Metabolism of Isolated Kidney Tubules. Oxygen Consumption, Gluconeogenesis and the Effect of Cyclic Nucleotides in Tubules from Starved Rats

Guder, W. G.; Wiesner, Wolfgang; Stukowski, Barbara; Wieland, O.

doi:10.1515/bchm2.1971.352.2.1319

Cited by 96 publications

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“…Renal 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].…”

Section: Preparation and Incubation Of Kidney-cortex Tubulesmentioning

confidence: 99%

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

Zabłocki¹,

Bryła²

1989

FEBS Letters

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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...

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Section: Preparation and Incubation Of Kidney-cortex Tubulesmentioning

confidence: 99%

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

Zabłocki¹,

Bryła²

1989

FEBS Letters

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“…Kidney tubules were prepared as described by Guder et al (1971), as modified by Baverel et al (1978).…”

Section: Preparation Ofkidney-cortex Tubulesmentioning

confidence: 99%

Fate of glutamate carbon and nitrogen in isolated guinea-pig kidney-cortex tubules. Evidence for involvement of glutamate dehydrogenase in glutamine sythesis from glutamate

Baverel¹,

Genoux²,

Forissier³

et al. 1980

Biochemical Journal

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1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[ 1-_4C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through a-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222,[813][814][815][816][817][818][819][820] and Preuss [(1972) Am. J. Physiol. 222, 1395-13971. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenousdifference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.Since the initial work of Krebs (1935), it is known that, in contrast with kidney-cortex slices of dog, cat, pig, rat, sheep and pigeon, slices of guinea pig and rabbit do not release ammonia when incubated with glutamate. Krebs concluded that this was so in both the latter species because the ammonia formed by the deamination of glutamate via glutamate dehydrogenase was used for glutamine synthesis (see Krebs, 1935). However, the role of glutamate dehydrogenase in the metabolism of glutamate by guinea-pig and rabbit kidney slices has been questioned by and Preuss (1972), because these authors observed that glucose was formed from glutamate without concomitant accumulation of Vol. 188 ammonia; they concluded that the initial step in glutamate metabolism was transamination rather than oxidative deamination. So the pathways of renal metabolism of glutamate in these species remain uncertain.In an attempt to clarify this subject, we studied glutamate metabolism in isolated guinea-pig kidney tubules. The data obtained support the view of Krebs (1935) that glutamate dehydrogenase plays a central role in the renal metabolism of glutamat...

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“…Confluent cultures were incubated for a further 72-hour period, and lactate and pyruvate concentra tions in culture media were determined at 24-hour intervals. The results are summarized in figure 5. O K gng+ consumed lactate very rap idly when supplied at 10 mM ( fig.…”

Section: Metabolism Ofokasa* Cellsmentioning

confidence: 93%

“…Mapping of the dis tribution of key gluconeogenic enzymes along the nephron [ 1.4] as well as metabolic studies using isolated tubular fragments [5][6][7] re vealed that in the kidney the gluconeogenic capacity is exclusively confined to proximal tubular cells. Main gluconeogenic substrates in vivo are lactate, pyruvate, glutamine, gluta mate, proline, and glycerol [1,3,5]. Fatty acids, like oleate, have been shown to exert a profound regulatory influence on renal lactate and pyruvate gluconeogenesis [8.…”

Section: Introductionmentioning

confidence: 99%

A Novel Gluconeogenic Strain of OK Cells with Metabolic Properties Different from Gluconeogenic LLC-PK<sub>1</sub> Cells

Gstraunthaler¹,

Thurner²,

Weirich‐Schwaiger³

et al. 1993

Cell Physiol Biochem

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By selective adaptation to glucose-free tissue culture conditions, a gluconeogenic substrain of the opossum kidney (OK) cell line, designated OK_gNg+, was established, analogous to the method recently employed for the isolation of gluconeogenic LLC-PK₁-FBPase+ cells [Gstraunthaler and Handler, Am J Physiol 1987;252:C232-C238]. OK_Gng+ cells express considerable amounts of fructose-1, 6-bisphosphatase activity, and metabolic flux through phosphoenolpyruvate carboxykinase (PEPCK), which is indicative for their gluconeogenic capacity, in contrast to OK wild-type cells. OK_gng+ cells also seem to exhibit a higher degree of differentiation indicated by an increased expression of γ-glutamyltranspeptidase activity. Karyotype analysis revealed that OK_gng+ cells are descendants of OK wild-type cells, exhibiting slight increases in tetraploidy. Remarkable differences between OK_gng+ and LLC-PK₁^-FBPase+ cells have been found in terms of substrate metabolism of the two gluconeogenic renal sub-strains. Cultures were incubated in culture medium containing either Na-L-lactate, Na-pyruvate or lactate/pyruvate mixtures (10:1, 5:5, 1:10) as main gluconeogenic substrates. OKg_gng+ cultures grew better in lactate than in pyruvate, whereas the opposite was observed with LLC-PK₁^-FBPase+ cells. This was further confirmed by the consumption rates of lactate and pyruvate by the substrains. When lactate or lactate/pyruvate were supplied, OKq_gng+ cells consumed lactate at much higher rates as compared to LLC-PK₁^-FBPase+ cultures. When pyruvate was added as the sole gluconeogenic substrate, pyruvate consumption rates by LLC-PK₁^-FBPase+ cells were linear over time, without considerable lactate production. However, pyruvate consumption by OK_gng+ cells was almost completed after 24-48 h, and resulted in an equimolar production and accumulation of lactate in the culture medium, respectively, which was consumed by the cells thereafter. In summary, a novel gluconeogenic strain of OK cells has been established (OK_gng+), as previously shown for gluconeogenic LLC-PK₁^-FBPase+ cells. Both cell strains, however, differ markedly in the metabolism of lactate and pyruvate, and thus obviously in the subcellular localization of PEPCK, in their regulation of gluconeogenesis by the intracellular NADH/NAD ratio, and/or in the rate of mitochondrial substrate fluxes and shuttle activities, respectively.

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Metabolism of Isolated Kidney Tubules. Oxygen Consumption, Gluconeogenesis and the Effect of Cyclic Nucleotides in Tubules from Starved Rats

Cited by 96 publications

References 0 publications

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

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

Fate of glutamate carbon and nitrogen in isolated guinea-pig kidney-cortex tubules. Evidence for involvement of glutamate dehydrogenase in glutamine sythesis from glutamate

A Novel Gluconeogenic Strain of OK Cells with Metabolic Properties Different from Gluconeogenic LLC-PK<sub>1</sub> Cells

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