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.     

Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway.

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2--3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.     

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.     

Control of the activity of phosphoenolpyruvate carboxykinase and the level of its mRNA in livers of newborn rats. Effect of diabetes, glucose load and glucocorticoids

Streptozotocin treatment produces a typical experimental diabetes in neonates exhibiting hyperglycemia, glucosuria, ketonemia and increased level of fatty acids in the blood. The liver is affected as well, with reduced activity of glycogen synthase and a corresponding decrease in the content of liver glycogen. In contrast, the activity of liver cytosolic phosphoenolpyruvate carboxykinase and the level of its mRNA are not affected. Using a cDNA containing P-pyruvate carboxykinase sequence, the relative abundance of the enzyme mRNA was estimated. The level of the mRNA was readily observed increasing by glucocorticoid treatment or decreasing in response to administered load of glucose. These parallel the changes observed in the activity of the enzyme under these treatments, indicating that the level of P-pyruvate carboxykinase mRNA actually determines that of the enzyme. The failure of diabetes to increase the level of enzyme mRNA and the limited response to glucose loading strongly suggest that the mechanisms controlling the level of P-pyruvate carboxykinase mRNA in neonates are relatively resistant to insulin. This is unique to neonates, since in both the adult and the fetal liver. P-pyruvate carboxykinase readily responds to insulin. The minimal levels of glucocorticoids characteristic of neonates may be associated with this phenomenon.     

Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue.

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used. Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine. The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 +/- 1% of leucine released from degradation of proteins in 30 subjects. A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed. The results indicate that biosynthesis and degradation of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly-turning-over proteins.     

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.     

Inactivation of phosphoenolypyruvate carboxykinase (GTP) by liver extracts.

1. The inactivation of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in liver extracts was catalysed by the microsomal fraction, and led to the enzyme becoming bound to the microsomal membranes. 2. Inactivation by microsomal fraction, typsin or heating at 48degreesC was accelerated by L-cystine, D-cystine and oxidized glutathione and decreased by dithiothreitol. 3. MnC1(2) and CoC1(2) protected the enzyme from inactivation by heat or microsomal fraction, but did not affect the inactivation caused by trypsin. 4. Several proteinase inhibitors had no effect on the microsomal inactivation reaction, suggesting that proteolysis was not involved. 5. It is argued that the initial step in the degradation of phosphoenolpyruvate carboxykinase (GTP) is an inactivation reaction, perhaps involving oxidized thiol compounds.     

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.     

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.     

Attempts to relate enzyme inactivation to degradation in vivo

Inactivation of liver cytosol proteins has been measured in vitro in the presence of various membranes and disulphides. Inactivation rates correlate with the known degradation rate constants of the enzymes in the intact liver. More extensive studies were carried out with glucose-6-phosphate dehydrogenase (G6PD) and phosphoenolpyruvate carboxykinase (PEPCK) using either cytosol as a source of these enzymes or alternatively highly purified preparations of each enzyme. All membranes purified from liver had a considerable capacity to inactivate the enzymes with higher activity found in the hepatocyte plasma membrane. Various lipid preparations or plasma membranes from other tissues were virtually ineffective. Inactivation was dependent on disulphides in the membranes as shown by the inhibition of activity if membranes were pretreated with thiols. Preliminary experiments of the fate of inactivated G6PD or PEPCK show binding to membranes and subsequent proteolysis. A model is proposed for the degradation of labile enzymes.     

Induction in primary culture of 'gluconeogenic' and 'glycolytic' hepatocytes resembling periportal and perivenous cells

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1. Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity 'periportal'. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply 'perivenous'. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2. Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3. Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 muM dexamethasone) gluconeogenesis was 1.5-fold faster in the 'periportal' cells and glycolysis was 2.4-fold faster in the 'perivenous' cells. 4. Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in 'periportal' cells containing high phosphoenolpyruvate carboxykinase activity, not in the 'perivenous' cells with a low level of this enzyme. 5. A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in 'periportal' cells and glycolysis was eightfold faster in 'perivenous' cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.     

Integration of insulin and amino acid signals that regulate hepatic metabolism-related gene expression in rainbow trout: role of TOR

Amino acids are considered to be regulators of metabolism in several species, and increasing importance has been accorded to the role of amino acids as signalling molecules regulating protein synthesis through the activation of the TOR transduction pathway. Using rainbow trout hepatocytes, we examined the ability of amino acids to regulate hepatic metabolism-related gene expression either alone or together with insulin, and the possible involvement of TOR. We demonstrated that amino acids alone regulate expression of several genes, including glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, pyruvate kinase, 6-phospho-fructo-1-kinase and serine dehydratase, through an unknown molecular pathway that is independent of TOR activation. When insulin and amino acids were added together, a different pattern of regulation was observed that depended upon activation of the TOR pathway. This pattern included a dramatic up-regulation of lipogenic (fatty acid synthase, ATP-citrate lyase and sterol responsive element binding protein 1) and glycolytic (glucokinase, 6-phospho-fructo-1-kinase and pyruvate kinase) genes in a TOR-dependent manner. Regarding gluconeogenesis genes, only glucose-6-phosphatase was inhibited in a TOR-dependent manner by combination of insulin and amino acids and not by amino acids alone. This study is the first to demonstrate an important role of amino acids in combination with insulin in the molecular regulation of hepatic metabolism.     

Amino acids and insulin are both required to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle

The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C(25) and C(0)) or blocked with diazoxide injections (diazoxide groups: DZ(25) and DZ(0)). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C(25) group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E. eIF4G complex. The p70 S6 kinase (p70(S6k)) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70(S6k) compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.     

Effect of insulin and prednisolone on cyclic nucleotides and phosphoenolpyruvate carboxykinase activity in brown fat and liver of developing rats

The concentrations of cyclic AMP and cyclic GMP in brown fat and liver of both suckling and adult rats at fixed times after injection of insulin (2.5 U/100 g body weight) or prednisolone (2.5 mg/100 g body weight) were compared with the activity of phosphoenolpyruvate carboxykinase assayed 24 h after the injections. A stimulus that produced an increase in cyclic AMP content also produced an increase in the enzyme activity. If the content of cyclic GMP was also increased there was no rise in phosphoenolpyruvate carboxykinase activity. A rise in the content of cyclic GMP alone was associated with a reduction in the activity of the enzyme. These preliminary results indicate that cyclic AMP could be involved in the induction of phosphoenolpyruvate carboxykinase and that cyclic GMP may somehow be related to its repression. The known differences in the response of phosphoenolpyruvate carboxykinase activity to insulin and prednisolone in different tissues and at different stages of ontogenic development may thus be linked to differences in the responsiveness of enzymes concerned with the metabolism of cyclic nucleotides.     

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions.

1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.