Liver Cytosolic Phosphoenolpyruvate Carboxykinase Research Articles

Effects of monensin on metabolic parameters, feeding behavior, and productivity of transition dairy cows

The effects of monensin on transition cow metabolism may be dependent on modulation of feeding behavior, rumen pH, and expression of key metabolic genes. Multiparous Holstein cows were used to determine the effects of monensin (400mg/cow daily) on these variables. Cows were randomly assigned, based on calving date, to control or monensin treatments (n = 16 per treatment) 21 d before their expected calving date, and cows remained on treatments through 21 d postpartum. Feeding behavior and water intake data were collected daily. Liver biopsies were conducted after assessing BCS and BW on d -21, -7, 1, 7, and 21 relative to calving for analysis of triglyceride (TG) content as well as mRNA abundance of cytosolic phosphoenolpyruvate carboxykinase, carnitine palmitoyltransferase 1a, and apolipoprotein B. Blood samples were collected 21, 7, and 4 d before expected calving and 1 (day of calving), 4, 7, 14, and 21 d postpartum for nonesterified fatty acid, β-hydroxybutyrate, glucose, insulin, and haptoglobin analyses. Ruminal pH was collected every 5 min on d 1 through 6 postpartum via a wireless indwelling probe. On d 7 postpartum, a caffeine clearance test was performed to assess liver function. Data were analyzed using mixed models with repeated measures over time. Monensin decreased mean plasma β-hydroxybutyrate (734 vs. 616 ± 41 μM) and peak concentrations (1,076 vs. 777 ± 70 μM on d 4 postpartum). Monensin also decreased time between meals prepartum (143 vs. 126 ± 5.0 min) and postpartum (88.8 vs. 81.4 ± 2.9 min), which was likely related to a smaller ruminal pH standard deviation in the first day after cows changed to a lactation ration (0.31 vs. 0.26 ± 0.015). Monensin also increased liver mRNA abundance of carnitine palmitoyltransferase 1a (0.10 vs. 0.15 ± 0.002 arbitrary units), which corresponded to a slower rate of liver TG accumulation from d -7 to +7 (412 vs. 128 ± 83 mg of TG/g of protein over this time period). No significant effects of monensin supplementation were observed on milk production, liver cytosolic phosphoenolpyruvate carboxykinase, apolipoprotein B, plasma nonesterified fatty acid, glucose, insulin, or haptoglobin. No effects on disease incidence were detected, but sample size was small for detecting such effects. Overall, results confirm that the effects of monensin on transition cows extend beyond altered propionate flux.

Effect of fasting on carbohydrate metabolism in frugivorous bats ( Artibeus lituratus and Artibeus jamaicensis)

The compensatory changes of carbohydrate metabolism induced by fasting were investigated in frugivorous bats, Artibeus lituratus and Artibeus jamaicensis. For this purpose, plasma levels of glucose and lactate, liver and muscle glycogen content, rates of liver gluconeogenesis and the activity of related enzymes were determined in male bats. After a decrease during the first 48 h of fasting, plasma glucose levels remained constant until the end of the experimental period. Plasma lactate levels, extremely high in fed bats, decreased after 48 h of fasting. Similarly, liver glycogen content, markedly high in fed animals, was reduced to low levels after 24 h without food. Muscle glycogen was also reduced in fasted bats. The expected increase in liver gluconeogenesis during fasting was observed after 48 h of fasting. The activities of liver glucose-6-phosphatase and fructose-1,6-bisphosphatase were not affected by food withdrawn. On the other hand, fasting for 24 h induced an increase in the activity of liver cytosolic phosphoenolpyruvate carboxykinase. The data indicate that liver gluconeogenesis has an important role in the glucose homeostasis in frugivorous bats during prolonged periods of food deprivation. During short periods of fasting liver glycogenolysis seems to be the main responsible for the maintenance of glycemia.

Happily at Work

Happily at Work

Effect of hydrazine sulfate on glucose-regulating enzymes in the normal and cancerous rat

Glucokinase, hexokinase, fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase specific activities were monitored in liver cytosol from rats that had been made cancerous with 1,2-dimethylhydrazine and then treated with hydrazine sulfate. The presence of intestinal cancer, specifically, was confirmed by laparotomy and by histological analysis. Sustained changes in hexokinase and glucokinase specific activities were first evident during the latter weeks that the carcinogen was being administered. Upon subsequent treatment with hydrazine sulfate, glucokinase activity further decreased, and liver cytosolic phosphoenolpyruvate carboxykinase activity increased. Liver cytosolic hexokinase and fructose 1,6-bisphosphatase specific activities were not appreciably affected by the hydrazine sulfate treatment. These results indicate that hydrazine sulfate may influence carbohydrate metabolism at the level of selected liver enzymes not only with respect to gluconeogenesis, but also in terms of glucose uptake.

Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase.

Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.

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.

Inactivation of phosphoenolpyruvate carboxykinase by the guanosine nucleotide analogue, 5′-p-fluorosulfonylbenzoyl guanosine

Abstract Rat liver cytosolic phosphoenolpyruvate carboxykinase is inactivated by incubation with 0.84 mM 5′-p-fluorosulfonylbenzoyl guanosine, but is not appreciably affected by the adenosine analogue, 5′-p-fluorosulfonylbenzoyl adenosine, in correspondance with the known nucleotide specificity of this enzyme. Marked protection against inactivation by 5′-p-fluorosulfonylbenzoyl guanosine is provided (either in the presence or absence of divalent metal cation) by GTP or GDP but not by ATP or phosphoenolpyruvate. The inactivation appears to be due to covalent reaction since radioactive reagent remains associated with the enzyme after extensive dialysis and gel filtration on Sephadex G-25. These results are consistent with affinity labeling of the nucleotide binding site of phosphoenolpyruvate carboxykinase by the guanosine nucleotide analogue 5′-p-fluorosulfonylbenzoyl guanosine.

Evidence for the Existence of Phosphoenolpyruvate Carboxykinase Ferroactivator Activity in Adult and Fetal Guinea Pigs

Newborn (24--72 h) guinea pig liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) activity is increased by incubation of the cytosol with the metal salts FeCl2, MnCl2, CoCl2 and CdCl2. FeCl2 at 30 micromol/l concentration is the most effective activator causing a 3.5-fold increase in activity. Purified rat liver cytosolic PEPCK is activated by 30 mumol/l FeCl2 in the presence of liver cytosol of fetal and newborn guinea pigs. These results confirm the existence of PEPCK ferroactivator in the guinea pig which has properties similar to the one found in rat liver. The tissue distribution of ferroactivator activity parallels that of cytosolic PEPCK, being highest in the gluconeogenic organs liver and kidney. Hepatic PEPCK ferroactivator activity can be demonstrated by day 45 of gestation, increasing linearly in specific activity to adult levels at term (65 days). The distribution and development of the ferroactivator is consistent with the hypothesis that it may play a role in the physiologic control of PEPCK.

Effects of adenosine cyclic 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.

Development of mitochondrial pyruvate metabolism in rat brain.

AbstractThe activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1‐14C]pyruvate to 14CO2 via pyruvate dehydrogenase and the fixation of H14CO3− in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four‐fold and 15‐fold respectively during 10–35 postnatal days. The rates of the fixation of H14CO3− by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD‐malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol‐pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three‐ to 10‐fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.