Permissive action of thyroid hormones in the cAMP-mediated induction of phosphoenolpyruvate carboxykinase in hepatocytes in culture

Thyroid hormones are known to stimulate in vivo the synthesis of several liver proteins. In order to determine their role in the regulation of important hepatic proteins at the cell level, the effect of T3 on the induction of the glucoregulatory enzyme phosphoenolpyruvate carboxykinase (PEPck) was investigated in cultured hepatocytes. The cells were isolated from adult hypothyroid rats and exposed to a chemically defined medium, devoid of serum supplement and hormone-free over a period of 48 h. Addition of T3 to the medium had only a minor effect on PEPck induction. Dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) or epinephrine provoked significant increase in PEPck synthesis within 4-6 h. Simultaneous addition of T3 was found significantly to promote the Bt2cAMP-mediated enzyme induction. Physiological concentrations of T3 (10 pM to 1 nM) were found to be effective in enhancing this cAMP-mediated signal. Exposure of the cells to T3 for only 90 min, 16 h prior to addition of Bt2cAMP was nearly as effective as continuous exposure to T3 in enhancing the cAMP-effect on PEPck synthesis. In contrast, no effect of T3 on the Bt2cAMP-induced tyrosine-aminotransferase activity could be detected. It is concluded that the role of T3 on the cAMP-elicited PEPck synthesis is selective, rapid, primarily permissive and significant within the physiological range of the hormone. In addition, these data indicate that such hepatocyte cultures prepared from hypothyroid rats are ideally suited to the analysis of the regulatory role of thyroid hormones on specific gene expression.     

Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP

Glucocorticoid hormones increase the activity of cytosolic aspartate aminotransferase (cAspAT) in the Fao rat hepatoma cell line. Maximal increase (6-10-fold) was observed 48 h following the addition of the glucocorticoid agonist dexamethasone at a concentration of 0.1 microM. The effect of dexamethasone was specific since it was not mimicked by sex steroids and was inhibited by the glucocorticoid antagonist RU 486. Insulin (0.1 microM) inhibited by more than 50% the induction of cAspAT by glucocorticoids. The cAMP analog, 8-bromoadenosine 3',5'-monophosphate (Br8cAMP, 0.5 mM), potentiated the effect of dexamethasone (2-3-fold) and partially relieved the inhibitory effect of insulin on the induction by dexamethasone. Both insulin and Br8-cAMP had no significant effect on basal activity. The mitochondrial isoenzyme was insensitive to the various hormonal treatments. Northern blot analysis revealed the presence of two major (2.1-kb and 1.8-kb) and one minor (4-kb) mRNA species hybridizing with a rat cAspAT probe. The regulation of these mRNAs by glucocorticoids, insulin and cAMP correlated with the variation of the cAspAT activity, suggesting that these hormones act at the pretranslational level. We compared the regulation of cAspAT mRNAs with those of tyrosine aminotransferase mRNA. Both were similarly increased by dexamethasone but the latter was also increased by cAMP even in the absence of the glucocorticoid agonist. In addition, the increase in tyrosine aminotransferase mRNA was inhibited by cycloheximide whereas the increase in cAspAT mRNAs was not. These results show that there are significant differences in the regulation of cAspAT and tyrosine aminotransferase by glucocorticoids and other hormones, although both enzymes probably contribute to the same metabolic pathway.     

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.     

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.     

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...     

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.     

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.     

Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7 alpha-hydroxylase.

To study the effect of steroid hormones on bile acid synthesis by cultured rat hepatocytes, cells were incubated with various amounts of these compounds during 72 h and conversion of [4-14C]cholesterol into bile acids was measured. Bile acid synthesis was stimulated in a dose-dependent way by glucocorticoids, but not by sex steroid hormones, pregnenolone or the mineralocorticoid aldosterone in concentrations up to 10 microM. Dexamethasone proved to be the most efficacious inducer, giving 3-fold and 7-fold increases in bile acid synthesis during the second and third 24 h incubation periods respectively, at a concentration of 50 nM. Mass production of bile acids as measured by g.l.c. during the second day of culture (28-52 h) was 2.2-fold enhanced by 1 microM-dexamethasone. No change in the ratio of bile acids produced was observed during this period in the presence of dexamethasone. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of the bile acid pathway, to bile acids was not affected by dexamethasone. Measurement of cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes, incubated with 1 microM-dexamethasone, showed 10-fold and 90-fold increases after 48 and 72 h respectively, as compared with control cells. As with bile acid synthesis from [14C]cholesterol, no change in enzyme activity was found in hepatocytes cultured in the presence of 10 microM steroid hormones other than glucocorticoids. Addition of inhibitors of protein and mRNA synthesis lowered bile acid production and cholesterol 7 alpha-hydroxylase activity and prevented the rise of both parameters with dexamethasone, suggesting regulation at the mRNA level. We conclude that glucocorticoids regulate bile acid synthesis in rat hepatocytes by induction of enzyme activity of cholesterol 7 alpha-hydroxylase.     

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.     

Regulation of messenger ribonucleic acid levels for five urea cycle enzymes in cultured rat hepatocytes. Requirements for cyclic adenosine monophosphate, glucocorticoids, and ongoing protein synthesis

In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor as a new regulator of induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids

Epidermal growth factor (EGF) dose-dependently enhanced the induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids in primary cultures of adult rat hepatocytes without itself having any effect on these enzymes in the absence of glucocorticoids. The amplifications were observed even with dexamethasone at high concentrations (10(-6) M-10(-5) M) that had a maximal effect. EGF had no effect on induction of tyrosine aminotransferase by glucagon or Bt2cAMP. The effect of EGF was also observed in adrenal-ectomized and submaxillary gland-ectomized rats. These results suggest that EGF is an endogenous amplifier of the action of glucocorticoids.     

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.     

The effects of glucocorticoids on insulin-stimulated lipogenesis in primary cultures of rat hepatocytes.

We used primary cultures of rat hepatocytes to evaluate the effects of glucocorticoids on insulin-responsive hepatic lipogenesis. The data indicate that hepatocytes incubated for 20 h with dexamethasone (0.1 microM) alone are profoundly resistant to the ability of insulin to stimulate lipogenesis acutely. In contrast, primary cultures of hepatocytes incubated with dexamethasone plus insulin are hyper-responsive to the ability of insulin to stimulate lipogenesis chronically. This potentiation of insulin action by a glucocorticoid occurs at physiological concentrations of the two hormones. Exposure to dexamethasone plus insulin for more than 4 h is required for the two hormones to enhance insulin action either by overcoming the insulin resistance induced by dexamethasone alone or by stimulating insulin action induced by insulin alone. Despite the marked potentiation of insulin action, hepatocytes exposed to dexamethasone plus insulin are less sensitive to insulin, as demonstrated by a shift to the right in the dose-response curve for insulin-stimulated lipogenesis. The resistance of hepatocytes to the acute effects of insulin after exposure to dexamethasone alone and the potentiation of insulin action and decreased sensitivity to insulin after exposure to insulin plus dexamethasone are all mediated by post-insulin-binding events. These studies demonstrate potentiation of insulin action in the liver by physiological concentrations of glucocorticoids and may have physiological significance for the regulation of normal hepatic lipogenesis, for the hyperlipidaemia observed with the pharmacological use of glucocorticoids, and for disease states in man associated with hyperinsulinaemia and hypercortisolism.