The Hormonal Control of Pyruvate Kinase Activity and of Gluconeogenesis in Isolated Hepatocytes

Abstract

Treatment of isolated rat hepatocytes with saturating concentrations of glucagon caused several modifications in the kinetic properties of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40): SO.5 (substrate concentration at half maximum velocity) for phosphoenolpyruvate was about doubled, whereas Vm.., was not changed; the activity measured at 0.15 mM phosphoenolpyruvate (physiological concentration) was reduced 65-80%; and there was also an increase in the Hill coefficient and in the affinity of the enzyme for the inhibitors Mg-ATP and alanine. Glucagon, 3':5'-cyclic AMP, and epinephrine caused an inactivation of pyruvate kinase together with a stimulation of gluconeogenesis. Insulin (10 nM) antagonized the effect of suboptimal doses of glucagon or cyclic AMP and of even maximal doses of epinephrine, on both pyruvate kinase activity and on gluconeogenesis. These observations can be explained by a phosphorylation of pyruvate kinase by cyclic-AMP-dependent protein kinase, as described by Ljungstrom et al. [(1974) Biochim. Biophys. Acta 358, 289-298] in a reconstructed system. They offer a molecular explanation for the hormonal control of gluconeogenesis. Glucose caused an inhibition of gluconeogenesis with no corresponding change in pyruvate kinase activity. The stimulation of gluconeogenesis by glucagon, 3':5'-cyclic AMP, and catecholamines, as well as the antagonistic effect of insulin against small doses of the hyperglycemic agents, has been described by several groups of investigators (for a review, see ref. 1). However, according to recent papers (2, 3), the sites of action of glucagon and catecholamines on hepatic gluconeogenesis are still unknown. A possible site of regulation of gluconeogenesis is at the level of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and it has indeed been shown by Krebs and Eggleston (4) that the activity of this enzyme was greatly increased in the livers of rats put on a high carbohydrate diet and was decreased upon fasting, when gluconeogenesis is enhanced. More recently, a rapid inactivation of pyruvate kinase by glucagon in isolated liver preparations has also been briefly reported (5, 6). (12). Two milliliters of cell suspension containing about 100 mg of cells were shaken (120 strokes/min) in stoppered 20 ml-vials at 370 in the presence of glucose (10 mM, unless otherwise stated), 0.1% bacitracin, and, when added, 10 nM insulin. Bacitracin was added to prevent degradation of the hormones (13). The gas phase was 95% 02 and 5% CO2. Glucagon, epinephrine, or cyclic AMP and 2 mM [U-14C]pyruvate, dissolved in isotonic NaCl, were added after 30 min of incubation. Five to 10 min later, 0.1 ml aliquots of the cell suspension were frozen at the temperature of solid CO2 in acetone for the determination of enzyme activities. Twenty minutes after the addition of the labeled precursor, 0.5 ml aliquots of cell suspension were taken in duplicate for the determination of radioactive glucose and glycogen formed from pyruvate.Measurement of Enz...

Regulation in vitro and in vivo of Adenosine 3′:5′‐monophosphate‐Dependent Inactivation of Rat‐Liver Pyruvate Kinase Type L

“…The main interest of this mechanism is to be under the control of several hormones such as glucagon and insulin which are involved in the control of gluconeogenesis [4]. It superimposes harmoniously on the control by allosteric effectors, since the metabolites that favour the active conformation of the enzyme are also those that prevent its inactivation by phosphorylation and conversely.…”

“…The activity of pyruvate kinase was measured as described previously, at 0.1 5 mM phosphoenolpyruvate [4]. This activity, referred as 2'0 15, represents 45 9', of V , measured at 5 mM phosphoenolpyruvate, when the enzyme is in its active form as obtained upon incubation of a cellular extract for 30 min at 25 "C in the prescnce of 5 mM MgC12.…”

“…Hepatocytes were isolated from livers of fed Wistar rats as previously described [4] and resuspended in a Krebs-Henseleit bicarbonate buffer [11 ] containing 10 mM glucose. 10 ml of cell suspension containing about 0.5 g of cells were shaken at 120 strokesimin for 30 min at 37 "C in 50-ml conical flasks stoppered with a rubber cap and filled with 95 "/; 0 2 and S COa.…”

“…Lactate dehydrogenase (EC 1. major role in the hormonal control of gluconeogenesis in the liver [4]. The purpose of the present work was to investigate some kinetic properties of the enzymatic system which, in a crude extract of rat hepatocytes, allows the interconversion of the two forms of pyruvate kinase L. Hepatocytes were used rather than the whole liver to avoid interference with an other pyruvate kinase, present in non-parenchymal liver cells [9].…”

“…In a paper published while this work was in progress, Berglund et al [lo] have reached a similar conclusion. Among the ligands that affect the activity of pyruvate kinase through this dual mechanism, phosphoeizolpyruvate was the most likely to play an important physiological role, since its concentration is assumed to control the rate of gluconeogenesis [4].…”