Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver

Abstract

A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors and gluconeogenic intermediates at near-physiological concentrations was shown to form hexose 6-phosphates linearly from lactate + pyruvate + glutamate at a rate of 0.82 + 0.05 ,umol/min per g of liver (mean + S.E.M., n = 8, 37°C). The indicated rates were measured between 20 min and 60 min incubation time, when the system was near steady state. Experiments with either [1-14C]lactate or [U-14C]glutamate revealed that the incorporation of radioactive label into hexose 6-phosphates was proportional to the utilization of lactate + pyruvate and of glutamate during incubation and that both served as gluconeogenic substrates at a ratio of about 2:1. When the [ATP]/[ADP] ratio was lowered from 60 to 19 by addition of ATPase, the rate of hexose 6-phosphate formation fell to one-third. This decrease in gluconeogenic flux was mainly due to a decreased flow through the phosphoglycerate kinase step. Hexose 6-phosphate formation could also be decreased by increasing the ratio [NADH]/[NAD+], either by addition of ethanol or by increasing the initial concentration of lactate+ pyruvate at a fixed ratio of 10:1. The observed inhibition was linked to a limitation in the availability of oxaloacetate for the phosphoenolpyruvate carboxykinase reaction and to an increased formation of sn-glycerol 3-phosphate. Finally, the rates of hexose 6-phosphate formation in incubations with cytosols from fed rats were only 50% of those observed with cytosols from animals starved for 48 h. One of the limiting steps was found to be the flow through the phosphoenolpyruvate carboxykinase step.

Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

“…The ratio of 19 calculated on the basis of the present results is considerably higher than the cytosolic ratio of ATPtota1/ADPtotal of 2.6-10 found by various fractionation techniques (Siess & Wieland, 1976;Tischler et al, 1977;Akerboom et al, 1978;Soboll et al, 1978). However, the present results agree well with previous findings showing that a ratio of at least 20 is a prerequisite for obtaining physiological rates of gluconeogenesis in cell-free systems (Morikofer-Zwez et al, 1982;Stoecklin et al, 1986).…”

“…However, cytosolic ratios of ATP/ADP reported for rat liver and rat hepatocytes are not higher than 10 (Siess & Wieland, 1976;Tischler et al, 1977;Akerboom et al, 1978;Soboll et al, 1978). This apparent discrepancy may stem from the fact that in cell-free, highly diluted, systems the adenine nucleotides are essentially present as free compounds (Stoecklin et al, 1986), whereas in the cell they may be bound in part to cytosolic proteins. Since metabolic regulation by adenine nucleotides depends on their free concentration, it seems essential to determine this parameter for cellular conditions.…”

“…In cell-free systems derived from rat liver, gluconeogenesis is strongly stimulated by increasing ratios of ATP/ADP (M6rikofer-Zwez et al, 1981(M6rikofer-Zwez et al, , 1982Stoecklin et al, 1986). A linear relationship between the gluconeogenic rate and the ratio of ATP/ADP, ranging from 20 to 60, was observed.…”

New kinetic parameters for rat liver arginase measured at near-physiological steady-state concentrations of arginine and Mn2+

“…of liver from rats killed by decapitation were immediately homogenized (Potter/ Elvehjem homogenizer) at 4°C with 15 ml of a buffer containing 0.14 M-sucrose, 50 mM-potassium phosphate buffer, pH 7.2, 2 mM-GSH and 0.1 mM-EDTA in order to bind bivalent cations. The homogenate was centrifuged, the cytosol was freed from EDTA and other low-Mr compounds by gel filtration and was further treated as described previously (Stoecklin et al, 1986).…”

“…Metabolites were determined in the neutralized extracts as described previously (Stoecklin et al, 1986). Urea was determined as described by Kersher & Ziegenhorn (1985).…”