The Anaplerotic Substrate Alanine Stimulates Acetate Incorporation into Glutamate and Glutamine in Rabbit Kidney Tubules

Abstract

Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [13 C]acetate and [ 14 C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO 2 ; with acetate plus alanine, the corresponding values were decreased to 49 and 25%. The only other important products formed from the acetate carbons were glutamine, and to a smaller extent, glutamate. By combining 13 C NMR and radioactive and enzymatic measurements with a novel model of acetate metabolism, fluxes through the enzymes involved were calculated. Thanks to its anaplerotic effect, alanine caused a stimulation of acetate removal and a large increase in fluxes through pyruvate carboxylase, citrate synthase, and the enzymes involved in glutamate and glutamine synthesis but not in flux through ␣-ketoglutarate dehydrogenase. We conclude that the anaplerotic substrate alanine not only accelerates the disposal of acetate but also prevents the wasting of the latter compound as CO 2 .Acetate is the main circulating volatile fatty acid in humans and other mammalian species. Its blood concentration is low (less than 0.2 mM) in fed and starved humans and starved herbivores but may reach the millimolar range in humans after alcohol consumption and in fed herbivorous species (1-7). The sources of blood acetate are on the one hand absorption of the acetate formed as a result of gastrointestinal bacterial fermentation, and on the other hand, the acetate formed and released by various tissues containing acetyl-CoA hydrolase activity (2,3,6,8).On the basis of experiments performed in vivo with labeled acetate, it has been shown that the turnover of circulating acetate is rapid and that, depending on the species and nutritional state, the oxidation of this compound provides from 6 to 70% of the whole body energy expenditure (3,4,7,9). This means that acetate is removed and metabolized by peripheral tissues. Indeed, acetyl-CoA synthetase, the enzyme that initiates acetate degradation, has been demonstrated to be active in many tissues including the liver, kidney, heart, brain, adipose tissue, and skeletal muscle (3). It has been found that, besides the heart, the kidney contains a high activity of this enzyme (3).In agreement with this observation, we have shown in a recent study that acetate is readily metabolized by suspensions of rabbit renal proximal tubules (10). In the same study (10), we have demonstrated that acetate significantly altered the metabolism of alanine, a major precursor of glutamine in these tubules. For this, we used 13 C-labeled alanine and unlabeled acetate in combination with enzymatic and 13 C NMR spectroscopy measurements to calculate metabo...

Complexity of glutamine metabolism in kidney tubules from fed and fasted rats

“…The model of glutamine metabolism used in the present study is based on principles that have been used and validated in previous work from this laboratory for studying glucose, glutamate, alanine and acetate metabolism in isolated rabbit kidney tubules [34][35][36][37]. It is applicable under conditions where the accumulation and labelling of products of glutamine metabolism are approximately linear with time.…”

“…However, given the potential importance of, and the renewed interest in, renal gluconeogenesis from glutamine, especially in the fasted state, we felt that it was especially important to re-examine and characterize thoroughly this metabolic process in both the fed and the fasted states. For this, we have incubated isolated kidney tubules in the presence of 13 C-labelled and 14 C-labelled glutamine, and have taken advantage of the use of 13 C NMR spectroscopy, which has allowed us to unravel the complexity of the renal metabolism of various substrates in previous studies from this laboratory [34][35][36][37]. With the data obtained, which were combined with a model of glutamate and glutamine metabolism developed previously [38], we were able to estimate the fluxes through enzymes of glutamine metabolism in kidney tubules from fed and fasted rats.…”

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable

“…These segments, made of the entire intestinal wall to mimic in vitro the structure of the small intestine in vivo, were incubated with 13 C-labelled glutamine. Taking advantage of the 13 C NMR spectroscopy and enzymatic approaches that enabled us to unravel the complexity of the renal metabolism of various substrates [17][18][19][20][21][22], we measured glutamine utilization and product accumulation. Despite high rates of glutamine metabolism, substantial glucose synthesis could not be detected.…”

Infusion of an acidified ethanolic—dextrose solution enhances urinary ammonium excretion and increases acid resilience in non—mechanically ventilated acidotic rabbits

“…Like the kidneys of other species, the rabbit kidney has a high capacity to utilize acetate as a substrate ( Chauvin et al, 1997 ). Following uptake by renal cells, acetate readily gains entry to mitochondria, and thereafter undergoes an intricate series of metabolic cycles during its metabolism, involving both the mitochondrion and cytosol ( Chauvin et al, 1997 ; Dugelay et al, 1999 ; Conjard et al, 2002 ). The oxidative metabolism of acetate would have generated considerable amounts of bicarbonate, which would have been returned to the blood stream, directly bolstering plasma bicarbonate levels.…”