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

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis 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-[U-14C]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 [U-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.     

Purinergic regulation of glucose and glutamine synthesis in isolated rabbit kidney-cortex tubules

The effects of extracellular purinergic agonists and their breakdown products on glucose and glutamine synthesis in rabbit kidney-cortex tubules incubated with aspartate + glycerol or alanine + glycerol + octanoate were investigated. A rapid extracellular degradation of ATP was accompanied by an accumulation of AMP, inosine, and hypoxanthine. Extracellular ATP and its breakdown products accelerated glucose synthesis in renal tubules, while ammonium released from adenine-containing compounds enhanced glutamine synthesis and diminished the degree of gluconeogenesis stimulation. In contrast to AMP and inosine, ATP evoked calcium signals, while both ATP and inosine decreased intracellular cAMP content and accelerated the flux through fructose-1,6-bisphosphatase as concluded from changes in gluconeogenic intermediates. Since (i) the activity of partially purified renal fructose-1,6-bisphosphatase was increased upon protein phosphatase-1 treatment and decreased following treatment of previously dephosphorylated enzyme with protein kinase A catalytic subunit and (ii) both 8-bromoadenosine 3',5'-cyclic monophosphate and 8-(4-chlorophenyltio)-cAMP inhibited renal glucose synthesis, it seems likely that in rabbit renal tubules ATP and inosine stimulate gluconeogenesis via cAMP decrease, which favors the appearance of a more active, dephosphorylated form of fructose-1,6-bisphosphatase, a key gluconeogenic enzyme.     

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.     

PPAR-gamma-independent inhibitory effect of rosiglitazone on glucose synthesis in primary cultured rabbit kidney-cortex tubules

Therapeutic effect of rosiglitazone has been reported to result from an improvement of insulin sensitivity and inhibition of glucose synthesis. As the latter process occurs in both liver and kidney cortex the aim of this study was to elucidate the rosiglitazone action on glucose formation in both tissues. Primary cultured cells of both liver and kidney cortex grown in defined medium were use throughout. To identify the mechanism responsible for drug-induced changes, intracellular gluconeogenic intermediates and enzyme activities were determined. In contrast to hepatocytes, the administration of a 10 micromol/L concentration of rosiglitazone to renal tubules resulted in about a 70% decrease in the rate of gluconeogenesis, accompanied by an approximately 75% decrease in alanine utilization and a 35% increase in lactate synthesis. The effect of rosiglitazone was not abolished by GW9662, the PPAR-gamma irreversible antagonist, indicating that this action is not dependent on PPAR-gamma activation. In view of rosiglitazone-induced changes in gluconeogenic intermediates and a diminished incorporation of 14CO2 into pyruvate, it is likely that the drug causes a decline in flux through pyruvate carboxylase and (or) phosphoenolpyruvate carboxykinase. It is likely that the hypoglycemic action of rosiglitazone is PPAR-gamma independent and results mainly from its inhibitory effects on renal gluconeogenesis.     

Differential effects of selegiline on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies

The action of selegiline, a selective and irreversible inhibitor of monoamine oxidase B, commonly applied in the therapy of Parkinson's disease, on glucose formation was investigated in isolated rabbit hepatocytes and kidney-cortex tubules, maintaining the whole body glucose homeostasis via gluconeogenic pathway activity. An intensive hepatic metabolism of selegiline resulted in formation of selegiline-N-oxide, desmethylselegiline, methamphetamine and amphetamine, whereas during slow degradation of the drug in freshly isolated renal tubules selegiline-N-oxide was mainly produced. At 100 μM concentration selegiline markedly diminished glucose synthesis in isolated renal tubules incubated with dihydroxyacetone or alanine + glycerol + octanoate (by about 60 and 30%, respectively), while at 5 μM concentration a similar degree of inhibition was achieved in renal tubules grown in primary culture under the same conditions (about 40 and 60%, respectively). Moreover, desmethylselegiline and selegiline-N-oxide considerably diminished glucose production in renal tubules whereas selegiline and its metabolites did not affect gluconeogenesis in hepatocytes. Contrary to control animals, following selegiline administration to alloxan-diabetic rabbits for 8 days (10 mg kg⁻¹ body wt. daily) the blood glucose and serum creatinine levels were significantly diminished, suggesting a decrease in renal gluconeogenesis and improvement of kidney functions.

Since in renal tubules selegiline induced a decline in the intracellular levels of gluconeogenic intermediates and ATP content accompanied by a decrease in oxygen consumption in both kidney-cortex and hepatic mitochondria it seems possible that its inhibitory action on renal gluconeogenesis might result from an impairment of mitochondrial function, while an intensive selegiline metabolism in hepatocytes causes decrease of its concentration and in consequence no inhibition of gluconeogenesis. In view of these observations it is likely that an increased risk of selegiline-induced hypoglycemia might be expected particularly in patients exhibiting an impairment of liver function and following transdermal administration of this drug, i.e. under conditions of increased serum selegiline concentrations.     

Differential effects of selenium compounds on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies

Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40-45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52+/-0.18 and 87.4+/-9.7 down to 1.63+/-0.11 and 39.0+/-2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.     

Diabetes-induced changes in glucose synthesis,intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules

Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals, diabetes-evoked increase in glucose formation in the presence of either aspartate + glycerol + octanoate or malate as gluconeogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione oxidised one (GSSG) ratio by about 30–40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relationship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover, diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis, providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway. (Mol Cell Biochem 261: 91–98, 2004)     

Stimulation of glutamine metabolism by 3-aminopicolinate in isolated dog kidney-cortex tubules

1. The effects of 3-aminopicolinate, a known hyperglycaemic agent in the rat, on glutamine metabolism were studied in isolated dog kidney tubules. 2. 3-Aminopicolinate greatly stimulated glutamine (but not glutamate) removal and glutamate accumulation from glutamine as well as formation of ammonia, aspartate, lactate, alanine and glucose. 3. The increased accumulation of aspartate from glutamine and glutamate, and the inhibition of glucose synthesis from various non-nitrogenous gluconeogenic substrates, as well as the increased accumulation of malate from succinate, support the proposal that 3-aminopicolinate is an inhibitor rather than a stimulator of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in dog kidney tubules. 4. With glutamine as substrate, the increase in flux through glutamate dehydrogenase (EC 1.4.1.3) could not explain the large increase in glutamine removal caused by 3-aminopicolinate. 5. Inhibition by amino-oxyacetate of accumulation of aspartate and alanine from glutamine caused by 3-aminopicolinate did not prevent the acceleration of glutamine utilization. 6. These data are consistent with a direct stimulation of glutaminase (EC 3.5.1.2) by 3-aminopicolinate in dog kidney tubules.     

Regulation of glucose formation from lactate and pyruvate in isolated tubules of chicken kidney.

1. Isolated kidney tubules from chicken have been used to study the actions of ethanol, ouabain and aminooxyacetate on glucose formation from lactate and pyruvate. 2. In kidney tubules from well-fed chickens the rate of glucose production from lactate was higher than from pyruvate. Ethanol (10 mM) and ouabain (0.1 mM) were found to increase glucose formation from pyruvate but not from lactate. 3. It is concluded that in the presence of ethanol the fluxes of pyruvate through pyruvate dehydrogenase are in favour of the pyruvate carboxylase reaction restricted. 4. Glucose formation from lactate is decreased by aminooxyacetate (0.1 mM) and ouabain (0.1 mM). 5. Aminooxyacetate inhibited glucose formation from lactate, although chicken phosphoenolpyruvate carboxykinase is located intramitochondrially. 6. The results indicate that the effect of aminooxyacetate like that of ouabain is caused by the restricted formation of pyruvate.     

Contribution of L-3,4-dihydroxyphenylalanine metabolism to the inhibition of gluconeogenesis in rabbit kidney-cortex tubules

The circulating L-3,4-dihydroxyphenylalanine, the drug of choice in the therapy of Parkinson's disease (PD), is efficiently extracted by kidney and converted to dopamine, known to control several renal functions. As: (i) in addition to liver, kidney is an important source of glucose in mammals and (ii) the action of this drug on renal gluconeogenesis has not yet been studied, the aim of the present investigation was to estimate the influence of L-3,4-dihydroxyphenylalanine metabolism on glucose formation in isolated kidney-cortex tubules incubated with various gluconeogenic substrates. The data indicate that a rapid intracellular degradation of L-3,4-dihydroxyphenylalanine and tyramine (at 100 and 200 microM concentrations) is accompanied by 25-40% decrease in glucose production from pyruvate, alanine + glycerol + octanoate and dihydroxyacetone due to augmented generation of hydrogen peroxide via monoamine oxidase B, resulting in a decline of glutathione redox state by 40%. Moreover, following inhibition of monoamine oxidase B by deprenyl or substitution of pyruvate by aspartate + glycerol + octanoate both L-3,4-dihydroxyphenylalanine and tyramine affect neither the rate of gluconeogenesis nor glutathione redox state. In view of: (i) L-3,4-dihydroxyphenylalanine- and tyramine-induced changes in intracellular levels of gluconeogenic intermediates, and (ii) a significant decline of phosphoenolpyruvate carboxykinase activity by 500 microM oxidized glutathione, it is likely that L-3,4-dihydroxyphenylalanine- and tyramine-evoked disturbances in the glutathione redox state might diminish flux through phosphoenolpyruvate carboxykinase and in consequence decrease glucose formation in renal tubules, suggesting a new potential side-action of L-3,4-dihydroxyphenylalanine treatment.     

Metabolism of acetaldehyde in human and baboon renal cortex. Ethanol synthesis by isolated baboon kidney-cortex tubules

Acetaldehyde (1-20 mM) was metabolized at high rates and in a dose-dependent manner in isolated human and baboon kidney-cortex tubules. Acetaldehyde removal was accompanied by a large accumulation of acetate in both human and baboon tubules. By contrast, a large synthesis of ethanol was observed only in baboon tubules. Consistent with the latter finding, ethanol was found to be metabolized at significant rates in baboon but not human tubules. In the tubules from both species, a significant fraction of the acetaldehyde removed was also completely oxidized to CO2 and H2O. These results suggest that, in both man and baboon, the kidneys participate in the in vivo metabolism of acetaldehyde; they also suggest that, in contrast with the human kidneys, the baboon kidneys contribute to the detoxication of circulating ethanol.     

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.     

Calcium transport in transverse tubules isolated from rabbit skeletal muscle

Isolated transverse tubule vesicles free of sarcoplasmic reticulum transport calcium with high affinity in the presence of ATP. The calcium transport by transverse tubules differs from calcium transport by sarcoplasmic reticulum. It is not increased by oxalate or phosphate, it has a different temperature dependence, it is inhibited by sub-micromolar concentrations of orthovanadate, it is stimulated by calmodulin, and is inhibited by quercetin without causing calcium release. The rates of calcium transport by transverse tubules are two orders of magnitude lower than those of sarcoplasmic reticulum, suggesting that the calcium pump protein of transverse tubules is a minor component of the membrane. Addition of calmodulin to transverse tubule vesicles--treated with high salt in the presence of EGTA to remove endogenous calmodulin--caused a marked stimulation of transport rates at low concentrations of calcium, and decreased from 1.0 to 0.3 microM the calcium concentration at which half-maximal rates of transport were obtained. A role for the transverse tubule calcium pump in maintaining low sarcoplasmic calcium concentrations is proposed.     

Riboflavin transport by isolated perfused rabbit renal proximal tubules

Rabbit renal proximal tubular transport of riboflavin(RF) was examined by using the in vitro isolated tubule perfusiontechnique. We found that proximal tubules actively reabsorbed(J_lb) and secreted (J_bl)RF. At 0.1 µM RF concentration, J_bl wassignificantly higher than J_lb, resulting in anet secretion. This net secretion of RF was decreased at 0.01 µM RFconcentration and increased at 1 µM RF concentration. BothJ_lb and J_bl wereinhibited by lowering temperature or by adding iodoacetate, a metabolicinhibitor, and lumichrome, an RF analog, suggesting the involvement ofcarrier-mediated transport mechanisms. J_bl wasinhibited by probenecid, an anion transport inhibitor, and bypara-aminohippuric acid, an organic anion, suggesting therelevance of RF secretion to renal organic anion transport.J_bl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca²⁺/calmodulin-dependent pathway on RFsecretion. Finally, we found that addition of chlorpromazine, aphenothiazine derivative, inhibited both J_lb andJ_bl, raising the concern about the nutritionalstatus in patients receiving such a type of medication.

     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

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.     

Receptor-mediated effect of a synthetic thromboxane-analogue on cytosolic calcium in isolated proximal tubules.

The aims of this study were to measure cytosolic calcium concentration -[Ca2+]i- under resting conditions in isolated renal proximal tubules and to analyze the effect of U-46619 (stable analogue of thromboxane A2/PGH2 on [Ca2+]i in a mammalian epithelium. Proximal tubules were dissected out from male New Zealand rabbits (2.5 to 3.0 kg). After isolation they were washed twice and resuspended in 2 ml phosphate buffer solution (PBS). Tubules were loaded with Quin 2-AM (25 microM) for 15 min. After washing with PBS to eliminate the excess of extracellular Quin 2, fluorescence was measured at 340 nm excitation and 490 emission, under resting conditions and after stimulation. U 46619 (from 10 nm to 10 mM) increased [Ca2+]i in a concentration-dependent pattern. Exposure to an antagonist of the thromboxane receptor (S-145) blocked the response to U-46619. Removal of external calcium abolished the response to U-46619. Change of PBS for Ringer-choline blunted the response to thromboxane analogue. Our results indicate that U-46619 increases cytosolic calcium through a receptor-mediated mechanism that requires external calcium to operate. Blockade of the response in the absence of external sodium suggests that Na+/Ca2+ exchanger participates in this response.     

Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator.

The methodology has been developed to measure cell chloride activity by fluorescence microscopy using the chloride-sensitive dye, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). SPQ was loaded into cells of the in vitro microperfused rabbit proximal convoluted tubule by a 10 min luminal perfusion with 20 mM SPQ at 38 degrees C. Fluorescence was excited with a broad band excitation filter (340 and 380 nm) and detected with a 435 nm cut-on filter. The signal to background (autofluorescence) ratio was 4.6 +/- 0.6. The halftime for SPQ leakage from cells at 38 degrees C was 8.6 +/- 1.1 min. In suspended tubules, SPQ did not affect O2 consumption significantly. Intracellular SPQ calibration was performed using the ionophores nigericin and tributyltin, high external potassium concentrations, and varying extracellular chloride concentrations. Cell fluorescence was related to intracellular chloride by a Stern-Volmer relation with a quenching constant of 12 M-1. Apparent chloride concentration in tubules perfused with solutions characteristic for the late proximal convoluted tubule was 27.5 +/- 5 mM (activity 20.6 mM). The halftime of the transient in cell chloride activity upon bath chloride addition was approximately 3 s (38 degrees C). Applications and limitations of this new fluorescence method to study cell chloride transport are discussed.