Cooperative effect of thyroid and glucocorticoid hormones on the induction of hepatic phosphoenolpyruvate carboxykinase in vivo and in cultured hepatocytes

The present study investigates the effect and interaction of glucocorticoid and thyroid hormones on the induction of phosphoenolpyruvate carboxykinase (PEPck) mRNA and enzyme protein under in vivo conditions and in serum-free cultured hepatocytes from hypothyroid rats. In hypothyroid/adrenalectomized rats T3 significantly enhanced the cAMP induced PEPck mRNA activity within 3-6 h. This effect was further enhanced by the presence of glucocorticoids. The half-life of PEPck mRNA, as determined after administration of cordycepin, was not affected by hypothyroidism or hyperthyroidism (t 1/2 approximately equal to 45 min), but considerably prolonged by the absence of glucocorticoid hormones (t 1/2 less than 80 min). In hepatocytes in culture Bt2cAMP (0.2 mM) provoked an increase in translatable PEPck mRNA within 2 h incubation time. Preincubation with either T3 (0.1 microM) or dexamethasone (0.1 microM) for 4 h significantly enhanced the cAMP response on PEPck mRNA. Addition of both, T3 plus dexamethasone further enhanced this Bt2cAMP-mediated effect. By measurement of PEPck synthesis corresponding findings were observed. It is concluded that glucocorticoid and thyroid hormones predominantly enhance the cAMP-provoked induction of hepatic PEPck mRNA and, consequently, of PEPck synthesis. Their effect is rapid, significant and additive, indicating an independent action. While glucocorticoids, in addition, accelerate PEPck mRNA degradation, the PEPck mRNA decay rate is similar in the presence and absence of thyroid hormones.     

Effect of thyroid state on cyclic AMP-mediated induction of hepatic phosphoenolpyruvate carboxykinase.

Hepatic phosphoenolpyruvate carboxykinase (PEPCK) is significantly increased in the hyperthyroid starved rat, and moderately decreased in the hypothyroid starved rat. As tri-iodothyronine by itself has only a small and sustained effect on the induction of this enzyme, as was previously shown in the isolated perfused organ, the effect of hypo- and hyper-thyroidism on the increase in cytosolic PEPCK provoked by dibutyryl cyclic AMP (Bt2cAMP) was investigated in vivo and in the isolated perfused liver. Compared with euthyroid fed controls, in hypothyroid fed rats Bt2cAMP provoked in 2 h only a small increase in translatable mRNA coding for PEPCK. In contrast, in hyperthyroid animals PEPCK mRNA as measured by translation in vitro was already increased in the fed state, and further enhanced by Bt2cAMP injection to values as in euthyroid controls. Under all thyroid states a close correlation between PEPCK mRNA activity and PEPCK synthesis was observed. In the isolated perfused liver from the hyperthyroid fed rat, the increase in PEPCK provoked by Bt2cAMP or Bt2cAMP + isobutylmethylxanthine was considerably enhanced compared with those obtained in livers of hypothyroid rats. Also, adrenaline provoked a stimulated induction of PEPCK in hyperthyroid rats compared with hypothyroid rats. To summarize, our data indicate that the primary action of thyroid hormones on the synthesis of hepatic cytosolic PEPCK is to accelerate the cyclic AMP- or adrenaline-induction of the enzyme, acting primarily at a pretranslational level.     

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.     

Interaction of glucocorticoid hormones and cyclic nucleotides in induction of tyrosine aminotransferase in cultured hepatoma cells.

Reproducible induction of the enzyme tyrosine aminotransferase by dibutyryl cAMP (Bt2cAMP) in a line of HTC hepatoma cells in suspension culture requires that the cells be preinduced with dexamethasone, a synthetic glucocorticoid which itself induces tyrosine aminotransferase. Concentrations of dexamethasone that do not induce tyrosine aminotransferase fail to support Bt2cAMP induction, removal of the steroid from the medium leads to a loss of the Bt2cAMP effect, and an HTC cell line whose aminotransferase is not steroid-inducible does not respond to the cyclic nucleotide. We show that the further induction of tyrosine aminotransferase by Bt2cAMP in dexamethasone-treated cells is due to an increased rate of enzyme synthesis. The cyclic nucleotide has no effect on aminotransferase synthesis in cells grown in the absence of steroid. Several lines of evidence suggest that dexamethasone acts at a step beyond the activation of protein kinase by cAMP: (a) basal levels of cAMP are not altered by growth of HTC cells in dexamethasone; (b) accumulation of cAMP from the medium is not enhanced; (c) the glucocorticoid does not induce cAMP-dependent protein kinase in HTC cells; and (d) there is no augmentation of cAMP binding to the regulatory protein, nor is there any change in cAMP activation of protein kinase caused by growth in dexamethasone. These results help define a system that should be useful in studying the interaction of cyclic nucleotides and steroid hormones.     

Hormonal effects on the biosynthesis of lactate dehydrogenase in rat hepatocytes.

Rat hepatocytes have been studied in suspension culture for 10-h periods. Levels of extractable lactate dehydrogenase (LDH) have been measured in these hepatocytes at hourly intervals in order to note the balance between biosynthesis and degradation of this enzyme. Newly synthesized LDH has been measured by following the rate of incorporation of [3H]leucine into radiochemically pure LDH of high specific catalytic activity as isolated by a rapid affinity chromatographic procedure. The effects of the addition of physiological concentrations of the following hormones at the beginning of 10-h culture periods immediately following preparation of the hepatocytes by the collagen perfusion procedure have been recorded. The hormones triiodothyronine (T3), insulin, glucagon, and dexamethasone have been added singly or in combination. The culture medium has supplied variable amounts of these hormones in the 10% of fetal calf (or other) serum added, and the hepatocytes themselves have provided intracellular amounts of hormones. In addition to the added hormones, N6,O2'-dibutyryl cyclic AMP (Bt2cAMP) has also been studied. Control suspensions of hepatocytes show reproducible initial levels of extractable LDH which are maintained or slightly increased during 10 h. Such control systems also incorporate [3H]leucine into total protein and into highly purified LDH at reproducible rates during 10 h of incubation. The effects of added hormones on LDH lavels are as follows: (a) T3 causes about a 2-fold increase in LDH at 7 to 8 h in hepatocytes from young adult animals, an effect which is lowered in either younger or older animals or in thyroidectomized animals. (b) Insulin leads to a similar increase in LDH at 5 to 6 h and a falling off at 8 to 10 h. (c) Glucagon also causes an approximate doubling of the amount of extractable LDH during a 10-doubling of the amount of extractable LDH during a 10-h period. (d) Dexamethasone does not produce an increase. (e) Bt2-cAMP produces an effect indistinguishable from that of glucagon. Paired combinations of these hormones fail to produce an additive response in any case. The combinations of T3 plus dexamethaseon and insulin plus dexamethasone lead to significant reductions in levels of extractable LDH when compared to the single hormone effects cited above. With respect to rates of synthesis of total protein as measured by [3H]leucine incorporation, only glucagon, glucagon plus Bt2-cAMP, glucagon plus insulin, T3 plus Bt2cAMP, and T3 plus insulin produce significant increases during a 10-h period. However, when [3H]leucine incorporation into highly purified LDH is measured as an index of LDH biosynthesis, T3, insulin, and glucagon consistently increase the biosynthetic rates during a 10-h period. Bt2cAMP produces a smaller increase. Dexamethasone fails to produce any significant change when compared to controls. Paired combinations of hormones again do not produce any additive effect on LDH biosynthesis when the hormone producing the higher level is taken as the reference...     

Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.     

cAMP-mediated upregulation of gelatinases in primary cultures of isolated rat hepatocytes

Matrix metalloproteinases (MMPs) play a major role in tissue remodelling and repair in pathophysiological conditions, such as liver fibrosis and regeneration. Regulation of the MMPs produced by liver cells is important in maintaining cell-matrix ratio in liver, which is a major target site for hormones that mediate their intracellular effects through cAMP. The possibility of cAMP affecting the activity of MMPs and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) was studied using isolated rat hepatocytes in culture. Zymographic analysis showed that treatment with hormones like epinephrine, thyroxine and dexamethasone and Bt2 cAMP increased 92 kDa MMP-9 activity. Bt2 cAMP caused upregulation of MMP-9 in a dose-dependent manner. The effect of hormones was less on MMP-2. ELISA using specific antibodies showed increase in levels of MMP-9 and TIMP-1 protein. Kinetic analysis of production of MMPs and TIMPs showed that the response to Bt2 cAMP was a delayed one, indicating its effect on de novo protein synthesis. These results suggest the possibility of cAMP dependent regulation of MMP-9 in the hepatocytes.     

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.     

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.     

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.     

Induction of ornithine decarboxylase in guinea pig lymphocytes by the divalent cation ionophore A 23187. Effect of dibutyryladenosine 3',5'-monophosphate

The divalent cation ionophore, A23187, at a concentration of 0.25 microgram/ml, enhanced influx of Ca2+, activity of ornithine decarboxylase and incorporation of [3H]thymidine into DNA of guinea pig lymphocytes. Combined treatment of cells with A23187 and dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) augmented these three events. A23187 at a concentration of 0.06 microgram/ml was insufficient for induction of ornithine decarboxylase stimulated neither Ca2+ influx nor [3H]thymidine incorporation, but stimulated Ca2+ efflux. A23187 (0.06 microgram/ml) in combination with Bt2cAMP caused a marked induction of ornithine decarboxylase and stimulation of [3H]thymidine incorporation into DNA. When the time of Bt2cAMP addition was delayed after A23187, the stimulation of ornithine decarboxylase activity decreased. Washout of Bt2cAMP from cell culture earlier than 4 h of incubation caused a reduction in the stimulatory effect of Bt2cAMP. These results suggest that raising concentrations of cytoplasmic Ca2+ and cellular cAMP are important to some initial events leading to induction of ornithine decarboxylase and these biochemical changes are obligatory sequential steps for stimulation of DNA synthesis.     

Induction of Cholinergic Expression in Developing Spinal Cord Cultures

The induction of choline acetyltransferase (ChAT) by cAMP derivatives was studied in dissociated spinal cord cultures. Dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (1 mM) produced a 2-3-fold stimulation of ChAT activity in developing cultures whereas 8-bromo cGMP had no effect. A phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, also increased (2-fold) ChAT activity in immature cultures. Significant elevations in ChAT were detected after 2 h incubation with dbcAMP. Maximum enzyme induction was observed 24 h after dbcAMP supplementation to the culture medium. Developmental studies revealed that ChAT could be induced on days 2-16 in culture. The largest induction of ChAT activity was observed on day 7 in culture. After day 19, when control enzyme activity attained levels of mature cultures, cAMP-mediated ChAT induction was no longer observed. Cycloheximide and actinomycin D blocked ChAT induction whereas basal enzyme activity remained unaffected. Culture protein content was not changed after 1-day exposure to dbcAMP. 125I-Tetanus toxin fixation after dbcAMP treatment revealed a 20% decrease from control in neuronal surface during days 7-9 in culture. These data indicated that cAMP derivatives produced a rapid increase in cholinergic expression during a specific period of development in spinal cord cultures. There appears to be specificity to this effect, as total neuronal surface does not respond in the same manner as ChAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of rat liver phosphoenolpyruvate carboxykinase (GTP) messenger ribonucleic acid activity by N6, O2'-dibutyryladenosine 3',5'-phosphate

N6,O2'-Dibutyryladenosine 3',5'-phosphate (Bt2cAMP) induces the synthesis of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.32), in rat liver by increasing the activity of messenger ribonucleic acid (mRNA) coding for this enzyme (mRNAPEPCK) more than 20-fold (from less than 0.01% to greater than 0.20% of total mRNA activity) as determined by using in vitro translation systems which measure only active mRNAPEPCK. The increase in mRNAPEPCK activity could result from increased synthesis, increased processing, or decreased inactivation rates. Actinomycin D and cordycepin inhibit mRNAPEPCK induction by 89% and 70%, respectively, a result that indicates a requirement for ongoing RNA synthesis but that does not distinguish which of these steps is regulated by cAMP. We have employed a kinetic approach, not involving RNA synthesis inhibitors, to determine the half-life of mRNAPEPCK both during a period of deinduction following glucose feeding and during a subsequent induction by Bt2cAMP. An estimated half-life of 20 +/-5 min during both of these periods indicates that Bt2cAMP has no effect on the rate of inactivation of mRNAPEPCK. We conclude that Bt2cAMP effects the increase in activity of mRNAPEPCK by promoting its synthesis or processing.     

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.