Download (,umoles/g. fresh wt./min. at 250)

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.