Stimulation by 6-N,2'-O-dibutyryladenosine 3' :5'-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.     

Stimulation by 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver (Short Communication)

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.     

Regulation of hormonal induction of chick liver cytosol-specific phosphoenolpyruvate carboxykinase

Administration of glucagon, epinephrine, or dibutyryl cAMP to chicks induced cytosol-specific phosphoenolpyruvate carboxykinase in liver. In vitro translation assay with poly(A)+RNA indicated that this induction was due to the increase in phosphoenolpyruvate carboxykinase-coding mRNA synthesis which resulted from an increased level of hepatic cAMP. Either hydrocortisone or alpha-adrenergic agonist was ineffective for the induction by itself, but showed a significant effect when administered together with one of the inducing agents given above. In particular, hydrocortisone enhanced the synthesis of phosphoenolpyruvate carboxykinase-specific mRNA without changing the profile of the time courses of the induction and of hepatic cAMP level. Those observations suggest that the fundamental machinery required for induction of cytosol-specific phosphoenolpyruvate carboxykinase in liver is shared in common between rat and chick, and that the absence of appreciable induction of cytosol-specific hepatic phosphoenolpyruvate carboxykinase in starved chicks is due to neither lack nor impairment of the hormone-mediated induction mechanism, but is due to the difference in usage of the genetic information between the two animal species.     

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.     

Induction of mitochondrial phosphoenolpyruvate carboxykinase in cultured human fibroblasts.

Mitochondria of cultured normal human fibroblast cells were found to contain the enzyme phosphoenolpyruvate carboxykinase. The activity of this enzyme in these cells is increased 2- to 3-fold by addition of 5 . 10(-4) M dibutyryl cyclic AMP, or 1.5- to 2-fold by the addition of dexamethasone (2 . 10(-7) M) or hydrocortisone (1.38 . 10(-6) M). These increases in enzyme activity were inhibited cycloheximide and actinomycin D, suggesting they are dependent upon de novo protein synthesis. Cultured human fibroblasts may thus provide a useful system for studying the regulation of mitochondrial phosphoenolpyruvate carboxykinase.     

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.     

Effect of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase in the newborn rat. Changes in the rate of synthesis of the enzyme and in the activity of its translatable messenger RNA.

The effects of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase activity in the developing rat were investigated. The hormone induced increases in pre-existing enzyme activity of both tissues in fetal and neonatal rats, yet did not cause the primary appearance of phosphoenolpyruvate carboxykinase activity in utero. Neonatal hepatic phosphoenolpyruvate carboxykinase activity was increased 2--3 fold by triamcinolone form the 3rd to the 15th postnatal day. This was shown to be additive to the effect of Bt2cAMP on enzyme activity. The increases in phosphoenolpyruvate carboxykinase activity were demonstrated to be due to increased synthesis of the enzyme, which was accompanied by a proportionate increase in the amount of functional phosphoenolpyruvate carboxykinase mRNA, as measured by the polyribosomal and poly(A)-containing RNA directed cell-free synthesis of the enzyme. The demonstration of a triamcinolone effect on kidney and liver phosphoenolpyruvate carboxykinase activity in fetal and neonatal rats provides support for a possible role of glucocorticoids in the regulation of phosphoenolpyruvate carboxykinase activity during development.     

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) in adipose tissue in vivo by glucocorticoids and insulin.

1. The regulation of the synthesis of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in epididymal adipose tissue, liver and kidney in vivo was studied immunochemically. 2. Phosphoenolpyruvate carboxykinase (GTP) synthesis in adipose tissue is increased by starvation, diabetes and noradrenaline, and decreased by re-feeding and insulin. These changes were also seen in adrenalectomized rats and are qualitatively similar to those observed for the liver enzyme. This indicates the involvement of cyclic AMP as an inducer and insulin as a de-inducer in the regulation of phosphoenolpyruvate carboxykinase (GTP) in both tissues. (Induction and de-induction are defined as selective increase and decrease respectively in the rate of enzyme synthesis, regardless of the mechanism involved.)3. Adrenalectomy had little effect on phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney, but increased the synthesis rate of the adipose-tissue enzyme. Starvation and adrenalectomy had additive effects in increasing the synthesis rate of adipose-tissue phosphoenolpyruvate carboxykinase (GTP). In adrenalectomized diabetic rats glucocorticoids increased phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney while decreasing enzyme synthesis in adipose tissue. De-induction of adipose tissue phosphoenolpyruvate carboxykinase (GTP) is therefore regulated independently by glucocorticoids and insulin. 4. Although liver, kidney and adipose-tissue phosphoenolpyruvate carboxykinases (GTP) are seemingly identical, there is an apparent tissue-specific differentiation in regulatory systems for the enzyme.     

Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver.

1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably less than the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.     

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 hepatic P-enolpyruvate carboxykinase (GTP) during development.

Hepatic gluconeogenesis in the rat does not begin until birth. The enzyme P-enolpyruvate carboxykinase appears initially at birth and is the final enzyme in the gluconeogenic sequence to develop. The appearance of this enzyme in the cytosol of rat liver is caused by the stimulation of enzyme synthesis, probably due directly to an increase in the hepatic concentration of cAMP. Enzyme degradation does not begin until 36 hours after birth. Studies with fetal rats in utero have shown that dibutyryl cAMP or glucagon will stimulate P-enolpyruvate carboxykinase synthesis and that this effect can be blocked by insulin. Insulin is known to depress the synthesis of P-enolpyruvate carboxykinase in adult rat liver and in Reuber H-35 liver cells in culture. The glucocorticoids are without effect on the synthesis of the enzyme in fetal rat liver. Work by Girard et al. (J. Clin. Invest. 52: 3190, 1973) has established that the molar ratio of insulin to glucagon drops from 10 immediately after birth, to 1 after one hour. This is due to both a rise in glucagon and a fall in insulin concentrations at birth. These studies, together with our work on the synthesis of P-enolpyruvate carboxykinase, indicate that the sharp drop in the concentration of insulin may relieve the normal inhibition of enzyme synthesis. This would allow the initial stimulation of enzyme synthesis by the glucagon-mediated rise in the concentration of CAMP.     

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.     

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.