Janet M. Wimhurst

Role of bivalent cations in the control of enzymes involved in gluconeogenesis.

Krebs et al. [1] found that gluconeogenesis by kidney slices was dependent on the availability of Ca 2+ in the medium. The function of Ca 2+ is uncertain but may be to prevent the breakdown of phosphoenolpyruvate by pyruvate kinase. Since the three key enzymes required for the reverse of the glycolytic sequence pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose1,6-diphosphatase are all activated by bivalent cations such as Mg 2+, it is likely that their action is also inhibited by Ca 2+ [2,3]. Thus the control of the relative rates of glycolysis and gluconeogenesis could result from translocation of Ca 2+ between mitochondrion and cytoplasm, differentially altering the extent of inhibition of pyruvate kinase and pyruvate carboxylase [4]. This would assume that phosphoenolpyruvate carboxykinase, which is cytosolic in species like the rat and fructose-l, 6-diphosphatase are not seriously inhibited by Ca 2+. Its effect on the activity of these enzymes in rat liver has not been systematically investigated, though inhibition of fructose-l,6-diphosphatase from trout liver and from muscle has recently been reported [5,6]. Mn 2+ can offset the inhibitory effect of Ca 2+ on mitoehondrial carboxylation of pyruvate [3] ; this may result partly because both Mn 2+ and Mg 2+ can activate carboxylase [3], which when activated by Mn 2+ is much less sensitive to inhibition by Ca 2+. In the present study, a comparison of the ability of Mn 2+ and Mg 2+, separately or together, to activate the three key gluconeogenic enzymes has been made and the effect of these ions on Ca 2+ inhibition has been investigated.

Comparison of ability of Mg and Mn to activate the key enzymes of glycolysis

Mg and Mn are activating ions for all enzymes employing adenine nucleotides and for many others. Ca by contrast is often an inhibitor. Interaction between the ions has been suggested as a possible mechanism involved in the control of metabolism including the pathways of glycolysis and gluconeogenesis. We have already published some kinetic parameters for the interaction of these ions with the enzymes of hepatic gluconeogenesis [ 11. The present study is concerned with the effects of bivalent ions on the activities of key glycolytic enzymes of liver.

The actions of insulin and glucagon on glucose metabolism and on related enzyme activities in the isolated perfused rat liver

Abstract 1. 1. The metabolic response of perfused livers to glucagon or insulin stimulation has been followed, measuring glucose, amino acid, urea, lactate, pyruvate, lipid and β-hydroxybutyrate levels in the perfusate. 2. 2. Analysis of these perfused livers shows changes in the activities of some of the key glycolytic, gluconeogenic and lipogenic enzymes. Lactate perfusion increases pyruvate carboxylase and decreases isocitrate dehydrogenase; these changes are prevented by insulin and glucagon respectively. Under these conditions, both hormones decrease phosphofructokinase, and insulin also increases fructose-1, 6-diphosphatase. At endogeneous substrate levels, insulin looses its effect on the latter enzyme. In both types of perfusion insulin produces a 50% drop in phosphoenolpyruvate carboxykinase and an increase in pyruvate kinase. 3. 4. Neither hormone has any effect on glucokinase, hexokinase, glucose-6-phosphatase, acetyl-CoA carboxylase, ATP-citrate lyase or malic enzyme.

Stimulation of mitochondrial pyruvate carboxylation by Mn2+ and the distribution of the products between mitochondrion and medium

The CO2 formed by the oxidative decarboxylation of pyruvate in mitochondria can be used to carboxylate more pyruvate to oxalacetate. The latter compound either condenses with the acetyl CoA formed as one of the decarboxylation products or is reduced to malate [ 1,2] . A third path, mediated by GTP, leads from oxalacetate to phosphoenolpyruvate with release of CO2 ; this process requires a supply of phosphate [3] and is stimulated by long chain fatty acids or uncouplers [4] , otherwise it is slow. Mehlman, Walter and Lardy [5], showed that the main products containing 14C when mitochondria were incubated with pyruvate and 14C-bicarbonate, were malate and citrate with fumarate equal to about 20% of the malate. They, as well as Stuart and Williams [2] , noted that some unlabelled precursor, suggested to be fatty acid, also fed carbon into the cycle. It seems valid to regard the increments in malate and citrate, in the presence of fluorocitrate to’block the aconitase, as a good measure of the pyruvate carboxylated when uncoupler is not added, and the experiment avoids ambiguity about the source of the carbon. The products distribute between the mitochondria and the medium with high internal/external ratios [681, and there is competition between the anions to neutralise the mitochondrial cations. It is important to know the distribution, because in the intact cell it is the cytoplasmic concentration of citrate which is one of the controls of phosphofructokinase and acetyl CoA carboxylase. This paper presents values for the distribution and shows that Mn2+ stimulates mitochondrial

Effects of manganese on the activity of glycolytic and gluconeogenic enzymes in the perfused rat liver

We have shown previously [l] that pyruvate carboxylase activity declines when liver from the fasted rat is perfused with glucose containing medium. Conversely addition of lactate to the perfusate enhances the activity of both pyruvate carboxylase and phosphoenolpyruvate carboxykinase [2]. It appears that the hepatic activity of these two enzymes may fluctuate depending on the concentration of the final product and on the supply of precursors for transformation. Administration of manganese results in a rapid increase in serum glucose [3] and is reported to enhance glucose production by the perfused liver [4]. It is a potent activator of several glycolytic and gluconeogenic enzymes including specifically pyruvate carboxylase and phosphoenolpyruvate carboxykinase [5,6]. We here report that addition of manganese to the perfused rat liver prevents the increase in activity of the two enzymes otherwise induced by lactate. This seemingly contradictory event is interpreted to mean that the extra functional capacity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase resulting from manganese activation minimises the influences which would otherwise induce extra enzyme synthesis.

Suppression of pyruvate carboxylase by glucose in the perfused rat liver.

The hepatic levels of three of the key enzymes of gluconeogenesis, pyruvate carboxylase (E.C. 6.4.1. l), phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) and glucose-6-phosphatase (E.C. 3.1.3.9) rise in fasting and conversely decrease on refeeding [l-5] , the fourth enzyme, fructose-l ,6-diphosphatase (E.C. 3.1.3.1 l), decreasing during an overnight fast [6-71 . The levels of these enzymes are also elevated in diabetes and lowered on treatment with insulin [8-121. It is thus likely that insulin rather than food per se is the suppressor of the enzyme levels in the fedstate, the glucose intake stimulating insulin secretion. If this is so, it is not to be expected that addition of glucose would influence the levels of these enzymes in the perfused liver. This appears to be the case for three of the four enzymes, but we find that the activity of pyruvate carboxylase does decline under such conditions.