Department of Metabolic Regulation, Institute of Biochemistry, Warsaw University, Warsaw, Poland
* Author to whom correspondence should be addressed at: Department of Metabolic Regulation, Institute of Biochemistry, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland. Tel.: +48 22 5543213; Fax: +48 22 5543221; E-mail: bryla{at}biol.uw.edu.pl
(Received 16 June 2003; first review notified 29 July 2003; in revised form 15 December 2003; accepted 20 December 2003)
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ABSTRACT |
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INTRODUCTION |
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According to recent reports, kidneys make a significant contribution to net glucose production, reaching 25% of total glucose output under normal conditions and increasing up to 50% during starvation, diabetes (Stumvoll et al., 1999) or liver damage (Cersosimo et al., 2000
). As (i) the role of renal gluconeogenesis in diabetes and alcohol disease has not been elucidated; (ii) previous investigations of the ethanol action on renal glucose production have been performed mainly with the use of lactate and pyruvate as substrates (O'Neill and Bannister, 1986
; Crabb and Sidhu, 1993
; Nikiforov and Ostretsova, 1994
); and (iii) amino acids are considered to be important precursors of glucose in kidney (Lietz et al., 1999
); we investigated the effect of ethanol on glucose synthesis in the presence of various amino acids in kidney-cortex tubules, both freshly isolated and grown in primary cultures.
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MATERIALS AND METHODS |
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Male white Termond rabbits were used throughout. They were maintained on the standard rabbit chow with free access to water and food. Alloxan-diabetes was induced according to Kiersztan et al. (2002). All procedures were approved by the First Warsaw Local Commission for the Ethics of Experimentation on Animals.
Isolation and incubation of kidney-cortex tubules
Kidney-cortex tubules were obtained and incubated according to Zablocki et al. (1983). Reactions were stopped following 60 min incubation by the addition of a 1-ml sample to 0.1 ml 35% PCA. Intracellular content of metabolites in renal tubules was measured in samples taken at 60 min incubation following centrifugation through silicone oil into 12% PCA solution. Samples used for GSSG determination were centrifuged through the silicone oil layer into the solution containing 50 mmol/l N-ethylemaleimide (NEM) in 12% PCA, in order to avoid the non-enzymatic GSH oxidation (Guthenberg and Rost, 1966
).
Isolation of kidney-cortex tubules for primary cultures
Kidney-cortex tubules were obtained from the left kidney perfused for 10 min through the left renal artery with sterilised Ca2+-free KrebsRinger bicarbonated buffer supplemented with penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Next, kidney was removed and washed with sterilized ice-cold and O2:CO2 (95%:5%) equilibrated KrebsRinger buffer containing 2.5 mmol/l Ca2+. The kidney-cortex tissue was mechanically minced and incubated at 37°C for 5 min in KrebsRinger buffer containing 2.5 mmol/l Ca2+ and 0.02% collagenase. This procedure was repeated three times. The suspension was then filtered through a 100-µm sieve and centrifuged at low speed (10 g for 30 s). The pellet was resuspended in basal medium and washed twice on the centrifuge to remove collagenase. Finally, the renal tubules were suspended in about 30 vol. of the basal medium containing serum-free DMEM without glucose and pyruvate, supplemented with 15 mmol/l HEPES, human transferrin (5 µg/ml), human insulin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (50 nmol/l), penicillin (100 U/ml), streptomycin (0.1 mg/ml), linoleic acid (150 nmol/l), thymidine (1.5 µmol/l), biotin (15 nmol/l) and vitamin B12 (0.5 µmol/l) as described previously by Nowak and Schnellmann (1995). (Substrates and ethanol were included at concentrations indicated in the legends of the Figures and Tables.)
The isolated renal tubules were immediately plated on 60-mm Corning tissue culture grade plastic dishes and grown in a humidified incubator under 95% air and 5% CO2 atmosphere at 37°C. Following 24 h incubation, unattached tubules were removed and the fresh medium (5 ml per 60 mm dish) was added. Dishes were placed on an orbital shaker and constantly swirled. The culture media supplemented with gluconeogenic substrates were changed every day.
Samples used for medium metabolite determinations were transferred into the 35% PCA solution (0.1 vol. of the sample) and immediately neutralized with 3 mol/l K2CO3. In order to measure intracellular metabolite levels kidney-cortex cells were washed twice with phosphate buffer saline (pH 7.4) and extracted with 12% PCA. For GSSG determination cells were treated with 12% PCA containing 50 mmol/l NEM. In order to measure reactive oxygen species H2DCF-DA was added to the cell culture medium at the final 5 µmol/l concentration. Stocks of H2DCF-DA were made in methanol and stored in the dark at 80°C under argon. To measure cell protein the cell residues were resuspended in 1 n NaOH.
Biochemical analysis
Glucose, pyruvate, gluconeogenic intermediates and GSSG levels were measured either spectrophotometrically or fluorimetrically by standard enzymatic methods (Bergmeyer, 1983). GSH level was determined by high performance liquid chromatography (HPLC) following the derivatization with N-(1-pyrenyl)maleimide (NPM) (Ridnour et al., 1999
). Amino acids were determined by HPLC as their DABS-derivatives according to Chang et al. (1983)
. Alanine and aspartate utilizations were measured as differences between their contents at 0 time and after 60 min incubation. ROS generation was assessed with fluorescent H2DCF-DA probe (Verzola et al., 2002
), while protein was measured according to Bradford (1976)
.
Measurement of enzyme activities
The alcohol dehydrogenase (ADH) activity was estimated according to Qulali et al. (1991) while aldehyde dehydrogenase (ALDH) activity was determined as described by Farres et al. (1994)
, but with acetaldehyde used instead of propionaldehyde as substrate. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate dehydrogenase activities were measured according to Lambeth et al. (1992)
and Lohr and Wahler (1974)
, respectively.
Expression of results
Data shown are means ± SD for the number of experiments indicated in the legends to the Tables and Figures. The statistical significance of differences was calculated by ANOVA.
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RESULTS |
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Glucose synthesis, lactate formation and substrate uptake
In contrast to rabbit hepatocytes (Zaleski and Bryla, 1978), freshly isolated kidney-cortex tubules produce glucose from amino acids efficiently only in the presence of glycerol or lactate and either fatty acids or ketone bodies (Lietz et al., 1999
). Therefore, in all experiments with the use of freshly isolated renal tubules, amino acids were applied at 2 mmol/l concentrations in the presence of 0.5 mmol/l octanoate and either 2 mmol/l glycerol or 2 mmol/l lactate. Alanine and glutamate have been chosen as glucose precursors feeding gluconeogenesis pathway following their entry into the mitochondrial compartment, while aspartate can be utilised in the cytosol.
As shown in Fig. 1, increasing ethanol concentrations resulted in a progressive decrease in the rate of glucose formation in freshly isolated renal tubules incubated with alanine plus glycerol plus octanoate. Because in some earlier works relative to ethanol action on renal cells 100 mmol/l ethanol was applied (Calvo et al., 1992; Rodrigo et al., 2002
), we have used the same alcohol concentration in subsequent experiments. Surprisingly, alcohol at this concentration did not affect gluconeogenesis in the presence of aspartate plus glycerol plus octanoate.
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As shown in Table 2, the addition of ethanol resulted in a decrease in alanine consumption by about 20 and 35% in the presence of glycerol and lactate, respectively, accompanied by accelerated uptake of both glycerol and lactate (by about 30 and 100%, respectively). However, when alanine was substituted by aspartate, ethanol changed neither amino acid uptake nor glycerol and lactate consumptions. Ethanol-evoked decline in alanine utilization was not due to a diminished alanine transport, as accumulation of this amino acid in renal tubules was not altered by alcohol in the presence of rotenon and aminooxyacetate (i.e. under conditions of inhibited alanine metabolism; data not shown).
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The redox state and ROS generation
In order to estimate ethanol-induced changes in the cytosolic NADH:NAD+ ratio, intracellular levels of lactate and pyruvate have been measured to calculate lactate:pyruvate ratios. Surprisingly, in renal tubules incubated with alanine plus glycerol plus octanoate, intracellular levels of lactate (3.74 ± 0.22 and 4.22 ± 0.24 nmol/mg protein without and with ethanol, respectively) and pyruvate (0.49 ± 0.07 and 0.56 ± 0.09 nmol/mg protein without and with ethanol, respectively) were similar in the absence and presence of ethanol, as estimated for three separate experiments. In view of no significant changes in the lactate:pyruvate ratio following alcohol administration (7.63 ± 0.38 and 7.53 ± 0.49 without and with ethanol, respectively), it is possible to conclude that ethanol-induced inhibition of glucose formation from alanine plus glycerol plus octanoate is not due to elevation of the cytosolic NADH:NAD+ ratio.
As shown in Table 3, in freshly isolated renal tubules the intracellular GSH and GSSG levels as well as GSH:GSSG ratios were similar in the presence of both alanine plus glycerol plus octanoate and aspartate plus glycerol plus octanoate. Although in renal tubules grown in the primary culture in the presence of either alanine plus lactate plus octanoate or aspartate plus lactate plus octanoate the intracellular GSH and GSSG levels were of an order of magnitude higher than in freshly isolated tubules probably due to cysteine supplementation, the GSH:GSSG ratios were similar to those estimated in freshly isolated tubules. Moreover, in the presence of alanine ethanol caused a decline in GSH:GSSG ratio by about 30%, accompanied by an elevation of ROS generation for about 40%, (Fig. 3). On the other hand, following substitution of alanine by aspartate ethanol changed neither GSH:GSSG ratio (cf. Table 3) nor ROS levels (cf. Fig. 3).
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Surprisingly, glucose-6-phosphate dehydrogenase activity was decreased following administration of 0.25 mmol/l acetaldehyde (from 7.03 ± 0.42 to 5.39 ± 0.32 nmol/min/mg protein without and with acetaldehyde, respectively; P < 0.05 vs. control values with no acetaldehyde, as determined for three separate experiments). The inhibitory action of this compound on the enzyme activity was completely abolished on the addition of 2 mmol/l aspartate into the reaction medium (7.122 ± 0.12 nmol/min/mg protein), indicating a protective effect of this amino acid against the inhibition by acetaldehyde.
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DISCUSSION |
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In healthy humans ethanol has been reported to reduce the incorporation of labelled lactate and alanine into plasma glucose (Kreisberg et al., 1972). Furthermore, following 5 h ethanol ingestion, plasma glucose level in humans has been observed to fall by about 50% (Siler et al., 1998
). Inhibition of glucose formation in the liver is thought to be responsible for hypoglycaemic ethanol action (Lieber, 2000
). Although glucose production in kidney is equally important to that in liver in view of the whole body glucose metabolism (Stumvoll et al., 1999
), data concerning regulation of renal glucose production as well as the action of ethanol on renal gluconeogenesis are scare and controversial. In isolated domestic fowl glucose production in renal tubules is decreased by ethanol in the presence of lactate, whereas it is stimulated with pyruvate as gluconeogenic substrate (O'Neill and Bannister, 1986
). No influence of ethanol on the rate of glucose production from lactate, pyruvate or glutamine has been reported in rat renal cortex fragment (Nikiforov and Ostretsova, 1994
) and rat tubule suspension (Crabb and Sidhu, 1993
). In view of the above data, Crabb and Sidhu (1993)
concluded that unlike in liver, ethanol has no effect on glucose formation in kidney, and that this phenomenon explains why alcoholic hypoglycaemia is rarely severe.
Results of the present investigation show that the specific ADH activity in rabbit kidney-cortex is similar to that observed in baboons (Lohr et al., 1998) and slightly lower than in rat kidney (Qulali et al., 1991
). The effect of ethanol on glucose synthesis in rabbit kidney tubules, both freshly isolated (cf. Table 1) and grown in primary culture (cf. Fig. 2), depends on substrates provided, with glucose formation being inhibited in the majority of cases (i.e. in the presence of alanine plus lactate/glycerol plus octanoate or glutamate plus lactate plus octanoate, whereas in the presence of aspartate, ethanol does not affect this process. An inhibition by ethanol of glucose formation from alanine plus glycerol plus octanoate was also observed in renal tubules derived from diabetic rabbits (cf. Table 1), suggesting that a decline of glucose formation in kidney may in part be responsible for the reported ethanol-induced decrease in gluconeogenesis from lactate, glycerol (Puhakainen et al., 1991
) and alanine (Clore and Blackard, 1994
) in patients with non-insulin-dependent diabetes mellitus. Thus, ethanol may be one of the putative risk factors responsible for hypoglycaemia among patients with diabetes, especially taking oral sulfonylurea medications (Burge et al., 1999
). Moreover, as in diabetic rabbits ALDH activity is lower than in control animals and concomitantly the ADH activity is increased, one may speculate that after alcohol consumption diabetic individuals are subjected to higher concentrations of toxic acetaldehyde than nondiabetic ones.
Gluconeogenesis in liver is decreased by ethanol mainly due to elevation of NADH:NAD+ ratio (Krebs et al., 1969; Crow et al., 1983
; Lieber, 2000
). In contrast to liver, ethanol effects on glucose synthesis in renal tubules are not mediated by excessive NADH formation in the reaction catalysed by ADH, as (i) there are no significant changes in NADH:NAD+ ratio reflected by lactate/pyruvate ratios in renal tubules following ethanol administration and (ii) the addition of 4-methylpyrazole, inhibitor of ADH activity (Julia et al., 1987
), does not ameliorate the alcohol action on glucose output. This is in agreement with data reported by Crabb and Sidhu (1993)
who have suggested that kidney ADH activity is probably too low to generate sufficient NADH to overwhelm shuttle mechanisms. Inhibitory action of alcohol in freshly isolated hepatocytes was considered to result from an inefficient transport of reducing equivalents through the mitochondrial membrane via malate-aspartate shuttle, as freshly isolated cells were depleted of shuttle metabolites (Crow et al., 1978
; Efthivoulou et al., 1995
). However, this does not seem to be the case in renal tubules, as follows. (1) Freshly isolated renal tubules have relatively high levels of glutamate, malate, 2-oxoglutarate and aspartate (see Results) probably due to smaller fluid volumes used for isolation of renal tubules in comparison with those applied for hepatocytes isolation (Zaleski and Bryla, 1978
). (2) Alcohol decreases glucose formation from alanine plus lactate plus octanoate in primary cultures (cf. Fig. 2), which were incubated with these substrates for more than 24 h, giving tubules enough time to replenish levels of all cellular metabolites. (3) Alcohol does not cause significant changes in glutamate and aspartate levels. (4) Ethanol does not evoke considerable changes in NADH:NAD+ ratio.
In view of ethanol-induced changes in gluconeogenic intermediates in the presence of alanine (cf. Fig. 4), disturbance of phosphoenolopyruvate-pyruvate futile cycle mediated by both PEPCK and PK seems to be responsible for the inhibitory alcohol action, while the ethanol-induced rise in lactate uptake (cf. Table 2) might be due to augmentation of lactate dehydrogenase activity (Olivares et al., 1997). Our data imply that in rabbit kidney, inhibition by ethanol of gluconeogenesis from alanine plus lactate plus octanoate and glutamate plus lactate plus octanoate might result from the generation of ROS and acetaldehyde during ethanol metabolism via the pyrazole-insensitive microsomal ethanol oxidising system (MEOS) (Lieber and DeCarli, 1970
). A decline in the intracellular glucose-6-phosphate content due to the decrease in gluconeogenesis by ethanol (cf. Fig. 4) and the inhibition of glucose-6-phosphate dehydrogenase activity by acetaldehyde (see Results); might cause a diminished NADPH generation and consequently decreased GSH:GSSG ratio (cf. Table 3). Ethanol-induced decline of rat liver glucose-6-phosphate dehydrogenase activity and renal GSH level have been reported previously by Oh et al. (1998)
and Scott et al. (2000)
, respectively. One may argue whether the measured malate:pyruvate ratios (cf. Fig. 4) imply a rise in NADPH:NADP+ ratios. In view of the observations that: (i) the KM value of the malic enzyme is far above malate intracellular concentration (Winiarska et al., 2003a
) and (ii) the malic enzyme activity in rabbit kidney tubules is very low and restricted to mitochondria (Winiarska et al., 2003a
), it does not seem to be rational to estimate NADPH:NADP+ ratio on the basis of changes in malate/pyruvate ratios in rabbit kidney tubules.
In view of the data of the present investigation, ethanol metabolism in kidney tubules might generate ROS and acetaldehyde and disturb the cellular defence mechanisms via inhibition of glucose-6-phosphate dehydrogenase, which is needed for NADPH formation and in consequence maintenance of high GSH:GSSG ratio (Winiarska et al., 2003b).
No inhibitory effect of ethanol on glucose formation in the presence of aspartate plus lactate plus octanoate remains to be elucidated. Aspartate does not seem to stimulate the malate-aspartate shuttle, as (i) the addition of 0.2 m mol/l aspartate to renal tubules incubated with alanine plus glycerol plus octanoate does not increase glucose synthesis (Lietz et al., 1999) probably due to its inability to penetrate the mitochondria (La Noue et al., 1973
), (ii) the intramitochondrial rather than cytosolic aspartate is required for malate-aspartate shuttle operation (La Noue et al., 1973
). This amino acid might ameliorate ethanol-induced gluconeogenesis decline in the presence of alanine or glutamate probably due to attenuation of both ROS generation (cf. Fig. 3) and inhibition of glucose-6-phosphate dehydrogenase by acetaldehyde (see Results), resulting in maintenance of high GSH:GSSG ratio (cf. Table 3). Aspartate has also been reported to normalise the lipid peroxidation as well as to restore NADH:NAD+ balance disturbed by ethanol in the liver (Park et al., 1998
). Moreover, aspartate administration to ethanol-treated rat testes has been reported to protect them against oxidative damage (Oh et al., 2002
).
In summary, data presented in this investigation with the use of freshly isolated and primary cultured renal tubules indicate that the action of alcohol on renal glucose synthesis depends on amino acids provided as glucose precursors. In the presence of both alanine and glutamate, ethanol-induced inhibition of glucose formation is likely due to a diminished flux through PEPCK, a key gluconeogenic enzyme and/or enhanced flux through pyruvate kinase, as concluded from an increased lactate formation. The ethanol action is mediated by ethanol metabolism via MEOS rather than by ADH, as pyrazol does not attenuate the inhibitory effect of alcohol. Moreover, both ethanol-induced increase in ROS generation and inhibitory effect of acetaldehyde on glucose-6-phosphate dehydrogenase activity may contribute to a limited generation of NADPH for glutathione reductase and in consequence to a decrease in intracellular GSH:GSSG ratio. However, the relationship between MEOS metabolism of alcohol, ROS and acetaldehyde generations and PEPCK and PK activities remains to be elucidated. In contrast to alanine and glutamate, aspartate may ameliorate metabolic action of ethanol due to: (i) decrease in ROS generation and (ii) protection against inhibitory effect of acetaldehyde on glucose-6-phosphate dehydrogenase, resulting in consequence in the maintenance of high GSH:GSSG ratio.
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ACKNOWLEDGEMENTS |
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