In human and primate liver, phenylacetate and glutamine form phenylacetylglutamine, which is excreted in urine. Probing noninvasively the labeling pattern of liver citric acid cycle intermediates with phenylacetylglutamine assumes that the labeling pattern of its glutamine moiety reflects that of liver alpha-ketoglutarate. To validate this probe, we infused monkeys with [U-13C3]lactate, [3-13C]lactate, [1, 2-13C2]acetate, [2-13C]acetate, [U-13C3]glycerol, or 2-[3-13C]ketoisocaproate and compared the labeling patterns of urinary phenylacetyl-glutamine with those of glutamate and glutamine in liver, plasma, muscle, and kidney and liver alpha-ketoglutarate. Only with [U-13C3]lactate or [3-13C]lactate does the labeling pattern of phenylacetylglutamine reflect patterns of liver alpha-ketoglutarate and glutamate. With [13C]acetate, muscle and kidney glutamate are more labeled than liver metabolites. This confirms that with [13C]acetate, the labeling pattern of liver metabolites is influenced by 13CO2 and [13C]glutamine made in peripheral tissues. Our data validate the use of phenylacetylglutamine labeled from [3-13C]lactate or [3-13C]pyruvate to probe noninvasively the pyruvate carboxylase-to-pyruvate dehydrogenase flux ratio in human subjects.
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.
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.
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