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L Reshef

Publications and source records attributed to L Reshef.

At least 37 records · Page 2Linked to original sources
PubMed1983

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.

Animals↗
PubMed1979

Premature appearance of hepatic phosphoenolpyruvate carboxykinase in fetal rats, not mediated by adenosine 3':5'-monophosphate.

Injection of streptozotocin in utero to fetuses elicited a premature appearance of cytosolic hepatic activity of phosphoenol pyruvate carboxykinase. This was due to a precocious initiation of the synthesis of the enzyme. The streptozotocin-induced appearance of enzyme activity was not mediated by adenosine 3':5'-monophosphate since the concentration of the cyclic nucleotide in the liver was unaffected by the antibiotic, the administration of dibutyryladenosine 3':5'-monophosphate to streptozotocin-treated fetuses elicited an additive increase in enzyme activity, and insulin administration in utero repressed the streptozotocin effect while the effect due to dibutyryladenosine 3':5'-monophosphate was not inhibited by simultaneous insulin injection. Streptozotocin treatment also caused a small but consistent retardation of fetal growth and a marked reduction of liver wet weight. Histological analysis of the liver demonstrated a premature loss of some hematopoietic elements, while hepatocytes appeared normal. Hepatic protein synthesis was unaffected by the streptozotocin treatment. Streptozotocin treatment had no effect on fetal renal phosphoenol pyruvate carboxykinase activity or kidney wet weight.

Animals↗
PubMed1979

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.

Aging↗
PubMed1976

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.

Adipose Tissue↗
PubMed1976

The effect of insulin and glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP) in rat adipose tissue cultured in vitro.

1. Epididymal adipose tissue from the rat was maintained in culture for periods of up to 96h. 2. After an initial decrease in protein synthesis during the first 24h of culture, the adipose tissue recovered its capacity to synthesize and accumulate proteins of a relatively large size. 3. The activity of phosphoenolpyruvate carboxykinase decreased in a parallel manner, but increased again after 24h of incubation of the tissue in culture, to a value twice that noted in the tissue in vivo. This increase in enzyme activity was due to an increase in its rate of synthesis. 4. Both insulin and dexamethasone (9alpha-fluoro-16alpha-methyl-11beta,17,-21-trihydroxypregna-1,4-diene-3,20-dione) inhibited phosphoenolpyruvate carboxykinase synthesis, but dexamethasone also decreased total protein synthesis. 5. The half-life of phosphoenolpyruvate carboxykinase in adipose tissue cultured in vitro was 5--7h and was not altered by insulin or dexamethasone. 6. It is concluded that both insulin and glucocroticoids lower the activity of phosphoenolpyruvate carboxykinase in rat adipose tissue by decreasing its rate of synthesis.

Adipose Tissue↗
PubMed1976

Partial isolation and translation in vitro of messenger ribonucleic acid for phosphoenolpyruvate carboxykinase (guanosine triphosphate).

1. mRNA was extracted from the livers of starved rats and incubated in a heterologous cell-free protein-synthesizing system derived from rabbit reticulocytes. The presence of newly synthesized phosphoenolpyruvate carboxykinase (GTP) was detected by immunoprecipitation with a specific antibody to the enzyme and analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The synthesis of the enzyme was dependent on the addition of rat liver RNA, whereas RNA isolated from rat spleen was inactive. If ovalbumin and anti-ovalbumin were used to form the immunoprecipitates, no radioactivity that migrated as phosphoenolpyruvate carboxykinase was detected. 3. The optimal concentrations of magnesium acetate and KCl for phosphoenolpyruvate carboxykinase synthesis were determined. 4. When polyribosomal RNA was separated by sucrose-gradient centrifugation, phosphoenolpyruvate carboxykinase mRNA migrated between 20 and 26 S in keeping with the high mol. wt. of the protein (85 000). 5. The presence of poly(A) in phosphoenolpyruvate carboxykinase mRNA was suggested by retention of mRNA activity on oligo(dT)-cellulose columns. 6. It was concluded that the cell-free synthesis of phosphoenolpyruvate carboxykinase can serve as a bioassay for intracellular phosphoenolpyruvate carboxykinase mRNA.

Animals↗
PubMed1975

Effects of cyclic adenosine monophosphate, dexamethasone and insulin on phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 hepatoma cells.

Antiserum prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) is shown to specifically precipitate the enzyme from Reuber H-35 cells. Synthesis of phosphoenolpyruvate carboxykinase, as measured immunochemically, is increased by dibutyryl cAMP and dexamethasone, the nucleotide maximally producing a sixfold and the glucocorticoid a threefold change in rate. Studies with actinomycin D, cordycepin, and cycloheximide suggest dibutyryl cAMP acts at a translational or post-transcriptional site. Insulin prevents the increase in synthesis of phosphoenolpyruvate carboxykinase produced by either dibutyryl cAMP or dexamethasone. This antagonism is concentration dependent and does not require the simultaneous presence of glucose, pointing to a direct effect of the hormone on liver enzyme induction. It is suggested that hepatic phosphoenolpyruvate carboxykinase activity is regulated predominantly by the antagonistic interaction of cAMP (glucagon) and insulin on enzyme synthesis.

Animals↗
PubMed1975

Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues.

Phosphoenolpyruvate carboxykinase (GTP) was induced by a combination of dibutyryl cyclic AMP, theophyline and dexamethasone in Reuber H35 hepatoma cells under conditions where an amino acid in the medium was replaced by an appropriate analogue. 2. With canavanine replacing arginine or with 5-fluorotryptophan or 6-fluorotryptophan replacing tryptophan the induced enzyme had a lower catalytic activity-relative to antibody reactivity. 3. These aberrant enzyme molecules were heat-labile in vitro. 4. Measurements of enzyme degradation in vivo indicated that the canavanine-containing enzyme and the 6-fluorotryptophan-containing enzyme were degraded more rapidly than the enzyme containing all natural amino acids.

Amino Acids↗
PubMed1975

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.

Animals↗
PubMed1974

Degradation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in vivo and in vitro.

1. Phosphoenolpyruvate carboxykinase (GTP) in the cytosol fraction of liver was labelled in young rats by the injection of [(3)H]leucine and then isolated with specific antibody. Antibody-antigen precipitates from ;pulse'-labelled animals and from animals in which the content of radioactive enzyme had been decreased by a period of degradation were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels. No radioactive breakdown products were found. 2. (3)H-labelled phosphoenolpyruvate carboxykinase (GTP) was purified from rat liver and used to measure degradation in vitro. There was first a loss of catalytic activity, then a disappearance of immunological activity and finally a loss of solubility before any evidence of proteolytic cleavage. Proteolytic-cleavage fragments, when found, were also insoluble. 3. An analysis of the subcellular location of enzyme inactivation showed that phosphoenolpyruvate carboxykinase (GTP) was stable when incubated with liver cytosol fraction and was inactivated most rapidly by the microsomal fraction. 4. We propose that denaturation of the enzyme is the rate-limiting step in degradation in vivo, and precedes proteolytic cleavage when the enzyme is incubated with liver preparations in vitro.

Animals↗
PubMed1974

Synthesis of phosphoenolpyruvate carboxykinase (guanosine triphosphate) by isolated liver polyribosomes.

1. Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was synthesized by postmitochondrial supernatants of rat liver in the presence of appropriate salts, an energy supply and [(3)H]leucine. Synthesis of enzyme released from polyribosomes was detected by immunoprecipitation with specific antibody followed by electrophoresis of the dissolved antibody-antigen precipitates on sodium dodecyl sulphate-polyacrylamide gels in the presence of a (14)C-labelled enzyme marker. 2. Enzyme synthesis in vitro occurs predominantly on free rather than bound polyribosomes. 3. Starved animals in which de-induction of phosphoenolpyruvate carboxykinase (GTP) had been initiated by re-feeding for 2h had a markedly decreased rate of enzyme synthesis, whether the measurements were made after injection of radioactive leucine into the intact animal or if synthesis was determined in vitro. 4. The low rate of enzyme synthesis by liver polyribosomes from re-fed animals was not due to the absence of soluble factors, nor could it be increased by the addition of cyclic AMP to the protein synthesis system. 5. Phosphoenolpyruvate carboxykinase (GTP) synthesis in vitro is diminished relative to total protein synthesis when the postmitochondrial supernatant is kept at 0 degrees C for several hours before measurement of protein synthesis. Since this effect is blocked by heparin, it is probably caused by selective ribonuclease attack on enzyme mRNA. 6. De-induction of phosphoenolpyruvate carboxykinase (GTP) is tentatively explained as being due to a transcriptional block in specific mRNA synthesis, followed by rapid degradation of existing message.

Animals↗
PubMed1974

Rapid loss of translatable messenger RNA of phosphoenolpyruvate carboxykinase during glucose repression in liver.

The rate of synthesis of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in rat liver decreased with a half-life of 30 min after fasted rats were refed either a chow diet or glucose. A requirement for both glucose and insulin to bring about this rapid deinduction was shown, as well as the ability of dibutyryl adenosine 3':5'-cyclic monophosphate to block the decrease in enzyme synthesis. Estimates of the stability of messenger RNA of phosphoenolpyruvate carboxykinase were made by using the inhibitors actinomycin D and cordycepin to block further messenger RNA synthesis, and then measuring the decrease in specific enzyme synthesis. It is suggested that the use of actinomycin D yields an overestimation of the template stability. The results with cordycepin imply that the enzyme messenger RNA has a short half-life of approximately 1 hr. Thus, it is possible that deinduction may proceed by way of a decrease in messenger RNA production, leading to a rapid fall in the synthesis of phosphoenolpyruvate carboxykinase.

Animals↗