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

S.H Lee,E.J Davis

doi:10.1016/s0021-9258(17)37934-6

Abstract

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.

Highlights

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
The value of about 24 pmol of propionate consumed (Table IX) corresponds very closely to an independent estimation (Table X) of 3-carbon products formed from propionate. This value is, substantially lower than the maximum rate at which the hindquarter is capable of removing citrate cycle intermediates, based on the following observations: (a) the tissue level of malate and fumarate is greatly diminished by arsenite, suggesting that when propionate is the sole source of 4-carbon acids, the rate of their production becomes ratelimiting for their subsequent decarboxylation; and (b) based on somewhat more indirect calculations, this rate is substantially higher when arsenite is not present
Garber et al [19] and Goldstein and Newsholme [21] have recently proposed schemes for alanine synthesis by muscle which are identical with that which we offered earlier [6, 15], except that these workers suggested that the NADP+-malic enzyme (EC 1.1.1.40) [19] or phosphoenolpyruvate carboxykinase (EC 4.1.1.32) plus pyruvate kinase (EC 2.8.1.40) [20] would provide pyruvate from 4-carbon dicarboxylic acids

Summary

AND METHODS

Perfusions-Female rats (180 to 200 g) fed a stock diet were used. Perfusions were performed as described by Ruderman et al [23], except as modified in the description below. In a glass container chilled with ice and water, the pluronic polyol solution in Krebs-Henseleit buffer was gassed with carbon dioxide for 5 to 10 min, after which the carbon dioxide entrance tube was raised about 0.5 cm above the surface to maintain a carbon dioxide atmosphere, and the l-cm diameter probe of the sonicator was placed into the above solution about 3 cm below the surface With this probe activated (100 watts), 62 ml of FC-47 fluorocarbon was slowly introduced to the solution through a long slightly bent hypodermic needle, the tip of which was positioned slightly below the end of the probe [24]. MO or from Boehringer Mannheim Corp., New York, NY

AND DISCUSSION

Glutamate

CONCLUSION