A role for bicarbonate in the regulation of mammalian glutamine metabolism

Abstract

1. The concentration of HCO3- (independent of any change of pH) exerts different effects on glutamine metabolism in rat kidney-cortex tubules, hepatocytes and enterocytes.2. In kidney tubules HCO3- (10.5-50 MM) has no effect on glutaminase (EC 3.5.1.2), whereas glutamate dehydrogenase (EC 1.4.1.3) is inhibited as HCO3- concentration is increased. The result is that flux through the entire glutamate-to-glucose pathway is inhibited by increasing HCO3- concentrations. A large proportion (more than 30%) of the glutamine removed undergoes complete oxidation. 3. In hepatocytes, and to a smaller extent in enterocytes, HCO3- is an accelerator of glutaminase. Synthesis of glucose and urea from glutamine in hepatocytes increases as HCO3- concentration is increased. Calculations show that fumarate, formed via aspartate aminotransferase and arginino-succinate lyase, is the precursor of the glucose. There is no complete oxidation of the carbon skeleton of glutamine in hepatocytes. 4. Leucine at near-physiological concentrations (0.1-1 mM) is an accelerator of glutaminase in hepatocytes, but not in kidney tubules or in enterocytes. 5. The results are discussed in relation to regulation of acid/base balance in vivo.

The Rabbit Kidney Tubule Simultaneously Degrades and Synthesizes Glutamate

“…Metabolite Assays-Glucose, glycogen, lactate, pyruvate, glutamate, glutamine, alanine, aspartate, citrate, ␣-ketoglutarate, fumarate, malate, glycerol, and glycerol 3-phosphate were determined by usual enzymatic methods, and the dry weight of tubules added to the flasks was determined as described previously (11,13). 13 C NMR Techniques-These were recorded at 100.6 MHz on a Bruker AM-400 WB spectrometer using a 10-mm broadband probe thermostated at 8 Ϯ 0.5°C.…”

“…13 C NMR Techniques-These were recorded at 100.6 MHz on a Bruker AM-400 WB spectrometer using a 10-mm broadband probe thermostated at 8 Ϯ 0.5°C. Magnet homogeneity was adjusted using the deuterium lock signal.…”

“…where L m is the amount of 13 C measured in the corresponding NMR resonance, l m is the natural 13 C abundance (1.1%) multiplied by the amount of the metabolite assayed enzymatically, E s is the 13 C enrichment of the C-1 or C-2, or C-3 or C-5 of glutamate, and e s is the natural 13 C abundance. Table I, rabbit kidney tubules metabolized glutamate at high rates; glutamate utilization was linear with time over 60 min.…”

“…In all experiments, zero time flasks, with and without substrates, were prepared by adding perchloric acid before the tubules. When radioactive glutamate or bicarbonate was present in the medium, incubation, deproteinization, collection, and measurement of the 14 CO 2 formed were performed as described by Baverel and Lund (13). After removal of the denaturated protein by centrifugation, the supernatant was neutralized with a mixture of 20% (w/v) KOH and 1% (v/v) H 3 PO 4 (8 M) for metabolite determination, measurement of bicarbonate fixation, and NMR spectroscopy measurements.…”

“…Modeling of the data obtained by 13 C NMR spectroscopy indicates that, during glutamate utilization via mainly glutamate dehydrogenase, glutamine synthetase, and alanine aminotransferase, glutamate synthesis also occurs at high rates. In addition, our study demonstrates that addition of acetate reduces net removal of glutamate by inhibiting not only net flux through glutamate dehydrogenase in the oxidative deamination direction but also by inhibiting glutamine, alanine, and serine synthesis.…”