Carnitine metabolism in isolated rat kidney cortex tubules

Renal carnitine metabolism was studied in isolated kidney cortex tubules from fed rats. The tubular distribution of free carnitine (C), acid-soluble short chain acylcarnitine (AcC), and total acid-soluble carnitine was measured. The content of the last-mentioned in rat cortical tubule suspensions was 2.85 +/- 0.15 nmol/mg protein, 46% representing AcC. In the absence of metabolic substrates the AcC/C ratio declined from 0.84 to 0.48 during incubation. The administration of 2mM acetoacetate or 2mM 3-hydroxybutyrate caused an increase in AcC by 45% and 51%, respectively. The rise in AcC was paralleled by a decrease in C, resulting in an increase of the tubular AcC/C ratio to 1.69 and 1.85, respectively. In the presence of 1 mM exogenous L-carnitine 35 +/- 6 nmol AcC/(mg protein X h) was formed. The addition of acetoacetate and 3-hydroxybutyrate led to a 3.5 to 3.8-fold rise in AcC formation. Other substrates which are likewise metabolized by proximal tubules were less effective. More than 90% of the formed AcC was recovered in the extracellular fluid. The results suggest that proximal renal tubule cells are the intrarenal site of carnitine acylation and may be involved in the regulation of blood and/or urinary carnitine acylation state.     

Triacylglycerol metabolism in isolated rat kidney cortex tubules

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-¹⁴C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-β-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-β-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.     

Fatty acids and glycerol or lactate are required to induce gluconeogenesis from alanine in isolated rabbit renal cortical tubules

Summary In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-¹⁴C]Alnine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in¹⁴CO₂ fixation and elevation of both cytosolic and mitochondrial NADH/NAD⁺ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.     

The intracellular compartmentation of metabolites in isolated kidney cortex tubules

• 1.1. A method is described to measure cytosolic and mitochondrial metabolites in isolated kidney tubule suspensions.
• 2.2. With the use of digitonin, 95% of cytosolic metabolites are released into the supernatant fraction whereas mitochondrial matrix enzymes are retained in the pellet fraction.
• 3.3. In a study of adaptation to acidosis, different α-oxoglutarate gradients between the tubular cell compartments are obtained, when medium pH is changed from 7.0 to 7.8.

     

Concomitant synthesis and degradation of glutamine in isolated rabbit kidney tubules

When rabbit kidney tubules were incubated with 1 mM [1-14C]glutamine as substrate, a release of 14CO2 together with a net production of glutamine were observed. That glutamine utilization was masked by higher rates of concomitant glutamine synthesis was demonstrated by: (i) inhibiting glutamine synthesis; and (ii) measuring the specific radioactivity of [1-14C]glutamine which fell during incubation.     

Glutamic acid decarboxylase in tubules and glomeruli isolated from rat kidney cortex

4-Aminobutyric acid (GABA) synthesis was examined in purified glomeruli and tubules of rat kidney cortex that were incubated in the presence of [2,3-3H2]glutamate. The GABA that was formed was separated from glutamate using anion-exchange resin, and identified by means of an automatic amino acid analyser. In the renal cortex only the tubules were able to form GABA (35.0 nmol mg-1 h-1); the remaining GABA synthesis found in the glomerular preparations can most probably be attributed to a contamination by cortical tubules (9%), as shown by determination of a known tubular marker enzyme (L-gamma-glutamyltransferase). Hydroxylamine (1 mM) and ethanolamine-O-sulfate (10 mM), well-known inhibitors of cerebral GABA formation and GABA catabolism respectively, inhibited renal tubular GABA formation at 100% and 44% respectively.     

Inhibitory effect of gentamicin on gluconeogenesis from pyruvate, propionate, and lactate in isolated rabbit kidney-cortex tubules

The effect of gentamicin on glucose production in isolated rabbit renal tubules was studied with lactate, propionate, malate, 2-oxoglutarate, and succinate as substrates. This antibiotic at 5 mM concentration inhibited gluconeogenesis from lactate by about 60% and that from either pyruvate or propionate by about 30%. In contrast, it did not alter the rate of glucose formation from other substrates studied. The rate of gluconeogenesis was higher at 1 mM propionate than at increasing concentrations of this substrate and was stimulated in the presence of 1 mM carnitine. However, the addition of carnitine did not affect the degree of inhibition of glucose formation by gentamicin. Since the mitochondrial free coenzyme A level was significantly lower in the presence of 10 than 1 mM propionate and increased on the addition of carnitine to the reaction medium, the inhibitory effect of propionate concentrations above 1 mM on gluconeogenesis in rabbit renal tubules may be due to a depletion of the free mitochondrial coenzyme A level, resulting in an inhibition of the mitochondrial coenzyme A-dependent reactions. In intact rabbit kidney cortex mitochondria incubated in State 4 as well as in Triton X-100-treated mitochondria, 5 mM gentamicin inhibited by about 30-40% the incorporation of 14CO2 into both pyruvate and propionate. The results indicate that the inhibitory effect of gentamicin on glucose formation in isolated kidney tubules incubated with lactate, pyruvate, or propionate is likely due to a decrease of the rate of carboxylation reactions.     

Effect of alloxan-diabetes on gluconeogenesis and ureogenesis in isolated rabbit liver cells.

1. Neither alloxan-diabetes nor starvation affected the rate of glucose production in hepatocytes incubated with lactate, pyruvate, propionate or fructose as substrates. In contrast, glucose synthesis with either alanine or glutamine was increased nearly 3- and 12-fold respectively, in comparison with that in fed rabbits. 2. The addition of amino-oxyacetate resulted in about a 50% decrease in glucose formation from lactate in hepatocytes isolated from fed, alloxan-diabetic and starved rats, suggesting that both mitochondrial and cytosolic forms of rabbit phosphoenolpyruvate carboxykinase function actively during gluconeogenesis. 3. Alloxan-diabetes resulted in about 2-3-fold stimulation of urea production from either amino acid studied or NH4Cl as NH3 donor, whereas starvation caused a significant increase in the rate of ureogenesis only in the presence of alanine as the source of NH3. 4. As concluded from changes in the [3-hydroxybutyrate]/[acetoacetate] ratio, in hepatocytes from diabetic animals the mitochondrial redox state was shifted toward oxidation in comparison with that observed in liver cells isolated from fed rabbits.     

Inhibition of 2-oxoglutarate metabolism by lithium in the isolated rat kidney cortex tubules

2-Oxoglutarate metabolism in the isolated rat kidney cortex tubules was inhibited by lithium at 5 mM concentration, and at pH increased from 7.1 to 7.6. The metabolism of pyruvate and acetylcarnitine to citrate and 2-oxoglutarate was enhanced by lithium and the increased pH value. The content of 2-oxoglutarate in the renal tubular cells was lowered by lithium but increased at elevated pH values. Both the intracellular pH value and bicarbonate ion concentrations in renal tubular cells were increased by lithium in the medium containing 2-oxoglutarate. The results obtained indicate that lithium disturbs renal metabolism by intracellular alkalization, with a simultaneous inhibition of the inflow of dicarboxylic substrates to the renal tubular cells.     

Phenylalanine hydroxylation in isolated rat kidney tubules

1. Phenylalanine hydroxylation has been demonstrated to occur in isolated rat kidney tubules under physiological conditions. 2. The hydroxylation flux response is hyperbolic with apparent Km and Vmax values of ca 85 microM phenylalanine and 49 nmol tyrosine formed/mg dry wt per hr respectively. 3. Hydroxylation in kidney tubules is substantially less sensitive to effectors of cyclic AMP turnover and Ca2+ mobilization than phenylalanine hydroxylation in isolated liver cells.     

Control of glycolysis and gluconeogenesis in rat kidney cortex slices

1. Glucose uptake or glucose formation has been studied in kidney cortex slices to investigate metabolic control of phosphofructokinase and fructose-diphosphatase activities. 2. Glucose uptake is increased and glucose formation is decreased by anoxia, cyanide or an uncoupling agent. Under these conditions the intracellular concentrations of glucose 6-phosphate and ATP decreased whereas that of fructose diphosphate either increased or remained constant, and the concentrations of AMP and ADP increased. 3. Glucose uptake was decreased, and glucose formation from glycerol or dihydroxyacetone was increased, by the presence of ketone bodies or fatty acids, or after starvation of the donor animal. Under these conditions, the concentrations of glucose 6-phosphate and citrate were increased, whereas those of fructose diphosphate and the adenine nucleotides were unchanged (see also Newsholme & Underwood, 1966). 4. It is concluded that anoxia and cell poisons increase glucose uptake and decrease gluconeogenesis by stimulating phosphofructokinase and inhibiting fructose diphosphatase, whereas ketone bodies, fatty acids or starvation increase gluconeogenesis and decrease glucose uptake through the citrate inhibition of phosphofructokinase.     

Stimulation of gluconeogenesis by somatostatin in rat kidney cortex slices.

The effect of somatostatin on gluconeogenesis was studied in kidney cortex slices. Addition of somatostatin (2 μg) stimulated gluconeogenesis from lactate, pyruvate and glutamine by 42%, 50% and 68% respectively. Stimulation of glucose synthesis from lactate by somatostatin was found to be linear with time and dose dependent between 0.1 and 20 μg. Somatostatin-stimulated gluconeogenesis was inhibited by phentolamine (10 μM) but not by propranolol (10 μM) suggesting that somatostatin action is mediated by α-adrenergic stimuli.