Utilization of alanine for glucose formation in isolated rabbit kidney‐cortex tubules

Abstract

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as giucose precursor, when added as a sole substrate. However, this amino acid decreases glucon#~nesis from low (up to 1 mM) '2-oxoglutarate concentrations and stimulates this process at higher (2.5-10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-I[J-V]alanine to glucose from 8-to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low p-14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis.Alanine; Gluconeogenesis; (Rabbit renal tubule) 1, INTRODUCTIONAlthough alanine aminotransferase is located in the straight portion of proximal nephron [l] it has been shown that in contrast to the rat perfused liver 121 and isolated hepatocytes [3,4], L-alanine is not used as glucose precursor in rat kidney cortex slices [5] and isolated renal cortex tubules derived from rat [6] and guinea pig [7]. Alanine does, however, exert a stimulatory effect on glucose synthesis from pyruvate and lactate in rat renal cortical tubules, probabIy due to an inhibition of pyruvate kinase activity by this amino acid [6], a potent inhibitor of the enzyme [8].It is established that the major role of alanine in renal metabolism reside in its production by renal tubules [9]. However, Michoudet et al. [IO] have recently reported that in guinea pig renal cortex tubules alanine may be converted into glutamine with the use of pyruvate carboxylase which catalyses the fixation of CO;! on pyruvate to give oxaloacetate for synthesis of citrate and then of aconitate and 2-oxoglutarate. Moreover, as concluded from 13C-NMR studies with the use of renal epithelial cells [ 111, 13C label from L-[13C]alanine may enter tricarboxylic acid cycle through both pyruvate dehydrogenase and pyruvate carboxylase pathways, giving rise to 13C-labelled glutamate. Since pyruvate carboxylase is the first enzyme of gluconeogenesis, studies were undertaken to establish conditions for MATERIALS AND METHODS AnimalsTermond strain, male white rabbits (2-3 kg body weight) were used throughout. Animals were maintained on standard rabbit chow. Preparation and incubation of kidney-cortex tubulesRenal cortex tubules from rabbits were isolated using basically the procedures of Burg and Orloff 1121 and Guder et al. [13] as described previously [14]. The dry weight of tubules was determined according to Krebs [IS]. Isolated kidney tubules (10-13 mg dry weight) were incubated at 37°C in 2 ml of Krebs-Henselei...

Glutamine synthesis is heterogeneous and differentially regulated along the rabbit renal proximal tubule

“…In agreement with previous findings, suspensions of rabbit proximal tubules, which contain all parts of the proximal tubules, have been shown to synthesize glutamine from alanine [11], aspartate [11][12][13], glucose plus ammonia [14] and glutamate [15]. However, despite the fact that glutamine synthetase activity was about the same in the three different parts of the rabbit proximal tubule [1], it remains at present unknown whether or not these three different segments of the proximal tubule synthesize glutamine from a given precursor at the same rates.…”

“…Since glutamine and to a lesser extent, glutamate, are almost the only non-volatile carbon end-products of alanine and aspartate metabolism in rabbit proximal tubules [11][12][13], one may determine, to a reasonable degree of accuracy, that the rates of alanine or aspartate utilization by the S1, S2 and S3 segments are twice the sum of the rates of utilization of glutamine and glutamate found (see Tables 1 and 2), because two alanine or two aspartate molecules are required for each glutamate or glutamine found. Given the high rates of alanine and aspartate utilization, which were of a similar magnitude as those of 5 mM alanine and 5 mM aspartate in a given segment of the proximal tubule, one may infer that high alanine\aspartate aminotransferase activities are present along the entire proximal tubule ; in this respect, the proximal tubule of the rabbit differs from that of the rat, in which a high alanine aminotransferase activity was only observed in the pars recta [29].…”

Glutamine synthesis is heterogeneous and differentially regulated along the rabbit renal proximal tubule

“…In agreement with previous findings, suspensions of rabbit proximal tubules, which contain all parts of the proximal tubules, have been shown to synthesize glutamine from alanine [11], aspartate [11][12][13], glucose plus ammonia [14] and glutamate [15]. However, despite the fact that glutamine synthetase activity was about the same in the three different parts of the rabbit proximal tubule [1], it remains at present unknown whether or not these three different segments of the proximal tubule synthesize glutamine from a given precursor at the same rates.…”

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

“…1.8-fold greater than that found by Lietz et al [7]. This is probably because our tubules utilized alanine at a rate that was more than twice that found in the other laboratory [24].…”