PEPCK Activity Research Articles

Insulin “inhibition” of gluconeogenesis by stimulation of protein synthesis

The effect of insulin on gluconeogenesis in diabetic rabbits has been studied by measurement of both pool size and specific activity of alanine, lactate, glucose, and protein during the infusion of labeled alanine, acetate, glycerol, and lactate. Insulin administration causes an immediate net drop in the pool sizes of alanine, lactate, glucose, pyruvate, and glycerol. The radioactivity of tracer alanine is found to be shunted from glucose to protein on insulin administration concurrent with the fall in the plasma levels of three-carbon compounds measured. This is consistent with the net pool size experiments and suggests that the major carbon flow is directed away from glucose toward protein synthesis by insulin. The recovery of most of the counts from alanine which did not go into glucose in protein also supports the proposition that the major effect of insulin on gluconeogenesis is to stimulate protein synthesis. Further experiments with [ 14C]acetate which can be only incorporated into glucose through the activity of PC and PEPCK show that although its incorporation into protein is increased significantly by insulin its incorporation into glucose is unaffected. All these results are consistent with the conclusion that insulin “inhibits” gluconeogenesis by stimulating resynthesis of amino acids into protein before they are converted to glucose. In this way insulin modulates hyperglycemia induced by gluconeogenetic hormones.

Stimulation of rat renal phosphoenolpyruvate carboxykinase activity after an intravenous lactic acid load

It is well known that metabolic acidosis attained by ammonium chloride administration leads to an increase in renal gluconeogenesis [ 1,2] through the activation of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (PEPCK) [3-51. We have reported that muscular exercise (swimming in water at 22” C for 2 h) results in an increase of renal gluconeogenic ability and PEPCK activity, most probably as a result of the metabolic acidosis caused by overproduction of lactate [6-91. To obtain further information about the relations between lactic acidosis and renal metabolism we produced experimental lactic acidosis which could mimic in some way the physiological acidosis accompanying exercise. Then we have studied the activity of PEPCK, the gluconeogenic ability in kidney cortex and the renal content of some intermediates of gluconeogenesis from rats loaded intravenously with lactic acid or sodium lactate. We have also studied the renal response to the infusion of adrenaline, because of the involvement of this hormone in the metabolic events during exercise [lo]. Our results indicate that induction of renal PEPCK is closely related to acidosis.

Ochratoxin A, an in vivo inhibitor of renal phosphoenolpyruvate carboxykinase

Ochratoxin A, a nephrotoxin produced as a secondary metabolite by A. ochraceus, is a potent inhibitor of renal PEPCK activity, in vivo. When fed orally to rats for 2 days, renal PEPCK activity is reduced 50% by a total dose of 0.3-0.5 mg toxin. Renal gluconeogenic capacity is reduced only after PEPCK activity is inhibited by 50%. Hepatic PEPCK activity is unaffected up to 1.5-2.0 mg ochratoxin A, which were the highest doses tested. Other enzymes located in proximal convoluted tubules, including phosphatedependent glutaminase, γ-glutamyl transpeptidase, pyruvate carboxylase, and Na,K-ATPase, are not affected. Renal protein synthesis from [ 3H]phenylalanine or [ 3H]leucine is inhibited 30–40% by ochratoxin A in vivo. By covalently coupling the toxin to albumin with carbodiimide or mixed anhydride, the inhibitory effect on renal PEPCK activity is retained, but protein synthesis is not affected and cytological evidence of nephrotoxicity is lost. Injection of the ochratoxin A-albumin carbodiimide complex results in a decrease of hepatic PEPCK activity as well. Removal of the phenylalanine group from the toxin prevents the in vivo inhibition of PEPCK activity, as well as protein synthesis. We conclude that the decrease in renal PEPCK activity, in vivo, requires the phenylalanine group of ochratoxin A, and occurs by a mechanism independent of the known nephrotoxicity effects.

239 THE EFFECT OF INTRAUTERINE GROWTH RETARDATION (IUGR) UPON HEPATIC AND RENAL PHOSPHOENOL PYRUVATE CARBOXY- KINASE (PEPCK) ACTIVITY IN THE NEONATAL RAT

The contribution of renal gluconeogenesis to glucose homeostasis in the newborn has been neglected despite its well-established role in fasting glucose homeostasis. We have studied the activity of the key gluconeogenic enzyme PEPCK (forward spectro-photometric reaction) in normal and IUGR rat neonates (uterine vessel ligation) fasted for 4 hours postpartum (34°C). Neonatal PEPCK activity was lower than maternal hepatic (1.14±.11 μmol/min/g) and renal (0.99±0.10 μmol/min/g) activity. Hepatic PEPCK activity was diminished in IUGR animals, but this was not significant. In contrast, renal PEPCK activity was significantly increased in IUGR neonates. This suggests independent regulation of neonatal renal and hepatic gluconeogenesis. The compensatory increase in renal PEPCK activity in growth-retarded newborns may contribute significantly to gluconeogenic capacity since PEPCK is the rate-limiting enzyme in the system.

Hormonal control of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities in the fetal rat kidney.

Triamcinolone (20 microgram per fetus) administered in utero to term rat fetuses (day 21 of gestation) increases the activities of renal G-6-Pase and PEPCK. The absence (or marked decrease) of circulating corticosteroids in fetuses from adrenalectomized, metopirone-treated mothers has, however, no clear effect on the enzyme activities. Therefore, it is doubtful that corticosteroids play a role in the development of G-6-Pase and PEPCK activities during the fetal period of life. In 21-day-old fetuses entirely decapitated on day 18, both enzyme activities are lower than intact fetuses of the same litter (G-6-Pase, -48%; PEPCK, -36%). In partially decapitated fetuses, the activity of G-6-Pase remains at the control level, whereas the PEPCK activity is clearly reduced (-39%). These results strongly suggest that on the last day of gestation the hormone group of parathormone, calcitonin, or thyroxine controls the G-6-Pase activity, whereas the hypothalamo-hypophysis system is implied in the development of PEPCK activity. Parathormone (1 unit per fetus) administered to decapitated fetuses completely restores the G-6-Pase activity. Neither rat growth hormone, synacthene, nor arginine vasopressin has significant effects on the activity of PEPCK in the kidneys of decapitated fetuses. The administration of 0.5 mumole of dibutyryl-cAMP or cyclic adenosine 3':5'-monophosphate (cAMP) to decapitated fetuses completely restores the activity of renal PEPCK, suggesting that the development PEPCK is controlled by cAMP-dependent hormone. The same dose of dibutyryl-cAMP has no effect on the activity of G-6-Pase; cAMP produces a slight but significant increase of this enzyme activity.

A study of phosphoenolpyruvate carboxykinase from Moniliformis dubius (Acanthocephala).

Procedures have been developed for the extraction and subsequent purification of the enzyme phosphoenolpyruvate carboxykinase (GTP) (PEPCK) from Moniliformis dubius (Acanthocephala), a parasite of the rat small intestine. This is believed to be the first purification of PEPCK from an invertebrate animal. The enzyme, when purified to homogeneity as indicated by electrophoretic criteria, has a molecular weight of 73 700. Kinetic studies indicated that the enzyme had a pH optimum of 5.5 and required Mn2+ as the divalent cation. The apparent Km values determined for the substrates of the carboxylation reaction were low compared with the published values for purified PEPCK from vertebrate sources. Several competitive inhibitors were found and their Ki values determined. The possible regulation of PEPCK activity in M. dubius is discussed with reference to the observed kinetic parameters.

Fetal metabolic response to phloridzin-induced hypoglycemia in pregnant rats.

Phloridzin, an inhibitor of renal sugar transport, produces an important loss of glucose in urine of treated animals. In order to reduce severely the maternal glucose supply to the fetuses in short-term experiments, we have combined phloridzin administration to pregnant rats with 18 h starvation. Fetuses from starved phloridzin-treated mothers were compared with fetuses from starved mothers. Combined treatment markedly decreases fetal blood glucose concentration (-36%) and fetal liver glycogen stores (-76%). These changes are associated with a decrease in plasma insulin (-25%), a rise in plasma glucagon (+120%) and a marked increase of hepatic PEPCK activity (+400%). It appears from these results that phloridzin treatment for a short duration is able to induce glycogenolysis and the premature appearance of PEPCK in the liver of rat fetuses.

Carrier detection of pyruvate carboxylase deficiency in fibroblasts and lymphocytes.

Pyruvate carboxylase (E.C. 6.4.1.1) activity was determined in the circulating peripheral lymphocytes and cultured skin fibroblasts from the family of a patient with hepatic, cerebral, renal cortical, leukocyte, and fibroblast pyruvate carboxylase deficiency (PC Portland deficiency). Lymphocyte activities were: mother, 33--39%; father, 11--29%; brother, 82--103%; and sister, 38--48% of the lowest normal. Fibroblasts from the patient's mother and father had 42 and 34%, respectively, of the activity of the lowest normal. These data demonstrate that the disease is inherited in an autosomal recessive manner and that lymphocytes and fibroblasts can be used to detect carriers. Neither pyruvate carboxylase nor mitochondrial PEPCK activity in lymphocytes was increased by a 21-hr fast.

Changes in activity of key enzymes of glyconeogenesis in the liver and kidney of rats exposed to subextremal and extremal factors

Activity of the key enzymes of glyconeogenesis-phosphoenolypyruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase (FDPase), and glucose-6-phosphatase (G6Pase) — in the liver and kidneys of rats was investigated during simultaneous exposure of the animals to subextremal and extremal factors. The low initial PEPCK activity in the liver and its high variability under the influence of extremal stimuli are evidence that this enzyme plays the role of limiting stage of glyconeogenesis. PEPCK activity in the kidneys was comparable with FDPase activity and significantly higher than G6Pase activity, PEPCK activity in the kidneys was 5–6 times higher than in the liver. Under the influence of extremal factors, PEPCK and G6Pase activity in the kidneys rose whereas FDPase activity was practically unchanged. The absence of any distinctly synchronized changes in the activity of the key enzymes of glyconeogenesis in the liver and kidneys indicates that there is no single operon in the cells of these organs for PEPCK, FDPase, and G6Pase with a common mechanism of regulation.

Carbohydrate metabolism in lean and obese Zucker rats

Carbohydrate metabolism was evaluated in lean and obese Zucker rats. Plasma glucose concentration, renal and hepatic gluconeogenesis, and hepatic glycogen content and rates of synthesis were investigated in 2-mo and 8-mo-old animals. Mild hyperglycemia was observed in obese Zucker rats compared to lean rats and was more pronounced in males than in females. Rates of glucose disappearance were normal in both female and male rats, although there was a trend toward decreased clearance in the male. Total organ hepatic and kidney PEPCK activity and kidney glucose production were elevated in obese compared to lean rats. Total organ hepatic glycogen levels and rates of glycogen synthesis were increased significantly in obese compared to lean, the increase being greater in males than females. The mild hyperglycemia present in obese Zucker rats is not associated with delayed disappearance of intravenously administered glucose, but may be due to the increased production of glucose by whole kidney and liver.

Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in leukocytes and fibroblasts from a patient with pyruvate carboxylase deficiency.

Normal values are given for the activities of pyruvate carboxylase (E.C.6.4.1.1), mitochondrial phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32, PEPCK), and citrate synthase (E.C. 4.1.3.7) in fibroblasts, lymphocytes, and leukocytes. Also given are values for these enzymes in the leukocytes and fibroblasts from a severely mentally and developmentally retarded patient with proximal renal tubular acidosis and hepatic, cerebral, and renal cortical pyruvate carboxylase deficiency. In normals, virtually all of the mitochondrial PEPCK and pyruvate carboxylase activity was present in the mononuclear leukocyte fraction of whole venous blood. Cellular fractionation studies with human lymphocytes and fibroblasts demonstrated that all of the PEPCK activity in these cells is mitochondrial. Normal values for pyruvate carboxylase in leukocytes were 0.092 (0.070--0.208) mU/mg protein (n=5), in lymphocytes 0.154 (0.092--0.262) mU/mg protein (n=5), and in fibroblasts 1.36 (0.778--2.19) mU/mg protein (n=5). The patient with hepatic, renal, and cerebral pyruvate carboxylase deficiency had no detectable activity (less than 0.009 mU/mg protein) in his leukocytes and 0.018 mU/mg protein in his fibroblasts. Data from an assay for pyruvate carboxylase activity in the patient's fibroblasts show that the activity observed is significant but very close to the lower limits of the assay. Values for PEPCK in normal lymphocytes were 1.42 (0.824--1.88) mU/mg protein (n=5), in leukocytes 1.68 (1.64--1.72) mU/mg protein (n=2), and in fibroblasts 5.49 (3.94--6.33) mU/mg protein (n=6).

Longitudinal Profiles of Carbon Dioxide Fixation Capacities in Marine Macroalgae

Fucus serratus L., Fucus spiralis L., and Fucus vesiculosus L. (Fucales, Phaeophyceae) as well as Laminaria digitata (Huds.) Lamour., Laminaria hyperborea (Gunn.) Fosl., and Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyceae) have been investigated for the distribution of enzymic CO(2) fixation capacities via phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (PEP-CK) and via ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) (RubP-C) in different regions of the thalli. The maximum of PEP-CK activity is found to be confined to the growing regions of the algae, while the activity of RubP-C achieves its highest values in the entirely differentiated parts of the fronds. These findings are confirmed by the results of photosynthetic and light-independent (dark) carbon assimilation as determined by in vivo(14)CO(2) fixation. The physiological significance of these differential patterns of carboxylation patterns is discussed with respect to the ontogenetic stage and the chemical constitution of the different thallus parts.

The potential for lactate utilization by red and white muscle of the eel Anguilla rostrata L.

The activities of lactate dehydrogenase (LDH) (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) can be divided into a lactate oxidase (lactate to pyruvate) and pyruvate reductase (pyruvate to lactate) component. These activities were examined in red and white muscle excised from the American eel Anguilla rostrata as an estimate of tissue lactate utilization, and compared with the kidney, gill, heart, and liver patterns. Phosphoenol pyruvate carboxykinase (PEP CK) activities in red and white muscle and liver were estimated as a marker for tissue gluconeogenic potential. Consistent with the possibility of lactate utilization for gluconeogenesis, both red muscle and liver possessed an active PEP CK and a bifunctional LDH, where oxidase activities were of the same magnitude as reductase activities. Kidney, which in mammals possesses gluconeogenic capabilities, together with heart and gill, also demonstrated LDH profiles consistent with the liver and red muscle enzymes. White muscle LDH was found to be essentially a pyruvate reductase and no PEP CK activity could be detected. These results suggest that eel red muscle has the potential to utilize lactate and has at least some of the enzymes required for gluconeogenesis. Therefore, the capacity of red muscle to perform a metabolic recycling function in addition to contraction cannot be excluded.

Implication of alanine and aspartate aminotransferases, glutamic dehydrogenase and phosphoenolpyruvate carboxykinase in glucose production in rats, chicken and pigs

1. 1. Blood glucose concentration in rat, pig and chicken was 88, 96 and 216 mg per 100 ml and hepatic glycogen content was 4.6, 2.6 and 2.4 mg/100 mg tissue, respectively. Except in pig, serum ASPTf activity was several times higher than ALAT activity. 2. 2. Total ASPT activity was 3 times higher than the total ALAT activity in rat liver, 14 times higher in chicken liver and 1.5 times higher in pig liver. In all species, ASPT activity in the muscle was roughly 50% of that in the liver. ASPT activity in chicken muscle was 70 times higher than the ALAT activity and 7 and 9 times higher in rat and pig muscle, respectively. 3. 3. ASPT activity in the liver was distributed between the mitochondria and the cytosol by 10% and 87% in rat, 14% and 78% in pig, and 14% and 82% in chicken, respectively, and ALAT activity in 13% and 83% in rat, 55% and 40% in pig and 90% and 6% in chicken, respectively. 4. 4. K m values for ASPT occurring in pig liver mitochondria and cytosol were 0.56 mM and 0.22 mM for α-ketoglutarate. 5. 5. Glutamic dehydrogenase was exclusively located in the mitochondria. Activity of this enzyme in pig liver mitochondria was approx 50 times higher than in the muscle mitochondria. PEPCK activity was distributed between the liver mitochondria and cytosol by 43% and 57%, respectively. 6. 6. Phosphorylase a, phosphoglucomutase, glucose-6-phosphatase, and fructose-1,6-diphosphatase activity in pig liver was not significantly different from the corresponding reported values for rat and chicken livers.

Schedule of protein ingestion and circadian rhythm of certain hepatic enzyme activities involved in glucose metabolism in the rat.

The circadian rhythms of liver glycogen, plasma glucose, corticosterone and insulin, and hepatic activity of PK, G6PDH, ME, Ac, CoA carbox. PEP-CK and GPT were studied in adult rats. Animals either received a mixed diet ad libitum (8% protein) or a protein meal (1.1 g protein) given at 05:00 or 17:00 h, with free access to a protein-free diet (separately fed). When the protein meal was ingested during the lighted period (17:00) the 24-hour average level of liver PEP-CK was greater than in rats consuming protein during darkness (05:00). In the latter case, modification of the circadian rhythm of liver glycogen and of circadian rhythm of liver PK, G6PDH, ME and Ac.CoA carbox. activity (increase of 24 h average level, extension of period of high activity, sudden increase after ingestion of protein meal) were observed. Conversely, the circadian rhythm of plasma insulin and corticosterone and of liver PEP-CK and GPT activity were only slightly affected by the mode of feeding.

Studies of the Effect of Dietary Cholesterol on Hepatic Protein Synthesis, Reduced Glutathione Levels and Serine Dehydratase Activity in the Rat

A basal diet or a basal diet plus 1% of cholesterol and 0.33% cholic acid was fed to rats for varying lengths of time and (1) the activities of liver phosphoenolpyruvate-carboxykinase (PEP-CK), tyrosine transaminase (TT), and serine dehydratase (SD); (2) the rate of total hepatic protein synthesis and (3) the concentration of hepatic reduced glutathione (GSH) were quantitated. The specific activity of PEP-CK was significantly depressed by cholesterol plus cholic acid feeding, while the specific activity of TT was unchanged. No significant effect of dietary cholesterol plus cholic acid was found on the total liver activities. In contrast, SD specific activity was increased 3-fold. The rate of (U-14C)-L-leucine incorporation into total TCA precipitable protein following ingestion of cholesterol plus acid was significantly reduced when the data were expressed as dpm (U-14C)-L-leucine/mg protein. After correcting this expression for specific radioactivity of the liver tissue free leucine pool, no significant effect of dietary cholesterol plus cholic acid on hepatic protein synthesis existed. In fact, the amount of 14C-leucine incorporated into protein on a total liver basis was 50% greater for the cholesterol group. On a per gram of liver basis, the concentration of GSH in the liver of rats fed a cholesterol plus cholic acid diet was significantly decreased. Considering the liver enlargement in rats fed cholesterol plus cholic acid, total organ GSH was found to be significantly greater than for rats fed a basal diet.

Metabolic control of enzymes in normal, Diabetic, and diabetic insulin-treated rats utilizing 1,3 butanediol

Diets containing 1,3 butanediol (BD) as a replacement of carbohydrate were fed to normal, diabetic, and diabetic insulintreated rats for two weeks. The diabetic animals fed BD survived longer than the diabetic animals on essentially the same diet without BD. The activities of malic enzyme in 105,000 × g. (1 hour) supernatant fractions of liver were determined. The PEPcK activity in liver was increased by 320 per cent in diabetic and by 240 per cent in diabetic rats fed the BD diet. Insulin decreased the PEPcK activity toward normal. In liver and adipose tissues malic enzyme activity was greatly decreased in diabetic and increased in diabetic insulin treated rats. The incorporation of radioactive bicarbonate into organic acids by liver mitochondrial pyruvate carboxylase and the formation of intermediates for gluconeogenesis were examined. The concentration of metabolites in liver was also examined and all changes were found to be minimal. Oral administration of BD greatly increased blood ketone levels and the ratio of β-hydroxybutyrate to acetoacetate. Normal animals fed BD had significantly lower blood glucose levels. Liver perfused with BD also showed decreased glucose production from lactate. Analysis of metabolites from livers perfused with BD showed a large increase in the lactate to pyruvate ratios (from 13.1 to 81.0). Malate and aspartate were increased, whereas pyruvate, PEP, 2 PGA, and 3 PGA were decreased. It is concluded that BD exerts its hypoglycemic effect at the conversion of malate to oxalacetate and hence to PEP.