Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study

Abstract

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

Cadmium chloride inhibits lactate gluconeogenesis in mouse renal proximal tubules: An in vitro metabolomic approach with 13C NMR

“…Metabolic rates were reported as means ± standard error of the mean (SEM) for four experiments and expressed as micromoles of substances removed or produced per gram dry weight per unit time. Enzymatic fluxes were calculated by combining enzymatic and NMR results with a mathematical model of renal lactate metabolism that has been derived from a model developed for 13 C alanine metabolism (Dugelay et al 1999) and used in previous publications (Faiz et al 2011;Renault et al 2010). Enzymatic fluxes were expressed as micromoles per gram dry weight per unit time.…”

“…These values are derived from the combination of the enzymatic and 13 C NMR spectroscopy results shown inTable 1and Tables 2-5. The calculations were derived from a mathematical model identical with one previously developed for 13 C-alanine(Dugelay et al 1999) and more recently used for other studies with 13 C-lactates as substrate(Faiz et al 2011;Renault et al 2010). The addition of 10 µM CdCl2 decreased fluxes through lactate dehydrogenase (-11%), pyruvate carboxylase (-19%), phosphoenolpyruvate carboxykinase (-28%) and glucose-6-phosphatase (-48%).…”

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.…”