Carboxylation and decarboxylation reactions. Anaplerotic flux and removal of citrate cycle intermediates in skeletal muscle.

A system for in situ perfusion of rat hindquarters using a fluorocarbon for oxygen and CO2 exchange, and a polyol to provide oncotic pressure is described. Perfusion with glucose plus insulin resulted in no significant change in the tissue level of citrate cycle intermediates, phosphocreatine, ATP, ADP, AMP, and glycogen. Glucose was consumed at a linear rate, and lactate, pyruvate, alanine, glutamine, glutamate, and citrate were released into the perfusing medium. Inclusion of pyruvate resulted in elevation of citrate cycle intermediates and alanine, whereas acetate elevated the level of cycle intermediates without significant effect on tissue alanine or its release. Radioactivity from NaH[14C]O3 was incorporated into citrate cycle intermediates, glutamate, aspartate, and lactate by glucose-perfused hindquarters, the extent of which was markedly elevated as the tissue pyruvate was increased. When pyruvate was in the physiological range, acetate caused elevation in incorporation of CO2 into these metabolites, increased the concentration of citrate, and doubled the concentration of acetyl-CoA. Thirty-five to forty-four per cent of 14C incorporated into citrate was retained after enzymic degradation to 2-oxoglutarate. Perfusion with [2-14C-]propionate led to elevation in the level of citrate cycle intermediates, and radioactivity was incorporated into the latter, as well as glutamate, aspartate, lactate, pyruvate, alanine, and CO2. Two independent calculations estimated the rate of flux of 4-carbon cycle intermediates to 3-carbon metabolites of about 68 mumol/h (approximately 38 nmol/min/g of tissue), a rate in excess of those reported for alanine release from human or rat muscle during starvation. Arsenite blocked carbohydrate flux through the citrate cycle and effected accumulation of lactate, pyruvate, alanine, and 2-oxoglutarate. Flux from 4- to 3-carbon acids was diminished by arsenite, apparently as a result of lowered substrate concentration for decarboxylation. 3-Mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase, was without effect on the parameters studied, suggesting that this enzyme is not involved in the decarboxylation reaction. It is concluded that (a) a constant level of citrate cycle intermediates is maintained in part by continuous flux of carbon into and out of the cycle by carboxylation and decarboxylation reactions; (b) the carbon skeleton of alanine released from skeletal muscle is derived in part from other amino acids which are catabolized to cycle intermediates; and (c) the subsequent removal of these intermediates is probably mediated by malic enzyme(s) (EC 1.1.1.40, or 1.1.1.36, or both.