Pyruvate and Glucose as Precursors of Acetoacetate

Abstract

Although carbohydrate is known to be "antiketogenic," pyruvate has been shown to form acetoacetate in cell-free liver preparations( 1,2,3). In the present series of studies, respiring homogenates of lactating guinea pig mammary gland were found to convert pyruvate and acetate to acetoacetate a t rapid rates, in contrast with a slow, but measurable rate of conversion of glucose to acetoacetate (4).The antiketogenicity of carbohydrate has been attributed to its ability to give rise to oxaloacetate, an explanation which is not accepted as entirely satisfactory( 5). Since ketogenesis is an alternative to lipogenesis (6), and the latter is a synthetic process requiring the participation of reduced pyridine nucleotide ( 7), the possibility was considered that the ability of glucose to regenerate reduced pyridine nucleotide in respiring systems might be a key factor in determining its low ketogenicity. This hypothesis could be tested by studying the behavior of lactate, a metabolite closely related to pyruvate, but whose oxidation, like the breakdown of glucose, is coupled with the reduction of DPN. Methods.Homogenates of mammary glands of lactating guinea pigs, prepared as described in a previous study of lipogenesis (8), and rabbit kidney cortex homogenates were incubated in a reaction mixture containing in addition to KCl: MgC12, 0.01 M; nicotinamide, 0.02 M; DPN, 3 x l P M ; ATP, 0.001 M ; phosphate buffer, pH 7.4, 0.01 M aminotrishydroxymethylmethane (tris) buffer pH 7.4, 0.01 M and carrier acetoacetate, 0.01 M. After incubation with C14-labeled substrates (0.005-0.01 M ) in the Warburg apparatus, the reaction mixtures were acidified and the bound C02 collected in the NaOH in the center wells which had served to absorb the COa during the measurements of O2 consumption. The contents of the center wells were then removed, replaced by fresh NaOH and the acetoacetate was decarboxylated by the addition of aniline citrate. After addition of carrier K2C03 the samples containing the respiratory COa and the terminal COZ of acetoacetate respectively were precipitated with BaCI2. Thin samples of BaC03 were mounted on planchets of 4.9 cm2 area and counted with a gas-flow counter with (hlicromil' window. After correction for self-absorption, the 'total counts' were calculated and converted to mpmole-equivalents of the radioactive substrate oxidized. Although the radioactivity of only the terminal carboxyl group of acetoacetate was determined, it was assumed that both halves of the molecule were equally labelled and the radioactive acetoacetate was calculated as being derived from two molecules of pyruvate, lactate or acetate and from one molecule of randomly labeled glucose respectively.Results. Experimental data representative of mammary and of kidney homogenates are shown in Table I. The high rates of conversion to acetoacetate of pyruvate and acetate are in contrast with the low ketogenicity of glucose and lactate. The addition of hexokinase, which accelerated the rate of aerobic glycolysis and increased the amount of glucos...