Regulation of ornithine aminotransferase mRNA levels in rat kidney by estrogen and thyroid hormone

The regulation of the mitochondrial matrix enzyme, ornithine aminotransferase, by estrogen and triiodothyronine (T3) in rat kidney was examined using a cloned cDNA probe and in vitro translation of poly(A+) RNA. After a single, acute dose of either 17 beta-estradiol or T3, the rate of enzyme synthesis and the levels of translatable and hybridizable ornithine aminotransferase mRNA all increase in parallel. Levels of hybridizable mRNA were estimated by hybridization of randomly 32P-labeled RNA to filter-bound plasmid DNA. Maximal levels of induction by estrogen and T3 were about 15- and 3-fold, respectively. Lag times of at least 5 h and less than 3 h were observed for induction by T3 and estrogen. T3 and estrogen exert a synergistic effect in increasing ornithine aminotransferase mRNA levels. 16 h after T3 administration and 24 h after estrogen administration, a 1.6- and 13-fold increase in mRNA levels were observed. Both of these treatments in combination for the indicated time periods resulted in a 21-fold increase in ornithine aminotransferase mRNA. From the mRNA accumulation curves, half-lives of 10 to 14 h and 12 to 16 h were estimated for the mRNA after estrogen and T3 induction, respectively. These similar half-lives suggest that an increase in the rate of mRNA production is primarily responsible for the induction observed after estrogen administration.     

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.     

Molecular mechanisms of the synergistic induction of ornithine decarboxylase by asparagine and glucagon in primary cultured hepatocytes

In primary cultures of adult rat hepatocytes maintained in a salts/glucose medium, a more than 100-fold increase in ornithine decarboxylase (EC 4.1.1.17) activity was caused by asparagine and glucagon in a synergistic manner. The synthesis rate of ornithine decarboxylase was determined by [35S]methionine incorporation into the enzyme protein, and the amount of ornithine decarboxylase-mRNA was measured by hybridization with a cloned rat liver ornithine decarboxylase-cDNA. The synthesis rate of ornithine decarboxylase was stimulated more than 20-fold by asparagine and glucagon together, but the amount of ornithine decarboxylase-mRNA was increased only 3-4-fold, indicating that translational stimulation was involved in the induction process. Asparagine alone stimulated the synthesis of ornithine decarboxylase without substantial effect on the amount of ornithine decarboxylase-mRNA, whereas glucagon alone increased the amount of ornithine decarboxylase-mRNA about 3-fold without a detectable change in either enzyme activity or enzyme synthesis. Asparagine, at least in part, also suppressed degradation of ornithine decarboxylase.     

Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.     

Regulation of ornithine aminotransferase by cyclic AMP and glucose in primary cultures of adult rat hepatocytes

Hepatic ornithine aminotransferase (EC 2.6.1.13) (OAT) is a mitochondrial matrix enzyme that plays a role in amino acid catabolism and in gluconeogenesis. In rats, the synthesis of hepatic OAT is regulated by glucagon, dietary protein, and glucose. Serum-free primary cultures of adult rat hepatocytes were used to demonstrate that glucagon, cyclic AMP, and glucose are able to alter OAT synthesis by a direct action on hepatocytes. The rates of OAT synthesis were measured by immunoprecipitation of pulse-labeled OAT with an affinity-purified monospecific antibody. Ten hours after cyclic AMP addition to the culture medium, the relative rate of OAT synthesis reached a peak value that was six- to eightfold above the control rate. OAT activity accumulated more slowly, reaching a level that was approximately threefold above the control by 24 h. The inclusion of glucose in the culture medium inhibited the increases in OAT synthesis and activity in a dose-dependent manner. Although synthesized as a precursor (pOAT), no pOAT was detected under control, induced, or carbohydrate-inhibited conditions; this suggests that pOAT processing may not be a regulatory site of OAT expression. By following the loss of labeled OAT, a half-life of 34 h in these cultures under all of the above conditions was observed. Regulation of OAT levels in cultured hepatocytes appears to be achieved primarily through changes in the rate of OAT synthesis.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat liver cells. Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone

Isolated rat liver cells maintained in suspension culture for 4 to 5 h synthesize the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase at a rate approximately 5-fold lower than the in vivo hepatic rate. Glucagon rapidly re-induces phosphoenolpyruvate carboxykinase synthesis in such cells. The rate of enzyme synthesis doubles in 40 min and plateaus at a level 6- to 13-fold higher than in control cells 120 min after glucagon addition at maximal concentration. Consistent with the presumed role of cyclic AMP as a mediator of enzyme induction, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, added simultaneously with glucagon, shifts the hormone dose-response curve 2 log units to the left. Moreover, cyclic AMP supplied exogenously to the cells mimics the inductive effect of glucagon. Total cellular RNA isolated from hepatocytes induced by glucagon contains an increased level of mRNA coding for phosphoenolpyruvate carboxykinase, as determined by translational assay. The kinetics and extent of the rise in mRNA level are adequate to explain the stimulation of enzyme synthesis. Although glucagon on its own induces a build-up of phosphoenolpyruvate carboxykinase mRNA and a commensurate stimulation of enzyme synthesis, the glucagon induction is very markedly amplified when the cells are first preincubated with dexamethasone. The glucocorticoid by itself, however, does not have any substantial effect on the level of phosphoenolpyruvate carboxykinase mRNA or on the rate of enzyme synthesis. Its role can therefore be characterized as permissive.     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.     

Hormonal regulation of lipogenic enzymes in chick embryo hepatocytes in culture. Expression of the fatty acid synthase gene is regulated at both translational and pretranslational steps

Mechanisms involved in the multihormonal regulation of fatty acid synthase have been investigated by comparing levels of its mRNA with rates of enzyme synthesis in chick embryo hepatocytes in culture. Triiodothyronine or insulin caused about a 2.5-fold increase in the relative rate of synthesis of fatty acid synthase. Together, these hormones were synergistic, stimulating enzyme synthesis by nearly 40-fold (Fischer, P.W.F., and Goodridge, A.G. (1978) Arch. Biochem. Biophys. 190, 332-344). Addition of triiodothyronine stimulated increases in mRNA levels comparable to increases in enzyme synthesis whether insulin was present or not. Thus, triiodothyronine regulates fatty acid synthase primarily by controlling the amount of its mRNA. Addition of insulin, in the presence of triiodothyronine, stimulated enzyme synthesis by 14-fold and mRNA levels by only 2-fold. In the absence of triiodothyronine, insulin had no effect on mRNA levels. Thus, insulin has a major effect on the translation of fatty acid synthase mRNA. After the addition of triiodothyronine, fatty acid synthase mRNA accumulated with sigmoidal kinetics, approaching a new steady state about 48 h after the addition of hormone. Puromycin, an inhibitor of protein synthesis, blocked the effect of triiodothyronine. We suggest that the abundances of both fatty acid synthase and malic enzyme mRNAs are regulated by a common triiodothyronine-induced peptide intermediate which has a relatively long half-life. Glucagon caused an 80% decrease in the synthesis of fatty acid synthase (Fischer, P.W.F., and Goodridge, A.G. (1978) Arch. Biochem. Biophys. 190, 332-344) and a 60% decrease in the level of fatty acid synthase mRNA. Thus, glucagon regulates fatty acid synthase by controlling the concentration of its mRNA. The synthesis of malic enzyme also was inhibited by glucagon at a pretranslational step, but the inhibition was almost complete. Thus, despite coordinated regulation of the concentrations of these enzymes during starvation and refeeding, individual hormones sometimes regulate synthesis of the two enzymes at the same step and to about the same degree and sometimes at different steps or to very different degrees.     

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.     

Catabolism of arginine and ornithine in the perfused rat liver: effect of dietary protein and of glucagon

The rates of oxidation of arginine and ornithine that occurred through a reaction pathway involving the enzyme ornithine aminotransferase (EC 2.6.1.13) were determined using (14)C-labeled amino acids in the isolated nonrecirculating perfused rat liver. At physiological concentrations of these amino acids, their catabolism is subject to chronic regulation by the level of protein consumed in the diet. (14)CO(2) production from [U-(14)C]ornithine (0.1 mM) and from [U-(14)C]arginine (0.2 mM) was increased about fourfold in livers from rats fed 60% casein diets for 3-4 days. The catabolism of arginine in the perfused rat liver, but not that of ornithine, is subject to acute regulation by glucagon (10(-7) M), which stimulated arginine catabolism by approximately 40%. Dibutyryl cAMP (0.1 mM) activated arginine catabolism to a similar extent. In retrograde perfusions, glucagon caused a twofold increase in the rate of arginine catabolism, suggesting an effect of glucagon on arginase in the perivenous cells.     

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.     

Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

The levels of functional mRNA encoding glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were examined in hepatocytes from fasted and fasted/carbohydrate-refed rats and in hepatocytes inoculated into primary culture. Functional G6PDH mRNA was assessed in a cell-free protein synthesis system in vitro. We observed that hepatocytes from fasted/carbohydrate-refed rats had a 12-fold higher level of mRNA than did hepatocytes from fasted rats. The possibility that the adrenal glucocorticoids and insulin were responsible for the increase in G6PDH mRNA in refed rats was examined by studying the effect of insulin and the synthetic glucocorticoid, dexamethasone, on the level of functional G6PDH mRNA in primary cultures of rat hepatocytes maintained in a chemically defined medium. Hepatocytes from fasted rats were inoculated into primary culture and maintained for 48 h either in the absence of hormones or in the presence of insulin alone, dexamethasone alone or both hormones together. We observed that dexamethasone alone caused a fourfold increase in G6PDH mRNA while insulin caused about a twofold increase. Both hormones together elicited an increase that was additive. A comparison of functional G6PDH mRNA levels with the effect of the hormones on G6PDH activity and relative rate of enzyme synthesis suggests that the glucocorticoid elevates the level of G6PDH mRNA within the cell without causing a concommitant increase in the rate of synthesis of the enzyme or the level of G6PDH activity. The results obtained with the primary cultures of hepatocytes indicate that insulin and the glucocorticoids are probably involved with the regulation of hepatic G6PDH mRNA. However, involvement of other hormones, such as thyroid hormone, seems likely since the induced levels of G6PDH mRNA in hepatocytes in culture was one-third of that observed in refed rats.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

Studies on the turnover rates of ornithine aminotransferase in Morris hepatoma 44 and host liver.

The ornithine aminotransferase [EC 2.6.1.13] content of Morris hepatoma 44 is about 15 times higher than that in normal liver. The turnover rates of ornithine amino-transferase in hepatoma 44 and host liver were determined using L-[14C]leucine. Studies on the incorporation of radioactive leucine into ornithine aminotransferase in rats bearing hepatoma 44 showed that the rate of synthesis of this enzyme in the hepatoma was about 5-fold higher than that in host liver. The half-life of ornithine aminotransferase in host liver was 0.98 day, which was the same as that in normal liver, whereas that in hepatoma 44 was 3.5 days. The rate constant of degradation of ornithine aminotransferase in hepatoma 44 was significantly less than that in host liver. These results show that the high ornithine aminotransferase content of hepatoma 44 is due to both increase in its rate of synthesis and decrease in its rate of degradation.     

Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, we focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and [35S] methionine incorporation into GAPDH protein increased 5-fold. The increase in GAPDH mRNA and GAPDH biosynthesis could be demonstrated using physiologic concentrations of insulin (0.24 nM), indicating that these effects are mediated through a specific interaction with the insulin receptor. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.     

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.