest that the substantial increase in the resting metabolic rate induced by CL 316,243 occurs in brown adipocytes in both BAT and WAT. The origin of the brown adipocytes that appeared in WAT is uncertain. They may have been small brown preadipocytes, expressing beta3-adrenoceptors but with few mitochondria and little or no UCP, that were induced to hypertrophy by the beta3-agonist.

Rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a unique bifunctional enzyme.

Fructose 2,6-bisphosphate is a potent allosteric activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. It potentiates the effect of AMP on both enzymes. A great deal of compelling evidence supports the hypothesis that fructose 2,6-bisphosphate plays a key role in the hormonal and substrate regulation of substrate cycling at the fructose 6-phosphate/fructose 1,6-bisphosphate level in liver. This regulation is exerted at the level of the enzyme activities responsible for the synthesis and degradation of fructose 2,6-bisphosphate. Synthesis of the compound is catalyzed by a unique enzyme which transfers the gamma-phosphate of ATP to the C2 position of fructose 6-phosphate (ATP:D fructose 6-phosphate 2-phosphotransferase) while degradation is catalyzed by a phosphohydrolase activity which is specific for the C-2 position of fructose 2,6-bisphosphate (D-fructose 2,6-bisphosphate 2-phosphohydrolase). These activities are distinct from the classical 6-phosphofructo 1-kinase and fructose 1,6-bisphosphatase with regard to molecular weight, interaction with ligands, and the efficiency with which phosphoryl transfer occurs. Both activities have been purified to homogeneity and have been shown to be present in a single enzyme protein, i.e. the enzyme is bifunctional. Incubation of the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase with cAMP-dependent protein kinase and ATP leads to phosphorylation of the enzyme resulting in inactivation of the phosphotransferase activity and stimulation of the phosphohydrolase activity. Since fructose 2,6-bisphosphate is not further metabolized and can only be recycled to fructose 6-phosphate, simultaneous modulation of the synthesis and degradation of the compound by covalent modification of a single protein provides a very efficient and sensitive regulatory mechanism. The bifunctional enzyme was also shown to possess an ATPase activity which was nearly equal to the activity of the kinase reaction. However, in the presence of fructose 6-phosphate the enzyme did not transfer phosphate to water but rather to the C-2 position of the phosphorylated sugar. The ability of the enzyme to catalyze a partial reaction at a rate nearly equal to that of the forward reaction suggested that the reaction mechanism of the kinase proceeds by a two step transfer, i.e. via a phosphoryl enzyme intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase.

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.

Modulation of the phosphorylation state of rat liver pyruvate kinase by allosteric effectors and insulin.

The regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone. In this case, flux through pyruvate kinase was increased. The increase in flux correlated with an elevation in fructose bisphosphate levels but not with P-enolpyruvate levels which were unchanged. Pyruvate kinase was activated and its affinity for P-enolpyruvate was increased 7-fold in hepatocyte homogenates. Precipitation of the enzyme from homogenates with ammonium sulfate removed fructose 1,6-bisphosphate and activation was no longer observed. These results indicate that flux through and activity of pyruvate kinase can be controlled by the intracellular level of fructose 1,6-bisphosphate. The effect of elevated fructose 1,6-bisphosphate levels on the ability of glucagon to inactivate pyruvate kinase was also studied where only covalent enzyme modification is observed. Inactivation by maximally effective hormone concentrations was unaffected by elevated levels of fructose 1,6-bisphosphate, but the half-maximally effective concentration was increased from 0.3 to 0.8 nM. Activation of the cyclic AMP-dependent protein kinase by 0.3 nM glucagon was unaffected, but the initial rate of pyruvate kinase inactivation was suppressed. These results suggest that alterations in the level of fructose 1,6-bisphosphate can affect the ability of physiological concentrations of glucagon to inactivate pyruvate kinase by opposing phosphorylation of the enzyme. Consistent with this view was the finding that physiological concentrations of fructose 1,6-bisphosphate inhibited in vitro phosphorylation of purified pyruvate kinase. Inactivation of pyruvate kinase by 0.3 nM glucagon or 1 microM phenylephrine was also suppressed by 10 nM insulin. Insulin did not act by increasing fructose 1,6-bisphosphate levels. The antagonism to glucagon correlated well with the ability of insulin to suppress activation of the cyclic AMP-dependent protein kinase. However, no such correlation was observed with phenylephrine in the absence or presence of insulin. Thus, insulin can enhance pyruvate kinase activity by both cyclic AMP-dependent and independent mechanisms.

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.

Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.

Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate-2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography, and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit of the cyclic AMP-dependent protein kinase from rat liver and [gamma-32P]ATP resulted in 32P incorporation into a protein with a subunit Mr of 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose-6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heat-stable protein kinase inhibitor. It is concluded that the glucagon-induced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cyclic AMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-kinase.

Regulation of rat liver fructose 2,6-bisphosphatase.

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase.

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.