Regulation of phosphofructokinase activity by glucagon in isolated rat hepatocytes

Pilkis, S J; Schlumpf, J.; Pilkis, J; Claus, Thomas H.

doi:10.1016/0006-291x(79)91501-8

Cited by 70 publications

(33 citation statements)

References 21 publications

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“…The concentration for half-maximal inhibition was around 0.2nM glucagon, independent of the substrate (glycogen or glucose) or the nutritional state. It is close to concentrations required for half-maximal effects on glycolytic enzymes (e. g. phosphofructokinase, inactivation at 0.2 nM [4] and phosphorylation at 0.06 nM [18]; pyruvate kinase, inactivation and phosphorylation at 0.3 nM [21]) and is within the range of glucagon concentrations reported for portal venous blood in viuo (i.e. 0.02 -0.8 nM [22 -241).…”

Section: Discussionsupporting

confidence: 64%

Actions of glucagon on flux rates in perfused rat liver

Mauch

Kerzl

Schwabe

et al. 1983

European Journal of Biochemistry

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Changes in the rates of glucose, lactate and pyruvate production following infusion of glucagon were studied in isolated livers from fed or fasted rats perfused with non-recirculating Krebs-Henseleit bicarbonate buffer.1. Evidence was presented that under these experimental conditions, the release of lactate plus pyruvate into the perfusate represents 80 -90 % of the total glycolytic flux; oxidation of pyruvate derived from glucose and/or glycogen accounts for the remaining 10 -20 %, whereas recycling of pyruvate to glucose is negligible.2. In the presence of glucagon, rates of lactate plus pyruvate production were diminished to less than 30 % in the fed state (glycogen as substrate) and to less than 10 % in the fasted state (glucose as substrate). Rates of pyruvate oxidation were unchanged. Although recycling of pyruvate to glucose was enhanced, it could account for not more than 20 % of the decrease in lactate plus pyruvate production. The data indicate a strong inhibition of the substrate flux through glycolysis.3. The glucagon concentration for half-maximal inhibition of glycolysis was 0.2 nM, independent of the substrate (glucose or glycogen) and the nutritional state. The effective concentrations were within the physiological range of glucagon concentrations reported for portal venous blood.4. Transient state analyses indicated that the inhibition of glycolysis precedes the stimulation of glycogenolysis. When after a delay glycogenolysis was accelerated, it was followed by a transient stimulation of glycolysis. The stimulatory component in the glucagon effect on glycolysis was diminished in glycogen-depleted livers and when glucose was the main substrate. The coordinated control of glycolysis and glycogenolysis by glucagon and the interaction of the two processes in the transient state are discussed.It is well known that glucagon stimulates hepatic glycogenolysis and that this phenomenon is due to a cyclic-AMPmediated activation of enzymes involved in glycogen breakdown [l -31. It was also reported that glucagon, in addition to its long-term effects, causes a rapid inactivation of key enzymes of glycolysis [4-71. Based on these findings, the inhibition of glycolytic flux by glucagon became textbook knowledge. A direct proof, however, was missing until now.In this communication, we describe the dose-response and the kinetics of the short-term effects of glucagon on rates of glycolysis and glycogenolysis in livers from fed and fasted rats. The isolated liver perfused with non-recirculating medium is a suitable model for the study of flux rates. Since metabolites do not accumulate in the perfusate, metabolite rates can be determined directly from the metabolic concentrations in the effluent perfusate. The release of glucose, lactate and pyruvate, therefore, should provide an estimate of rates of carbohydrate degradation. We present evidence that under our experimental Enzymes. Phosphofructokinase or ATP : ~-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.1 1); phosphofructokinase 2, PFK2 or ATP : ~-fructo...

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Section: Discussionsupporting

confidence: 64%

Actions of glucagon on flux rates in perfused rat liver

Mauch

Kerzl

Schwabe

et al. 1983

European Journal of Biochemistry

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“…In this communication we show that phosphofructokinase isolated from the livers of diabetic mice is more sensitive to ATP inhibition than is the enzyme isolated from the livers of normal controls and therefore may represent a physiologically less active form of phosphofructokinase. These results are entirely in accord with three recent independent reports which have indicated decreased liver phosphofructokinase activity after either administration of glucagon to rats (8) or addition of glucagon to isolated rat hepatocytes (9,10 Normal C57BL/KsJ (misty m+/m+, or m+/db+) and genetically diabetic C57BL/KsJ-db (db+/db+) mice were obtained at 7-8 weeks of age from The Jackson Laboratory (Bar Harbor, ME). The animals were housed in plastic cages in a Puffer-Hubbard Environator chamber maintained at 21°C with a 12-hr light (0800-2000)/12-hr dark (2000-0800) cycle.…”

supporting

confidence: 79%

Increased ATP inhibition of liver phosphofructokinase from genetically diabetic mice.

Hosey¹,

Chatterjee²,

Cohen³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Phosphofructokinase (ATP:D-fructose-6phos-phate 1-phosphotransferase, EC 2.7.1.11) was partially purified the livers of genetically diabetic mice (C57BL/Ksj-db) and their lean littermates (C57BL/KsJ). These genetically diabetic mice have been shown to be hyperglucagonemic and to exhibit symptoms resembling those of maturity-onset diabetes in humans. Two isozymes of phosphofructokinase were obtained after DEAE-Sephadex chromatography of extracts of livers from either normal or diabetic animals. One of these isozymes, peak II, from the genetically diabetic mice was shown to be more sensitive to ATP inhibition at physiological pH than the peak II isozyme from the normal animals. In addition, the peak II isozyme from the diabetic mice exhibited decreased affinity for fructose 6-phosphate. The (4). Enhanced gluconeogenesis appears to be a major factor in the hyperglycemia of diabetic mice. Studies of gluconeogenic fluxes and metabolite levels in diabetic mice indicate that the increased liver gluconeogenesis is likely to be due to a facilitation of the fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) reaction or a restraint of the phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) reaction or both (5). Studies on gluconeogenic rates in isolated rat liver cells also led to the proposal that glucagon enhances gluconeogenesis and inhibits glycolysis at the site of fructose 1,6-bisphosphatase and phosphofructokinase (6).With this background it appeared to us that a comparative study of both phosphofructokinase and fructose 1,6-bisphosphatase from the livers of C57BL/KsJ normal and diabetic hyperglucagonemic mice could provide an insight into the mode of action of glucagon at the metabolic steps catalyzed by the above enzymes.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2497A previous study of fructose 1,6-bisphosphatase isolated from the livers of diabetic mice and from the normal controls showed no difference in either molecular or enzymatic properties between the two purified enzymes (7). In this communication we show that phosphofructokinase isolated from the livers of diabetic mice is more sensitive to ATP inhibition than is the enzyme isolated from the livers of normal controls and therefore may represent a physiologically less active form of phosphofructokinase. These results are entirely in accord with three recent independent reports which have indicated decreased liver phosphofructokinase activity after either administration of glucagon to rats (8) or addition of glucagon to isolated rat hepatocytes (9, 10). MATERIALS AND METHODSFructose 6-phosphate, fructose 1,6-bisphosphate, ATP, NADH, DEAE-Sephadex A-25, and aldolase were purchased from Sigma. Triose-phosphate isomerase and a-glycerophosphate dehydrogenase were from Boehringer Mannheim. Ammonium sulfate was from ...

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“…These effects account in part for the stimulation of gluconeogenesis by the hormone. The inhibition of both enzymes is characterized by increases in the substrate concentration needed for half-maximal activity (So.5) (1,2,(4)(5)(6)(7)(8) and in sensitivity to inhibition by ATP (3,5, 7). Glucagon also stimulates the in vivo phosphorylation of both enzymes (3, 9), and this has led to the hypothesis that enzyme inhibition by phosphorylation is the mechanism by which the hormone stimulates gluconeogenesis (10).…”

Section: Introductionmentioning

confidence: 99%

“…The cells were suspended to a final concentration of 50 mg of liver/ml in Krebs-Henseleit buffer that contained 0.5% bacitracin. In experiments in which only phosphofructokinase activity was measured, heated extracts from 5-ml aliquots of cell suspension were prepared as described (1), except that the cells were homogenized in 1.5 ml of buffer (50 mM potassium phosphate, pH 7.5/100 mM NaF/1 mM EDTA/20 mM 3-mercaptoethanol/0. 1 mM phenylmethylsulfonyl fluoride).…”

mentioning

confidence: 99%

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Mechanism of action of glucagon on hepatocyte phosphofructokinase activity.

Claus¹,

Schlumpf²,

el-Maghrabi³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Addition of glucagon to isolated hepatocytes reduced the activity of 6-phosphofructokinase (ATP:D-fructose--phosphate 1-phosphotransferase, EC 2.7.1.11) and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) Phosphorylation contributed to the inhibition of pyruvate kinase, but several lines of evidence indicated that this reaction was not responsible for the inhibition of phosphofructokinase. First, the increase in phosphorylation in intact cells induced by increasing the concentration of glucagon did not correlate well with the decrease in enzyme activity. Second, phosphorylation of phosphofructokinase induced by addition of cyclic AMP and Mg2 -ATP or by addition of Mge+-ATP and the catalytic subunit of the cyclic AMP-dependent protein kinase to hepatocyte extracts had no effect on enzyme activity. Third, ammonium sulfate precipitation of the enzyme from extracts of cells incubated with glucagon abolished the hormone effect. The effect could be restored, however, by the addition of a phosphofructokinase-free extract from glucagon-treated cells to the ammonium sulfate-treated enzyme from either untreated or glucagontreated cells. These results suggest that the inhibition of phosphofructokinase by glucagon is due to changes in the level of an allosteric effector(s). The addition of glucagon to isolated rat hepatocytes inhibits the activity of both phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.11.1) (1-3) and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) (4-8). These effects account in part for the stimulation of gluconeogenesis by the hormone. The inhibition of both enzymes is characterized by increases in the substrate concentration needed for half-maximal activity (So.5) (1,2,(4)(5)(6)(7)(8) and in sensitivity to inhibition by ATP (3,5, 7). Glucagon also stimulates the in vivo phosphorylation of both enzymes (3, 9), and this has led to the hypothesis that enzyme inhibition by phosphorylation is the mechanism by which the hormone stimulates gluconeogenesis (10). The results we report here support this hypothesis in the case of pyruvate kinase, but not in the case of phosphofructokinase.MATERIALS AND METHODS Preparation and Incubation of Hepatocytes. Isolated hepatocytes were prepared from fed rats (male SpragueDawley, 200-300 g) as described (8). The cells were suspended to a final concentration of 50 mg of liver/ml in Krebs-Henseleit buffer that contained 0.5% bacitracin. In experiments in which only phosphofructokinase activity was measured, heated extracts from 5-ml aliquots of cell suspension were prepared as described (1), except that the cells were homogenized in 1.5 ml of buffer (50 mM potassium phosphate, pH 7.5/100 mM NaF/1 mM EDTA/20 mM 3-mercaptoethanol/0. 1 mM phenylmethylsulfonyl fluoride). In experiments in which 32p incorThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this...

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Regulation of phosphofructokinase activity by glucagon in isolated rat hepatocytes

Cited by 70 publications

References 21 publications

Actions of glucagon on flux rates in perfused rat liver

Actions of glucagon on flux rates in perfused rat liver

Increased ATP inhibition of liver phosphofructokinase from genetically diabetic mice.

Mechanism of action of glucagon on hepatocyte phosphofructokinase activity.

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