Expression of human IMP dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.

Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an important target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP dehydrogenase activity and were partially separated from endogenous E. coli IMP dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP dehydrogenase was similar in these cells, however, indicating that the increase in IMP dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP dehydrogenase molecular species.     

Effect of alimentary thiamine deficiency on the activity of gluconeogenic key enzymes in rat liver and kidney

Activity of the key enzymes of gluconeogenesis under alimentary thiamine deficiency (15 days of dietary treatment) was studied in the liver and kidney of fed and 48 h starved rats. As compared to pair-fed controls vitamin B1-deficiency was followed by a decrease of glucose 6-phosphatase and fructose 1,6-bisphosphatase activities in both organs; the activity of phosphoenolpyruvate carboxykinase was diminished only in the liver. Starvation of thiamine-deficient rats (as compared to pair-fed starved group) resulted in lower activation of these enzymes. The decrease of the enzyme activities in thiamine-deficient animals indicates that de novo glucose synthesis in the tissues is depressed, though thiamine-requiring enzymes are not directly involved in this process. Possible mechanisms of alterations described are discussed.     

Down-regulation of c-myc and c-Ha-ras gene expression by tiazofurin in rat hepatoma cells

There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.     

Increased activity, amount, and altered kinetic properties of IMP dehydrogenase from mycophenolic acid-resistant neuroblastoma cells

Mouse wild-type neuroblastoma cells (NB cells) were stepwise selected for 10,000-fold increased resistance to mycophenolic acid (NB-Myco cells), an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). IMP dehydrogenase activity was increased 25-fold, from 3.1 to 75 nmol/min.mg of protein; and a 56.7-kDa peptide was increased in abundance 200-500-fold in NB-Myco as compared to NB cells. Purification and sequence analysis confirmed that the abundant protein was IMP dehydrogenase. The stepwise selection, increased activity and protein abundance, and unstable phenotype are indirect evidence for a process of gene amplification. Kinetic findings consistent with an Ordered Bi Bi mechanism were indicative of IMP dehydrogenase having undergone mutation. The Michaelis constants were unchanged for IMP (14 and 13 microM) and increased 4-fold for NAD from 25 to 94 microM for NB and NB-Myco cells, respectively. The Ki for mycophenolic acid was increased 2400-fold from 1.4 nM to 3.4 microM for the enzyme from NB versus NB-Myco cells, and the Ki for XMP was increased 4-fold from 78 to 336 microM. Mycophenolic acid exhibited uncompetitive inhibition with IMP, consistent with the formation of a dead end E-XMP-inhibitor complex. The cellular GTP concentration was increased 2-fold in resistant cells and, upon removal of mycophenolic acid, further increased to 4.5-fold that of NB cells.     

Dichloroacetate-induced changes in liver of normal and diabetic rats.

Dichloroacetate (DCA) was administered orally to normal (nondiabetic) and streptozotocin-diabetic rats in a dose of 1000 mg/day/kg rat wt. One group of diabetic animals received DCA both orally and intraperitoneally. DCA therapy lowered the blood glucose values of diabetic animals but did not alter values in nondiabetic rats. The hepatic activity of glucokinase and pyruvate kinase were significantly lower in both DCA-treated nondiabetic and DCA-treated diabetic animals than values observed for untreated animals. However, DCA therapy was accompanied by remarkable increases in the activities of glucose-6-phosphate dehydrogenase and malic enzyme in both nondiabetic and diabetic animals. Glucose-6-phosphate dehydrogenase was 3-fold higher in DCA-treated nondiabetic animals whereas malic enzyme activity was 10-fold higher in the treated animals than observed in the untreated animals. Similar changes, although smaller in magnitude, were observed for these enzymes in the DCA-treated diabetic animals. Although DCA therapy was accompanied by a significant increase in the wet weights of the liver for both nondiabetic and diabetic animals, no morphological changes were seen by light or electron microscopy. Our observations coupled with those of previous investigators suggest that DCA therapy may have an important role in pyruvate metabolism and may lower the blood glucose concentration by inhibiting hepatic gluconeogenesis.     

Behavior of L-threonine-degrading enzymes during liver regeneration

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.     

Purification of IMP dehydrogenase from rat hepatoma 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

Colon tumor: enzymology of the neoplastic program

The biochemical strategy of colon tumor was investigated by comparing the enzymic programs of glycolysis, pentose phosphate production and purine and pyrimidine biosynthesis and degradation in liver, normal colon mucosa and transplantable colon adenocarcinoma in the mouse. In normal colon mucosa the carbohydrate and pentose phosphate enzymes were 2- to 9-fold higher in specific activity than those in liver. Among the enzymes of CTP synthesis, CTP synthetase was the rate-limiting one in both liver and colon. In colon tumor CTP synthetase, OMP decarboxylase, uracil phosphoribosyltransferase and thymidine kinase activities increased to 927, 863, 597 and 514% of activities of normal colon. In contrast, the activity of the catabolic enzymes, dihydrothymine dehydrogenase and uridine phosphorylase, decreased to 51 and 25%. The ratios of activities of uridine kinase/uridine phosphorylase and thymidine kinase/dihydrothymine dehydrogenase were elevated 6- and 10-fold. The activity of the key purine synthetic enzyme, glutamine PRPP amidotransferase, increased 7-fold and the opposing rate-limiting enzyme of purine catabolism, xanthine oxidase, decreased to 7%. The ratio of amidotransferase/xanthine oxidase was elevated to 8, 150%. Activities of glucose-6-phosphate dehydrogenase and transaldolase did not increase, but that of pyruvate kinase was elevated to 154%. Similar enzymic programs were observed in a transplantable adenocarcinoma of the colon in the rat. The alterations in gene expression in colon tumor manifested in an integrated pattern of enzymic imbalance indicate the display of a program, a segment of which is shared with rat and human liver and kidney tumors. These alterations in gene expression should confer selective advantages to colon tumor cells. The striking increases in the activities of CTP synthetase, OMP decarboxylase, glutamine PRPP amidotransferase and thymidine kinase mark out these enzymes as potentially sensitive targets for combination chemotherapy by specific inhibitors of these enzyme activities.     

Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.     

Peroxisomal beta oxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase.

Activity of enoyl-CoA hydratase in rat liver was elevated about 6-fold by the administration of di-(2-ethylhexyl)phthalate, a hepatic peroxisome proliferator. Almost all of the increased activity was the peroxisomal enzyme, which was distinguished by its heat-lability from mitochondrial one. Heat-labile enoyl-CoA hydratase was copurified with peroxisomal 3-hydroxyacyl-CoA dehydrogenase. The purified enzyme corresponded to a peroxisome specific peptide with a molecular weight of 80,000.     

Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.     

The association of lysosomal enzymes with nuclei during enzyme induction in rat liver.

Male rats of the Holtzman strain were fasted for 3 days and refed a diet high in carbohydrate (68.9%). The induction of liver glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was monitored for up to 48 h after refeeding. Induction occurred by 24 h, and by 48 h, 4.2- and 1.5-fold increases were observed for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, compared with that of livers of pellet-fed rats. After refeeding, lysosomes increased in fragility as judged by an increased release of acid phosphatase activity during standard homogenization. Fragility was greatest 3 h after refeeding, but normal fragility was observed 24 h after refeeding. Nuclei were isolated from the liver samples before and after refeeding. Those isolated just before refeeding revealed small latent acid phosphatase activity (4–6%). However, after refeeding the carbohydrate-rich diet, a transient and significant (P < 0.01) increase in the latent activity occurred that was maximal (20%) at 1 h, returning to normal by 24 h. Cross-mixing the 800g nuclear pellet from livers of animals starved for 3 days with the 800g supernatant fraction from livers of animals refed the carbohydrate-containing diet did not alter the nuclear lysosomal-free (overt) or latent (detergent-released) enzyme activity. Similarly, mixing the 800g nuclear pellet from livers of animals refed for 1 h with the 800g supernatant fraction from livers of animals starved for 3 days, but not refed, did not change the nuclear lysosomalfree or latent enzyme activity. Purified nuclei, further washed in hypotonic buffer, lost acid phosphatase activity, but those isolated from livers of rats refed for 1 h retained 10% of the enzyme latency, whereas all latency was lost from those isolated from uninduced rats. A second lysosomal enzyme, β-galactosidase, became associated with the nuclei with the same temporal pattern as for acid phosphatase. However, no variation in nuclear content of cytosolic lactate dehydrogenase occurred as a result of feeding the high-carbohydrate diet to starved rats. When similarly starved rats were refed a diet high in lipid and carbohydrate-free, no induction of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was observed. Lysosomes were not temporarily fragile and purified nuclei did not exhibit increased latency of acid phosphatase activity. Though the evidence presented does not establish a direct correlation between lysosome migration to nuclei as a required function in enzyme induction, the temporal and specific nature of the phenomenon support the hypothesis that liver lysosomal enzymes participate in early signals in the induction of enzymes of lipogenesis.     

Lipid metabolism by rat lung in vitro. Utilization of citrate by normal and starved rats

1. The utilization of [1,5-(14)C(2)]citrate by lung slices and cell cytosol preparations, and the activities of liver and lung cytosol citrate-cleavage enzyme (EC 4.1.3.8), l-malate-NAD oxidoreductase (malate dehydrogenase, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (EC 4.1.1.32) were examined in normal and starved rats. 2. Lipogenesis from citrate was decreased by approx. 70% in both the phospholipid and neutral lipid fractions of lung slices from starved rats as compared with fed controls. 3. Incorporation of citrate by lung cytosol preparations into fatty acids was decreased by approx. 35% in the starved rats. The apparent inhibition by avidin of fatty acid synthesis was overcome partially by preincubation of lung cytosol preparations with biotin. These results are consistent with the presence in lung tissue of the malonyl-CoA pathway for fatty acid synthesis. 4. Lung citrate-cleavage enzyme activity decreased in rats that had been starved for 72h whereas malate dehydrogenase and phosphoenolpyruvate carboxylase activities remained unchanged. The results suggest that the pattern of utilization of lipid precursors by rat lung may be altered during various nutritional states.     

Coordinate control of rat liver lipogenic enzymes by insulin

Recent evidence has established that insulin is required for the dietary induction of rat liver fatty acid synthetase [Proc. Nat. Acad. Sci. USA69, 3516 (1972)]. Since other hepatic lipogenic enzymes as well as fatty acid synthetase exhibit coordinate adaptation to nutritional changes [Advan. Enzyme Regul.10, 187(1972)], the role of insulin in the dietary induction of these enzymes has been investigated. When a high-carbohydrate, fat-free diet was fed to diabetic rats previously fasted for 48 hr, insulin was shown to be required for the dietary induction of acetyl-CoA carboxylase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fatty acid synthetase, and glucokinase. Activity of serine dehydrase, selected as a model gluconeogenic enzyme, was increased in diabetic rats, whereas insulin treatment reduced the activity of this enzyme during the course of refeeding. The behavior of serine dehydrase was consistent with its gluconeogenic role. The activity of the cytosol isocitrate dehydrogenase did not change during refeeding in the diabetic or insulin-treated diabetic rat. Glucagon, the physiological antagonist of insulin, inhibited the increase in activity of each of the lipogenic enzymes requiring insulin for induction. Our results indicate that insulin is required for the coordinate regulation of the lipogenic enzymes of mammalian liver.     

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide)

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.     

Levels of mRNAs coding for lipogenic enzymes in rat lung upon fasting and refeeding and during perinatal development

The relative amounts of mRNAs coding for fatty-acid synthase (EC 2.3.1.85), acetyl-CoA carboxylase (EC 6.4.1.2), ATP citrate lyase (EC 4.1.3.8) and malic enzyme (EC 1.1.1.40) were determined in lungs and livers of adult rats that were normally fed, starved for 48 h or starved for 48 h and subsequently refed for 72 h with a carbohydrate-rich, fat-free diet. In the liver, starvation caused a small decrease in the relative abundance of the mRNAs which was not statistically significant. Subsequent refeeding caused a statistically significant increase in mRNAs for all of the enzymes studied. In the lung, no significant changes were found, indicating that the regulation of the abundance of mRNAs encoding the lipogenic enzymes in the lung differs from that in the liver. In the developing rat lung, mRNA for fatty-acid synthase increased 3-fold in abundance between fetal days 18 and 20 and decreased directly after birth (at day 22 of gestation). A similar pattern was observed for ATP citrate lyase mRNA. The level of acetyl-CoA carboxylase mRNA decreased significantly after birth. These observations indicate that in perinatal rat lungs, pretranslational regulation is involved in the control of the synthesis of these enzymes. The abundance of acetyl-CoA carboxylase mRNA did not change in the prenatal period, a time during which the specific activity of this enzyme increases. This lack of correlation between the specific activity of acetyl-CoA carboxylase and the abundance of its mRNA may indicate that translational regulation of the synthesis of the enzyme or post-synthetic regulatory effects on enzyme molecules are involved in the control of this enzyme in the prenatal period. No changes in the abundance of lung malic enzyme mRNAs were observed throughout the perinatal period.     

The level of beta-alanine aminotransferase activity in regenerating and differentiating rat liver

beta-Alanine aminotransferase from rat liver was purified to electrophoretic homogeneity. The immunological and kinetic properties of this enzyme were similar to those of the enzyme from rat brain. However, the liver enzyme transaminates from beta-alanine to 2-oxoglutaric acid, while the brain enzyme transaminates from gamma-aminobutyric acid. beta-Alanine aminotransferase activity in regenerating rat liver was lower than that in control rat liver. Activity of this enzyme, as well as of other uracil-catabolizing enzymes (Weber, G., Queener, S.F. and Ferdinandus, A. (1970) in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 9, pp. 63-95, Pergamon Press, Oxford), was low in newborn rat liver and increased about 5-fold, reaching the level observed in adult rat liver. beta-Alanine and prednisolone induced beta-alanine aminotransferase in rat liver.     

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat.

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.