Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver.
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 mumol/min per g of liver (mean +/- S.E.M., n = 8, 37 degrees 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.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology; D005943:Gluconeogenesis; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006600:Hexosephosphates / Q000096:biosynthesis*; D007773:Lactates / Q000378:metabolism*; D019344:Lactic Acid; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D011773:Pyruvates / Q000378:metabolism*; D019289:Pyruvic Acid; D051381:Rats; D011919:Rats, Inbred Strains
D005971:Glutamates; D006600:Hexosephosphates; D007773:Lactates; D011773:Pyruvates; D019344:Lactic Acid; D000431:Ethanol; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate