Liver cytosol preparations from 48 h starved rats, supplemented with 3 mM ATP, 0.5 mM sn-glycerol 3-phosphate, 10mM MgSO4 and a NADH-regenerating system showed a net glucose 6-phosphate formation from 2mM 3-phosphoglycerate of 0.72 micromol x min-1 x (g liver)-1. Glycerol 3-phosphate slightly increased when added at 0.5 mM; in its absence, dihydroxyacetone-phosphate formed from 3-phosphoglycerate was mainly reduced to glycerol 3-phosphate at the expense of fructose 1,6-bisphosphate and glucose 6-phosphate synthesis; when glycerol 3-phosphate was added at higher concentrations, net utilization occurred. The rate of glucose 6-phosphate formation positively correlated with the concentration of 3-phosphoglycerate and with the ratio of ATP/ADP. Increasing NADH concentrations stimulated the flow from 3-phosphoglycerate to glyceraldehyde 3-phosphate but also enhanced the reduction of dihydroxyacetone phosphate to glycerol 3-phosphate; as a result, the concentration of free NADH had little effect on the rate of glucose 6-phosphate formation. Lowering the concentration of magnesium from 10mM to 4mM, corresponding to free Mg2 concentrations of 7 and 0.6 mM respectively, resulted in a 30% increase of the rate of glucose 6-phosphate formation. The physiological relevance of the results presented is discussed.

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.

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A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors at near physiological concentrations was shown to form glucose 6-phosphate from malate + 3-phosphoglycerate at a rate of 1.11 0.09 pmol . min-' . g liver-' (mean f SEM, n = 9, 30°C). At least 70% of glucose 6-phosphate formed was derived from malate as calculated from experiments with [U-14C]malate. The indicated rates were measured between 10 min and 30 min incubation time when the system was near steady state with respect to the lactate/pyruvate ratio and to most of the gluconeogenic intermediates.In the absence of mitochondria, the rate of formation of glucose 6-phosphate from malate was about seven times lower than in their presence. A comparison between incubations carried out in presence or absence of mitochondria revealed that mitochondria decreased the lactate/pyruvate ratio and increased the ratio of (ATP + ITP)/(ADP + IDP). It could be shown that under the present incubation conditions, formation of glucose 6-phosphate was closely linked to the ratio of (ATP + ITP)/(ADP + IDP) whereas changing redox ratios had little influence on the gluconeogenic rate.Regulation of gluconeogenesis in liver is known to involve mitochondrial, cytosolic as well as microsomal reactions and has mainly been studied in liver slices, perfused liver or hepatocytes (for a review see [I]). Two early attempts have, however, been made to set up gluconeogenic systems which are readily accessible to low molecular weight effectors in order to obtain more detailed information on regulatory mechanisms. Mendicino and Utter [2] showed that a recombination of purified gluconeogenic enzymes was able to form glucose 6-phosphate from 3-phosphoglycerate or fumarate in the presence of chicken liver mitochondria. Furthermore, the group of Krebs [3,4] described a cell-free system of pigeon liver homogenate which formed glucose from lactate at physiological rates. In both cases, systems were employed where phosphoenolpyruvate carboxykinase is localized within the mitochondria [S,6] which is not the case in rat liver [7]. Attempts of Krebs et al. [3] to obtain gluconeogenesis in liver homogenates from rats and several other species were not successful and this system as well as the one of Mendicino and Utter [2] has not been used for further studies. Beside these investigations on the whole chain of gluconeogenesis in cellfree systems, some studies on partial sequences in subcellular fractions of rat liver have been made by the group of Lardy [8,9], by McDermott and Veneziale [lo] and by ourselves Recently, liver cells rendered permeable to substrates and effectors by treatment with filipin were proposed as a possible [I 1,121. model for studying metabolic regulation by low molecular weight compounds [13]. A different approach to the same problem is described in the present study which demonstrates the conversion of malate to glucose 6-phosphate at rates comparable to those observed in rat hepatocytes in a cell-free system derive...