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Biochem. J. (1965) 94, 3c 3a The Role of Pyruvate Kinase in the Regulation of Gluconeogenesis By H. A. KREBS and L. V. EGGLESTON Medical Re8earch Council Unit for Re8earch in Cell Metabolism, Department of Biochemi8try, University of Oxford (Received 22 October 1964) During systematic assays of rat-liver enzymes related to gluconeogenesis it was noted that the activity of pyruvate kinase (ATP-pyruvate phosphotransferase, EC 2.7.1.40) showed exceptionally large variations when the dietary regime was changed. These are illustrated by Table 1. On changing from a standard diet to a low-carbohydrate diet the enzyme activity fell to about onethird, and on a high-carbohydrate (sucrose-casein) diet it rose more than threefold. Thus the difference in enzyme activity between diets low and high in carbohydrate was about tenfold. It is very probable, as the following considerations show, that these variations of pyruvate-kinase activity play a key role in the regulation of gluconeogenesis, and account in particular for the fact that gluconeogenesis ceases when the stores of carbohydrate in the liver, muscle and other tissues are 'full' to capacity, and when any surplus of carbohydrate is either oxidized to completion or converted into, and deposited as, fat. The mechanism by which the 'switch-over' from glycogen storage to fat storage is regulated has so far been obscure. Phosphoenolpyruvate, one of the reactants of the pyruvate-kinase system, is placed at a branching point of intermediary metabolism. In the liver it can be formed by glycolysis, though the rate of this process is usually slow and becomes zero when gluconeogenesis prevails. In this latter situation phosphoenolpyruvate, being an intermediate in the degradation of all major potential precursors of carbohydrate except glycerol, arises from the glucogenic amino acids, propionate, citrate and various other precursors. The phosphoenolpyruvate formed can enter two reactions: (1) The pyruvate-kinase reaction, requiring ADP and leading to pyruvate and subsequently acetylCoA. (2) The enolase reaction leading to 2-phosphoglycerate and subsequently carbohydrate. The continual occurrence of this reaction requires a supply of ATP to convert 3-phosphoglycerate into 1,3-diphosphoglycerate. The enzymes catalysing the above two reactions are both cytoplasmic, and the relative activity of the two enzymes therefore decides the extent of the two pathways. When the diet is low in carbohydrate, or in starvation, i.e. when gluconeogenesis occurs on a major scale, pyruvate-kinase activity is low. This stops, or slows down, the conversion of phosphoenolpyruvate into pyruvate. On the other hand, when the diet contains an excess of carbohydrate, the greatly increased pyruvate-kinase activity promotes the degradation of phosphoenolpyruvate to pyruvate, and subsequently acetyl-CoA, which may either be oxidized or form fatty acids. As shown in Table 1, the changes in pyruvatekinase activity that follow modifications of the Table 1. Effect of nutritional condition on the pyruvate-kina8e activity of rat liver Male Wistar rats weighing about 300 g. were used. Pyruvate kinase was assayed according to the method of Bucher & Pfleiderer (1955), slightly modified in that potassium phosphate, pH 7-4 (final conen. 67 mm), was used instead of triethanolamine plus KCI, and the concentration of ADP was 1X3 mm. The standard diet was Spiller's Small Laboratory Animal Diet, which contains about 55% of carbohydrate. The 'low-carbohydrate diet' was that used by Krebs, Bennett, de Gasquet, Gascoyne & Yoshida (1963), consisting of 75% of casein and 25% of margarine supplemented with minerals and vitamins, as were the sucrose-casein mixtures used. The results are given as means+ S.E.M., with the numbers of observations in parentheses. Pyruvate-kinase activity Diet (,umoles/g. fresh wt./min. at 250) Standard diet 27-2+2*6 (7) Low-carbohydrate diet 9.4+0.9 (7) Starvation (for 24 hr.) 11*9+0-8 (5) Starvation (for 48 hr.) 9*9+0*7 (7) Sucrose (70%)-casein (30%) (for 3 days) 83-7+5-6 (8) Sucrose (80%)-casein (20%) (for 1 day) 67 Sucrose (80%)-casein (20%/1) (for 2 days) 106 Sucrose (80%)-casein (20% ) (for 3 days) 93 4c H. A. KREBS AND L. V. EGGLESTON diet require a few days to reach a maximum. This suggests that they are due to changes in the rate of enzyme synthesis. However, variations of the rate of enzyme synthesis are not likely to be the only regulatory mechanism at the pyruvatekinase stage. This enzyme is inhibited by physiological concentrations of ATP (McQuate & Utter, 1959; Reynard, Hass, Jacobsen & Boyer, 1961; Lowry & Passonneau, 1964) and dependent on the supply of ADP, and, as mentioned above, ATP favours indirectly the enolase reaction. One can thus visualize that the relative concentrations of ATP and ADP also play a role in deciding the fate of phosphoenolpyruvate. Other intracellular constituents that can affect the activity of the enzyme are Ca2+ ions, which are powerful inhibitors, and K+ ions, which are activators (Kachmax & Boyer, 1953). Kidney-cortex pyruvate-kinase activity showed only small increases when the casein-sucrose diet was given, and no fall on the change from the standard diet to the low-carbohydrate diet. The activity of liver and kidney-cortex enolase underwent no measurable changes under the dietary regimes tested. The change in pyruvate-kinase activity is not the only component of the mechanisms that regulate the switch-over from glycogen storage to fat storage. Another component is the relative activity of the enzymes initiating the degradation of amino acids, especially the transaminases, serine dehydrase and threonine dehydrase (for details see Krebs, 1964). A third component is the activation and reactivation of glycogen-UDP glycosyltransferase (Danforth & Harvey, 1964), which is probably a major factor in the control of the fate of excess of dietary glucose, regulating the choice between deposition as glycogen and conversion into fat. Pyruvate-kinase deficiency may possibly play a part in some forms of glycogen-storage disease. Specific enzyme defects have been established for most forms of the disease, but not for all, and multiple enzyme defects cannot be excluded (Schmid, 1964; Hers, 1964; Lehoczky, Halasy, Simon & Harmos, 1964). Only one enzyme related to carbohydrate metabolism, other than pyruvate kinase, is so far known to undergo comparably drastic changes on feeding carbohydrate. Fitch & Chaikoff (1960) observed that the activity of glucose 6-phosphate 1965 dehydrogenase increased nine- to ten-fold on a diet rich in glucose or fructose. We have confirmed this for rats fed on a sucrose (80%)-casein (20%) mixture. Other enzymes of the oxidative pentose phosphate cycle show parallel but less marked changes. As Fitch & Chaikoff (1960) have pointed out, the increased activity of enzymes ofthe pentose phosphate cycle is most likely related to the increased need for NADPH2 when carbohydrate is converted into fat, a process that readily occurs on high-sugar diets (Hill, Bauman & Chaikoff, 1957). To sum up, the activity of pyruvate kinase of rat liver was found to be tenfold greater when a high-carbohydrate diet instead of a low-carbohydrate diet was given. A low pyruvate-kinase activity favours gluconeogenesis from amino acids and other precursors, and a high pyruvatekinase activity favours the degradation of carbohydrate and its conversion into fat. The activity of pyruvate kinase can thus play a key role in the switch-over from glycogen storage to fat storage. This work was aided by grants from the Rockefeller Foundation and the U.S. Public Health Service. Buicher, Th. & Pfleiderer, G. (1955). In Method8 in Enzymology, vol. 1, p. 435. Ed. by Colowick, S. P. & Kaplan, N. 0. New York: Academic Press Inc. Danforth, W. H. & Harvey, P. (1964). Biochem. biophy8. Re8. Commun. 16, 466. Fitch, W. M. & Chaikoff, I. L. (1960). J. biol. Chem. 235, 554. Hers, H. G. (1964). Ciba Found. Symp.: Control of Glycogen Metaboli8m, p. 318. Ed. by Whelan, W. J. & Cameron, M. P. London: J. and A. Churchill Ltd. Hill, R., Bauman, J. W. & Chaikoff, I. L. (1957). J. biol. Chem. 228, 905. Kachmar, J. F. & Boyer, P. D. (1953). J. biol. Chem. 200, 669. Krebs, H. A. (1964). Proc. Roy. Soc. B, 159, 545. Krebs, H. A., Bennett, D. A. H., de Gasquet, P., Gascoyne, T. & Yoshida, T. (1963). Biochem. J. 86, 22. Lehoczky, T., Halasy, M., Simon, G. & Harmos, G. (1964). Brit. med. J. ii, 802. Lowry, 0. H. & Passonneau, J. V. (1964). J. biol. Chem. 239,31. McQuate, J. T. & Utter, M. F. (1959). J. biol. Chem. 234, 2151. Reynard, A. M., Hass, L. F., Jacobsen, D. D. & Boyer, P. D. (1961). J. biol. Chem. 236, 2277. Schmid, R. (1964). Ciba Found. Symp.: Control of Glycogen Metabolism, p. 305. Ed. by Whelan, W. J. & Cameron, M. P. London: J. and A. Churchill Ltd.