Mitochondrial isocitrate dehydrogenase and isocitrate oxidation of rat ventral prostate.

Mitochondrial preparations isolated from rat ventral prostate were capable of oxidizing isocitrate by way of NADP isocitrate dehydrogenase (NADP-IDH) and NAD-IDH. NAD-IDH activity required ADP for activation. The pH responses for NAD-IDH and NADP-IDH were quite different. The results indicated that two different enzymes were involved in the NAD- and NADP-IDH activities. Indirect evidence indicated that NADPH-NAD transhydrogenase activity might also be involved in the mitochondrial pathway for isocitrate oxidation. NADP-IDH activity was significantly greater than NAD-IDH activity. The oxidation of isocitrate through IDH activity was coupled to the cytochrome system by NADPH- and NADH-cytochrome c reductase activities. Citrate, via isocitrate, oxidation proceeded at a much slower rate suggesting that aconitase activity could be limiting in the oxidation of citrate. In comparison to other tissues, the prostate oxidative enzyme activities are considerably lower. The results suggest that the accumulation of high prostate citrate levels is not due to a limitation imposed by a lack of IDH activity in prostate mitochondria.     

NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.     

Role of NADPH in the regulation of NADP-specific isocitrate dehydrogenase from pig heart.

The present results show that the NADP specific isocitrate dehydrogenase from pig heart exhibits a time lag before the reaction rate approaches a constant value at low metal ion concentrations. Addition of NADPH or EDTA to the assay mixture abolished the lag, and will under certain conditions activate the enzyme.The lag time increased with increasing concentrations of isocitrate and decreased with increasing enzyme concentration. The NADP and metal ion concentration affected the lag in a complex manner. At low NADP and isocitrate concentration, the lag was reduced 50% by an NADPH concentration of less than 2 μm. Stopped flow experiments showed that premixing of NADP or NADPH with the enzyme abolished the effect of NADPH on the lag time. NADPH activated the enzyme at high NADP concentrations. This activating effect could be accounted for by removal of substrate inhibition by NADP.Evidence was obtained to show that the effect of NADPH on the activity was caused by binding of the reduced coenzyme to a site separate from the normal coenzyme binding site. Binding of metal ions by the reduced coenzyme is probably of importance as EDTA affects the lag time and activity in a manner similar to NADPH. The NADPH effect seems to be a general property of NADP-linked isocitrate dehydrogenases.     

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.     

Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes.

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.     

The effect of glucagon on ureagenesis from ammonia by isolated rat hepatocytes

Glucagon, at a maximally effective concentration of 1 μM, stimulated by 35% the rate at which rat hepatocytes synthesized urea from 10 mM NH₄Cl in the presence of 10 mM ornithine. The rate at which citrulline accumulated in the incubations was relatively unchanged by the presence of glucagon.Mitochondria isolated from glucagon treated hepatocytes were observed to synthesize citrulline from 10 mM NH₄Cl and 10 mM ornithine more rapidly than did mitochondria isolated from untreated hepatocytes.The role of the intracellular malate concentration in the regulation of the rate of urea synthesis, and the changes observed in the cellular content of malate in response to glucagon are discussed.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Extracellular metabolites in suspensions of isolated hepatocytes.

The activity of lactate dehydrogenase and the concentration of several metabolites were measured in a suspension of isolated hepatocytes and in the extracellular medium, obtained after elimination of the cells by centrifugation for 15 s. The initial proportions of ATP, fructose 2,6-bisphosphate and glycogen present in the medium were similar to that of lactate dehydrogenase, and were therefore explained by unavoidable cell breakage occurring during resuspension of the hepatocytes. ATP disappeared from the medium in less than 10 min, being presumably destroyed by membrane nucleotidases. By contrast, the proportions of hexose 6-phosphates and of glycerol 3-phosphate in the medium were several-fold in excess over that of lactate dehydrogenase; under certain conditions, the extracellular value accounted for 80-90% of the metabolite present in the total suspension, and there was no relationship between the extra- and intracellular concentrations of these metabolites. A potential source of external glycerol 3-phosphate was the hydrolysis of glycerophosphocholine by membranous enzymes. The main conclusion of this work is that the measurement, in isolated hepatocytes, of hexose 6-phosphates, glycerol 3-phosphate and possibly other metabolites that were not investigated, requires the previous separation of the cells from the incubation medium. This conclusion may apply to other cellular suspensions.     

Hippurate synthesis and ammonia metabolism in isolated hepatocytes

The incorporation of benzoate into hippurate by isolated hepatocytes was limited by the availability of glycine. This limitation was unaffected by the addition of ammonia. When isolated hepatocytes were incubated in a solution containing urease, benzoate was completely ineffective in combating ammonia accumulation, while a mixture of α-keto analogues of several amino acids reduced ammonia accumulation by 50%. These findings do not support the use of benzoate to combat hyperammonemia, but they do support the current use of α-keto analogues of amino acids for that purpose.     

Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes

Polychlorinated biphenyl (PCB) and PCB metabolites are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. The molecular cytotoxic mechanisms of these metabolites are still not understood. The aim of the present study was to compare the cytotoxicity and toxicological properties of six dihydroxylated metabolites using isolated rat hepatocytes. All of the metabolites were more cytotoxic than 4-chlorobiphenyl (4-ClBP) and less cytotoxic than phenyl hydroquinone (PHQ). The order of cytotoxic effectiveness of catecholic metabolites expressed as LC(50) (2h) was 3',4'-diCl-2,3-diOH-biphenyl>PHQ>4'-Cl-2,5-diOH-biphenyl, 4'-Cl-2,3-diOH-biphenyl>2',5'-diCl-3,4-diOH-biphenyl>2',3'-diCl-3,4-diOH-biphenyl>3',4'-diCl-3,4-diOH-biphenyl>4'Cl-3,4-diOH-biphenyl>4'-Cl-biphenyl; showing that the positions of hydroxyl and chlorine groups were important for their hepatotoxicity and that the two 2,3-diOH congeners were the most cytotoxic. Cytotoxicity for 3,4-diOH metabolites correlated with the number and position of chlorine atoms with the more chlorine atoms being more cytotoxic. The cytotoxic order of metabolites with two chlorine atoms being 2',5'>2',3'>3',4'. Borneol, an uridine diphosphate glucuronosyltransferases (UGT) inhibitor, increased the cytotoxicity of all tested metabolites; suggesting that glucuronidation was a major mechanism of elimination of these compounds. On the other hand entacapone, a catechol-O-methyl transferase (COMT) inhibitor, only increased the cytotoxicity of 3',4'-diCl-3,4-diOH-biphenyl, 3',4'-diCl-2,3-diOH-biphenyl and 4'-Cl-2,3-diOH-biphenyl. Hepatocyte GSH was depleted (oxidized and conjugated) by these metabolites before cytotoxicity ensued in a similar order of effectiveness to their cytotoxicity with PHQ being the most effective. Hepatocyte mitochondrial membrane potential also decreased before cytotoxicity ensued with a similar order of effectiveness as their cytotoxicity. These results suggest that catecholic cytotoxicity can be attributed to mitochondrial toxicity and oxidative stress. Semiquinone or benzoquinone species were also important in the cytotoxicity of catecholic metabolites.     

Mitochondrial and cytosolic ATP/ADP ratios in isolated hepatocytes. A comparison of the digitonin method and the non-aqueous fractionation procedure.

The ratio of ATP content/ADP content in the mitochondrial matrix was found to be 2.07 +/- 0.21 and 2.26 +/- 0.22 as determined with six different preparations of isolated hepatocytes subfractionated with the digitonin and non-aqueous-fractionation procedures, respectively. In contrast, the mitochondrial matrix ATP/ADP determined with isolated haemoglobin-free perfused liver by using the non-aqueous-fractionation procedure was about 0.2, whereas the cytosolic values obtained with isolated cells and with the intact organ were similar. It is concluded that the relatively higher ATP/ADP ratio in the mitochondrial matrix of isolated hepatocytes represents a biochemical difference due to properties of the model rather than a methodological artifact.     

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.     

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.     

Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.