Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis

Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis.     

Effect of high- and low-protein diets on the regulation of rat liver tyrosine aminotransferase and tryptophan oxygenase activities by l-tyrosine and l-tryptophan

Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryptophan was studied in groups of rats fed on diets containing 18 or 5% protein. The basal activity of hepatic tyrosine aminotransferase of rats receiving 5% protein gradually increased with the age of the animals but that of rats receiving 18% protein did not. l-Tyrosine induced hepatic tyrosine aminotransferase in rats receiving 18% protein when tested at ages from 4 to 20 weeks. When induction by l-tyrosine was carried out in rats receiving the 5% protein diet, significant induction of tyrosine aminotransferase occurred only in 4- or 6-week-old rats. Induction by l-tryptophan of tryptophan oxygenase in liver or the basal activity of this enzyme in liver did not differ between the groups fed on 5 and 18% protein. On changing the diet from 0 to 18% protein, the above-mentioned effects on the induction of hepatic tyrosine aminotransferase were reversed.     

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.     

Effect of vitamin B6 on the synthesis and degradation of aspartate aminotransferase in chicken embryo fibroblasts

The effect of pyridoxal depletion and supplementation on the intracellular level of mitochondrial and cytosolic aspartate aminotransferase in cultured chicken embryo fibroblasts was examined. No apoenzyme was detected in cells grown in the presence of pyridoxal, and the specific activity of total enzyme did not vary profoundly from primary to quaternary cultures. Under pyridoxal depletion, up to 40% apoenzyme was found in tertiary cultures which was entirely due to the mitochondrial isoenzyme. Cytosolic apoenzyme was never detected. Total aspartate aminotransferase relative to total protein was increased 2-fold in secondary cultures; only the mitochondrial isoenzyme contributed to the increased specific activity. The cytosolic isoenzyme decreased steadily and was below the limit of detection in quaternary cultures. The changes are attributed to an increased and decreased synthesis of mitochondrial and cytosolic isoenzyme, respectively. No induction of either isoenzyme was observed after incubating the cells with different hormones and substrates. In secondary cultures, no degradation of mitochondrial isoenzyme could be detected under pyridoxal deficiency or supplementation during 4.4 days, an interpassage duration. The cytosolic aspartate aminotransferase was degraded initially with an apparent half-life of approximately 0.9 day under both sets of conditions. The pronounced stability of mitochondrial aspartate aminotransferase, even though one-third of it was present as apoenzyme, excludes the formation of the apoform to be the rate-limiting step in its degradation. The present results show that pyridoxal affects the synthesis of mitochondrial and cytosolic aspartate aminotransferase, but differently.     

Effects of dietary protein of proso millet on liver injury induced by D-galactosamine in rats

In this paper, we examined the effects of dietary protein from proso millet on liver injury induced by D-galactosamine or carbon tetrachloride in rats using serum enzyme activities as indices. D-galactosamine-induced elevations of serum activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were significantly suppressed by feeding the diet containing 20% protein of proso millet for 14 days as compared with those of rats fed a 20% casein diet, but not in the case of carbon tetrachloride. The results showed that proso millet protein is effective at lower dietary protein levels than that of dietary gluten reported previously. Therefore, the findings reported here may suggest that proso millet protein is considered to be another preventive food for liver injury.     

Translational and pretranslational control of ornithine aminotransferase synthesis in rat liver

The mitochondrial matrix enzyme, ornithine aminotransferase, is induced in rat liver by the administration of a diet high in protein and by glucagon. The rate of synthesis of the enzyme is increased 100-fold in the livers of rats maintained on a 60% relative to a 0% protein diet, whereas the levels of functional and hybridizable mRNA measured by in vitro translation and through the use of a cloned cDNA probe increased by only 2- to 6-fold and 2- to 3-fold, respectively. Under conditions of glucagon induction that resulted in a 10- to 12-fold increase in the rate of enzyme synthesis, the relative level of functional ornithine aminotransferase mRNA increased by only 2-fold, and the level of hybridizable mRNA actually decreased. The rate of polypeptide chain elongation and the relative number of ornithine aminotransferase nascent chains on polysomes were 2-fold and 23-fold greater, respectively, in hepatocytes derived from 60% relative to 0% protein-fed rats. Using these data, a 23-fold increase in the translational efficiency of the mRNA was calculated. This increase, along with a 2-fold increase in the mRNA level, completely account for the 40-fold increase in the rate of ornithine aminotransferase synthesis observed in hepatocytes derived from 60% protein-fed rats. We conclude that ornithine aminotransferase synthesis is regulated at both a translational and a pretranslational level in rat liver.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.     

Selective determination of fish aspartate aminotransferase isoenzymes by their differential sensitivity to proteases

Various proteases (proteinase K, subtilisin, trypsin and chymotrypsin) were used to study the selective inactivation of the aspartate aminotransferase (EC 2.6.1.1) isoenzymes of grey mullet (Mugil auratus Risso; Osteichthyes). The cytosolic isoenzyme was significantly inactivated by proteinase K, subtilisin and chymotrypsin, while the mitochondrial isoenzyme was sensitive only to proteinase K and to high doses of trypsin. Further identification of the aspartate aminotransferase isoenzymes was based on their discrete sensitivity toward chymotrypsin. Chymotrypsin (1 mg/ml) successfully inhibited purified cytosolic aspartate aminotransferase as well as cytosolic isoenzyme from plasma, whereas the mitochondrial form persisted unaffected. Similar results were obtained when examining liver and red muscle homogenates. This method revealed that the increased total activity of aspartate aminotransferase in fish plasma with induced acute liver injury, was partially a result of the mitochondrial isoenzyme leakage from damaged tissue.     

Metabolic studies in experimental liver disease resulting from d(+)-galactosamine administration

1. In confirmation of previous work, administration of d(+)-galactosamine (0.5-0.75g/kg body wt.) to rats caused a hepatitis with histological evidence of liver damage and a 9-fold rise in aspartate aminotransferase activity in serum. 2. There was a significant elevation of blood lactate and pyruvate concentrations in 24h-starved rats treated with galactosamine but no change in the [lactate]/[pyruvate] ratio. 3-Hydroxybutyrate and acetoacetate concentrations in blood were decreased. 3. The changes in the concentrations of lactate, pyruvate and ketone bodies in the freeze-clamped liver were parallel to those observed in the blood. 4. In the livers of 24h-starved galactosamine-treated rats there were large increases in the concentrations of alanine (3-fold), citrate (5-fold), 2-oxoglutarate (4-fold), with smaller increases in malate, glutamate and aspartate. There was a 4-fold rise in the value of the mass-action ratio of the alanine aminotransferase system in the livers of galactosamine-treated rats when compared to controls. 5. There was a significant decrease in the activities of aspartate and alanine aminotransferases in the cytoplasm and the soluble fraction of sonicated homogenates of the livers of rats treated with galactosamine. The activity of phosphoenolpyruvate carboxylase was decreased by 75% of the control value. 6. Glucose synthesis from lactate in perfused livers from galactosamine-treated rats was inhibited 39% when compared with controls. 7. The results indicate that the conversion of lactate into glucose is decreased in the livers of galactosamine-treated rats and that this decrease may be due to the loss of phosphoenolpyruvate carboxylase from damaged hepatocytes.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.     

Functional inhibition of cytosolic and mitochondrial aspartate aminotransferase by l-2-amino-4-methoxy-trans-3-butenoic acid in isolated rat hepatocytes and mitochondria

The transaminase inhibitor l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) decreased aspartate aminotransferase activity by approximately two-thirds in isolated rat liver mitohondria incubated with succinate, ammonia, and ornithine. Aspartate production by the mitochondria was unaffected over the 30-min incubation period, indicating that mitochondrial aspartate aminotransferase activity is normally far in excess of that required for maximal rates of aspartate production. In rat hepatocytes incubated with lactate, ammonia, and ornithine the inhibition of both the cytosolic and mitochondrial isozymes of aspartate aminotransferase by AMB was partially blocked by the presence of ammonia and ornithine. When pyruvate was substituted for lactate as a carbon source with isolated hepatocytes, the presence of ammonia and ornithine blocked the inhibition by AMB of the mitochondrial but not the cytosolic isozyme of aspartate aminotransferase. Urea formation by cells incubated with lactate, ammonia, and ornithine was unaffected by AMB unless the cells were preincubated with the inhibitor prior to the addition of substrates. However, urea formation by cells incubated in the presence of pyruvate, ammonia, and ornithine was inhibited strongly by AMB even without preincubation. The results suggest that the stimulation of ureogenesis from ammonia and ornithine by pyruvate involves the cytosolic isozyme of aspartate aminotransferase. In contrast, the stimulation of ureogenesis elicited by lactate primarily involved mitochondrial aspartate aminotransferase.     

Short-term metabolic fate of [13N]ammonia in rat liver in vivo

The short-term metabolic fate of [13N]ammonia in the livers of adult male, anesthetized rats was determined. Following a bolus injection of tracer quantities of [13N]ammonia into the portal vein, the single pass extraction was approximately 93%, in good agreement with the portal-hepatic vein difference of approximately 90%. High performance liquid chromatographic analysis of deproteinized liver samples indicated that labeled nitrogen is exchanged rapidly among components of: mitochondrial aspartate aminotransferase and glutamate dehydrogenase reactions and cytoplasmic aspartate aminotransferase and alanine aminotransferase reactions (t1/2 for the exchange of label toward equilibrium is on the order of seconds). Comparison of specific activities of glutamate and ammonia suggests that at 5 s most labeled glutamate was mitochondrial, whereas at 60 s approximately 93% was cytosolic; this change is presumably brought about by the combined action of the mitochondrial and cytosolic aspartate aminotransferases and the aspartate carrier of the malate-aspartate shuttle. Specific activity measurements of glutamate, alanine, and aspartate are in accord with the proposal by Williamson et al. (Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497-502) that the components of the aspartate aminotransferase reaction are in thermodynamic equilibrium, whereas the components of the alanine aminotransferase reaction are in equilibrium but compartmented in the rat liver. Despite considerable label in citrulline at early time points, no radioactivity (less than or equal to 0.25% of the total) was detected in carbamyl phosphate, suggesting very efficient conversion to citrulline with little free carbamyl phosphate accumulating in the mitochondria. Our data also show that some portal vein-derived ammonia is metabolized to glutamine in the rat liver, but the amount is small (approximately 7% of that metabolized to urea) in part because liver glutamine synthetase is located in a small population of perivenous cells "downstream" from the urea cycle-containing periportal cells. Finally, no tracer evidence could be found for the participation of the purine nucleotide cycle in ammonia production from aspartate. The present work continues to emphasize the usefulness of [13N]ammonia for short-term metabolic studies under truly tracer conditions, particularly when turnover times are on the order of seconds.     

THE EFFECT OF ELEVATED AMINO ACIDS ON AMINOTRANSFERASE LEVELS IN BRAIN AND LIVER OF THE MOUSE

Abstract— Adult mice were fed standard diets that were enriched with selected amino acids, i.e. 3% methionine, 6% valine, or 8% lysine. These diets caused the following changes in the amino acid pool of the brain measured at 7 and 21 days. The high methionine diet resulted in 50-fold higher levels of methionine and cysteine and somewhat lower levels of serine and glutamine. The valine and lysine-enriched diets also caused 2- to 4-fold increases in valine and lysine contents of brain, respectively. In spite of the large changes in amino acid levels, however, there were essentially no changes in aspartate: α-ketoglutarate, alanine: α-ketoglutarate, ornithine: α-ketoglutarate, methionine: α-ketoglutarate, and the branched chain aminotransferase activities of brain 3, 10, and 21 days after the onset of the dietary regimen. In contrast, these diets produced significant changes in some of these enzyme activities in liver. Changes in liver included a 2-fold increase in ornithine and alanine aminotransferase activities with the methionine-enriched diet. Liver ornithine aminotransferase activity also increased slightly in animals fed the valine-enriched or lysine-enriched diet.     

Marginal iron deficiency enhances liver triglyceride accumulation in rats fed a high-sucrose diet

ABSTRACT

We investigated whether marginal iron-deficiency (MID) without anemia influences liver lipid accumulation in rats. Ingestion of a MID diet in which the iron concentration was half of AIN-93 formulation (iron-adequate, IA) for 3 weeks decreased liver iron concentration without anemia. We then evaluated the influence of the MID diet on liver lipid accumulation in combination with a high-sucrose (HS) diet and confirmed that the HS-MID diet successfully decreased liver iron concentration without anemia. Additionally, a significant increase in liver triglyceride concentration was found, accompanied by upregulation of hepatic fatty acid synthase expression in the rats fed the HS-MID diet compared to those in the rats fed an HS-IA diet, although no difference was observed in plasma transaminase activity and hepatic interleukin-1β expression. These results suggest that MID enhances de novo lipid synthesis via upregulation of lipogenic gene expression in combination with sucrose in the diet.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HS, high sucrose; IA, iron adequate; ID, iron deficiency; MID, marginal irondeficiency; NAFLD, non-alcoholic fatty liver disease     

Regulation of aspartate aminotransferase activity associated with change of pyridoxal phosphate level.

The activities of aspartate aminotransferase (EC 2.6.1.1) in the cytosol fractions of the liver and kidney of rats fed pyridoxine-deficient or control diet for 3 weeks were determined. In the absence of pyridoxal phosphate, the activities in the liver and kidney preparations of deficient rats were both abnormally low. The activity in the kidney fraction of deficient rats was restored to almost the control level by addition of pyridoxal phosphate, whereas that of the liver was only partially restored. The antigen activity, however, measured using anti-aspartate aminotransferase, was similar in liver fractions from deficient and control rats. These findings suggest the existence of a form of transaminase with little or no activity in the liver of deficient rats. The properties of the crude enzymes from deficient and control rats were indistinguishable by immunodiffusion, and the enzymes had the same subunit size and heat stability under the conditions tested. However, purified enzyme from deficient rat liver had a different specific activity and absorption spectrum from purified enzyme from normal liver.     

Effect of rhodium (III) complex on experimental carcinogenesis in the livers of rats treated with thioacetamide

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats. The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration. The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.     

Similarities between rat liver mitochondrial and cytosolic glutathione reductases and their apoenzyme accumulation in riboflavin deficiency

Glutathione reductase has been purified 5,500-fold from rat liver mitochondrial matrix in a yield of 30%. The mitochondrial enzyme was immunochemically indistinguishable from that of the cytosol and the subunit molecular weight was apparently similar to that of the cytosolic enzyme, that is, 50,000 daltons. The optimum pH and kinetic properties investigated were not significantly different from those of the cytosolic enzyme. When rats were fed a riboflavin-deficient diet, the enzyme activity in the mitochondria decreased to a greater extent than that in the cytosol, and greater accumulation of apo-enzyme in the former than that in the latter was confirmed by the amount of immunoprecipitable protein, activation by FAD addition in vitro, and the enzyme activity recovery after injection of riboflavin, into riboflavin-deficient rats.