Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.     

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.     

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.     

Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids

The effects of branched-chain alpha-ketoacids on flux through and activity state of the branched-chain alpha-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of alpha-ketoisocaproate caused a dramatic stimulation (50% activation at 20 microM) of alpha-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. alpha-Keto-beta-methylvalerate was also effective, but less so than alpha-ketoisocaproate. alpha-Ketoisocaproate did not stimulate alpha-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate stimulated alpha-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain alpha-ketoacid dehydrogenase was alpha-ketoisocaproate greater than alpha-keto-beta-methylvalerate greater than alpha-ketoisovalerate, i.e., the same order of potency of these compounds as branched-chain alpha-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain alpha-ketoacid dehydrogenase phosphatase, largely prevented alpha-ketoisocaproate and alpha-chloroisocaproate activation of flux through the branched-chain alpha-ketoacid dehydrogenase. Assay of the branched-chain alpha-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that alpha-ketoacids affected the activity state of the complex. Branched-chain alpha-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain alpha-ketoacid dehydrogenase kinase activity by branched-chain alpha-ketoacids is important for regulation of the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase.     

Effects of diet and of alloxan-diabetes on the activity of branched-chain 2-oxo acid dehydrogenase complex and of activator protein in rat tissues.

The total activities (sum of active and inactive forms) of branched-chain 2-oxo acid dehydrogenase complex in tissues of normal rats fed on a standard diet were (unit/g wet wt.): liver, 0.82; kidney, 0.77; heart, 0.57; hindlimb skeletal muscles, 0.034. Total activity was decreased in liver by 9%- or 0%-casein diets and by 48 h starvation, but not by alloxan-diabetes. Total activities were unchanged in kidney and heart. The amount of active form of the complex (in unit/g wet wt. and as % of total) in tissues of normal rats fed on standard diet was: liver, 0.45, 55%; kidney, 0.55, 71%; heart, 0.03, 5%; skeletal muscle less than 0.007, less than 20% (below lower limit of assay). The concentration of the active form of the complex was decreased in liver and kidney, but not in heart, by low-protein diets, 48 h starvation and alloxan-diabetes. In heart muscle alloxan-diabetes increased the concentration of active complex. The concentration of activator protein (which activates phosphorylated complex without dephosphorylation) in liver and kidney was decreased by 70-90% by low-protein diets and 48 h starvation. Alloxan-diabetes decreased activator protein in liver, but not in kidney. Evidence is given that in tissues of rats fed on a normal diet approx. 70% of whole-body active branched chain complex is in the liver and that the major change in activity occasioned by low-protein diets is also in the liver.     

Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart

Branched-chain alpha-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart. The initial step was the purification of rat liver and heart branched-chain alpha-ketoacid dehydrogenase complex with high kinase activity by a modification of a method described previously. Preservation of high kinase activity during purification of the complex required the presence of fresh dithiothreitol throughout the procedure. The kinase was released from the complex by oxidation of dithiothreitol with potassium ferricyanide and purified by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography. Both kinase preparations gave only one polypeptide band with a molecular weight of 44,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex by the purified kinase was inhibited by alpha-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex. The kinase did not exhibit autophosphorylation and does not correspond to the same protein as pyruvate dehydrogenase kinase. The kinase phosphorylated histone (type II-S), but this reaction was slow relative to the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex and was not inhibited by alpha-chloroisocaproate.     

Effect of diet and starvation on the activity state of branched-chain 2-oxo-acid dehydrogenase complex in rat liver and heart

In rats fed a high-protein diet, the branched-chain 2-oxo-acid dehydrogenase complex in liver was essentially fully acitve and its activity state was unaffected by subsequent starvation for 48 h. Feeding with a low-protein diet led to a decrease in the activity state which was essentially reversed by 48 h of starvation. In heart, the enzyme was primarily inactive (activity state 18%) in rats fed a high-protein diet, with both low-protein diet and starvation leading to a further decrease in the activity state.     

Effect of diet and starvation on the activity state of branched-chain 2-oxo-acid dehydrogenase complex in rat liver and heart

In rats fed a high-protein diet, the branched-chain 2-oxo-acid dehydrogenase complex in liver was essentially fully active and its activity state was unaffected by subsequent starvation for 48 h. Feeding with a low-protein diet led to a decrease in the activity state which was essentially reversed by 48 h of starvation. In heart, the enzyme was primarily inactive (activity state 18%) in rats fed a high-protein diet, with both low-protein diet and starvation leading to a further decrease in the activity state.     

Purification of branched chain alpha-ketoacid dehydrogenase complex from rat liver

A new method using hydrophobic interaction chromatography on phenyl-Sepharose was developed to purify branched chain alpha-ketoacid dehydrogenase complex from commercially available frozen rat liver. Yields of greater than 50% were routinely achieved. The purified enzyme, composed of E1 alpha, E1 beta, and E2 subunits, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained endogenous kinase activity for phosphorylation and inactivation of the complex.     

Reversion of the maple syrup urine disease phenotype of impaired branched chain alpha-ketoacid dehydrogenase complex activity in fibroblasts from an affected child

Branched chain alpha-ketoacid dehydrogenase is a heteroprotein complex of mitochondria and commits the branched chain alpha-ketoacids to their catabolic fate. Inherited nuclear mutations in humans decrease the activity of this complex and result in maple syrup urine disease. Here we demonstrate the restoration of branched chain alpha-ketoacid dehydrogenase activity to fibroblasts from a child with this disorder by transfection with a cDNA for the prebranched chain acyltransferase. Prior to transfection these fibroblasts contained the prebranched chain acyltransferase gene but failed to transcribe the gene and thus lacked the protein. Regulation of the restored complex by phosphorylation mechanisms resembles that of wild-type cells. These results describe a human cell modeling system for testing engineered proteins and support the possibility of gene replacement therapy for this human disorder.     

Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism

The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated hepatocytes

Hepatocytes, isolated from rats fed a low-protein diet, were incubated with [32P]Pi and the phosphoproteins analysed. Immunoprecipitation using antibody against El of branched-chain 2-oxo acid dehydrogenase complex demonstrated phosphorylation of the alpha-subunit of El. Analysis of the tryptic phosphopeptides from the alpha-subunit indicated that two sites were phosphorylated. 4-methyl 2-oxopentanoate and DL-2-chloro 4-methylpentanoate decreased labelling of both sites. No major direct effects of several hormones on phosphorylation of branched-chain 2-oxo acid dehydrogenase was observed.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells.

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.     

Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone

The lipogenic capacity of rat liver is increased in animals fed a high carbohydrate, fat-free diet or by the administration of 2,2',5'-triiodo-L-thyronine. Underlying this change is a generalized induction of the enzymes involved in lipogenesis, including glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, which together serve to generate the additional NADPH required for increased fatty acid synthesis. This report presents evidence indicating that induction of the hexose-shunt dehydrogenases involves increased enzyme synthesis secondary to elevated enzyme specific mRNA levels, as has previously been shown for malic enzyme. Activities of specific mRNAs, estimated by cell-free translation of hepatic poly(A)-containing RNA in the mRNA dependent rabbit reticulocyte lysate, were compared with enzyme specific activities and relative rates of specific enzyme synthesis. The 2-fold increase in glucose-6-phosphate dehydrogenase specific activity in hyperthyroid rats and the 13-fold increase in rats fed a high carbohydrate, fat-free diet, relative to euthyroid, chow-fed controls were paralleled by comparable increases in the synthetic rates and mRNA levels of this enzyme. Similarly, consonant changes in the rate of enzyme synthesis and concentration of 6-phosphogluconate dehydrogenase mRNA accompanied the 2.5- and 3-fold increases in specific activity of this enzyme observed in response to hormonal and dietary induction, respectively. Thus, both thyroid hormone and carbohydrate feeding appear to induce glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase primarily by increasing the effective cellular concentrations of their respective mRNAs and, consequently, their rates of synthesis.     

Restoration by dietary glutamine of reduced tumor necrosis factor production in a low-protein-diet-fed rat model

Tumor necrosis factor-alpha (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.     

Activation of branched-chain alpha-keto acid dehydrogenase complex by exercise: effect of high-fat diet intake

The effect of exercise on the activity of branched-chain alpha-keto acid dehydrogenase complex in liver and muscle was studied in rats fed a high-fat (FAT) or a high-carbohydrate (CHO) diet. Both diet groups of rats were offered isoenergetic diets by a meal-feeding method and were trained by treadmill running. On the final day of the experiment, half of the rats in each diet group were exercised by 2 h of running just before they were killed. The activity state of the enzyme complex was elevated maximally by exercise in liver of rats fed the FAT diet but not in liver of rats fed the CHO diet, suggesting that catabolism of branched-chain amino acids in rat liver during exercise was enhanced by the FAT diet. The activity state of the enzyme complex in muscle was enhanced by exercise in both groups of rats, but a significant difference was not observed between the groups. The concentration of branched-chain amino acids was elevated in liver and muscle by exercise in both groups of rats, but the elevated levels in liver were lower in rats fed the FAT diet than in those fed the CHO diet. Serum branched-chain amino acid concentrations were significantly lower in rested rats fed the FAT diet than in those fed the CHO diet, and the leucine and isoleucine concentrations in the former were elevated by exercise, but the serum concentrations in the latter were not significantly affected by exercise. ATP and ADP concentrations in muscle were not significantly affected by either diet or exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.     

Tert-butyl hydroperoxide selectively inhibits mitochondrial respiratory-chain enzymes in isolated rat hepatocytes

Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate + malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I).