Enzyme Phosphoenolpyruvate Carboxykinase Research Articles

Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p.

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

Effect of methadone addiction on glucose metabolism in rats

Female albino rats were exposed to methadone over a 35-day period by addition of the drug in their drinking water. The final dose of the drug was 1.8 mg/kg body weight per day. After this period, the drug was withdrawn from some animals for 30 days (postexposure). Compared to unexposed controls, serum glucose levels rose during exposure and returned to control levels postexposure. Oral glucose tolerance tests showed impairment in 35-day drug-exposed animals compared to controls and postexposure. The activities of three key enzymes of glycolysis and three key enzymes of gluconeogenesis were measured in liver during and at the end of the exposure period, as well as postexposure. Compared to unexposed controls and postexposure, specific activities of two glycolytic enzymes in livers of exposed animals—hexokinase and phosphofructokinase 1—were significantly reduced, whereas the activity of a third glycolytic enzyme—pyruvate kinase—was unchanged. The specific activities of two gluconeogenic enzymes—glucose-6-phosphatase and fructose-1,6-biphosphatase—were significantly elevated in the drug-exposed animals compared to controls, whereas the activity of a third enzyme—phosphoenolpyruvate carboxykinase—was unchanged. These data indicate that methadone addiction produces a metabolic state similar to insulin-resistant diabetes.

“Spot 14” Protein Functions at the Pretranslational Level in the Regulation of Hepatic Metabolism by Thyroid Hormone and Glucose

"Spot 14" protein appears rapidly in nuclei of hepatocytes exposed to glucose and thyroid hormone. Exposure of glucose- and T3-treated hepatocytes to a spot 14 antisense oligonucleotide inhibited induction of mRNAs encoding malic enzyme, ATP citrate-lyase, fatty acid synthase, liver-type pyruvate kinase, phosphoenolpyruvate carboxykinase, and type I deiodinase but not hydroxymethylglutaryl-CoA reductase, cytochrome c, and actin mRNAs. Induction of spot 14, ATP citrate-lyase, and fatty acid synthase polypeptides, but not propionyl-CoA carboxylase and mitochondrial pyruvate carboxylase, was inhibited. Antisense treatment of hepatocytes transfected with a reporter controlled by a glucose- and T3-inducible fragment of the pyruvate kinase gene promoter inhibited reporter activity, as did cotransfection of the reporter and a spot 14 antisense plasmid. Spot 14 protein acts in the induction of mRNAs coding for key lipogenic (malic enzyme, ATP citrate-lyase, fatty acid synthase), glycolytic (pyruvate kinase), and gluconeogenic enzymes (phosphoenolpyruvate carboxykinase), as well as the diet-responsive type I deiodinase, but not those involved in mitochondrial respiration (cytochrome c) or cholesterol synthesis (hydroxymethylglutaryl-CoA reductase). Transfection experiments indicated that these effects are mediated at the transcriptional level. The protein functions in the activation of genes involved in metabolic switching between the fasted and fed states in liver.

CHANGES IN HEPATIC REGULATORS AND GENE EXPRESSION CAN ACCOUNT FOR GLUCONEOGENIC AGING BUT NOT TRAINING EFFECTS 590

We have consistently found evidence of an age-related decline in hepatic gluconeogenic rate in rats; furthermore, chronic dynamic exercise can partially offset this decline. The purpose of the present investigation was to examine changes in several potential regulators of gluconeogenesis with aging and training. Sixty-six male Fischer 344 rats 4 mo (young), 12 mo(middle-aged) and 22 mo (old were pair-matched and assigned to the trained or sedentary group. Trained animals ran on a motorized treadmill 1 hr/day, 5 days/wk at 75% of maximal capacity for 10 wk. Animals were sacrificed at rest, sections of liver removed and immediately frozen in liquid nitrogen. Fructose 2,6-bisphosphate (an inhibitor of fructose 1,6-bisphosphatase in liver) concentration was significantly greater in old compared to young rats in both training groups (54 and 44% increase in old trained and untrained animals, respectively). No changes were found with training. Phosphoenolpyruvate carboxykinase (PEPCK) gene expression (mRNA), determined by Northern blot analysis, decreased 31% in both the trained and untrained old animals compared to their young counterparts; training had no effect in any age group. In similar fashion, PEPCK activity declined in the old animals compared to young and middle-aged animals regardless of training condition. Elevated levels of fructose 2,6-bisphosphate in old animals may stimulate the glycolytic pathway via phosphofructokinase-1 and inhibit fructose 1,6-bisphosphatase thereby reducing gluconeogenic rates. Furthermore, decreased mRNA and activity of the critical gluconeogenic enzyme PEPCK may also contribute to the decline in hepatic gluconeogenesis found with advancing age. Possible mechanisms/regulators responsible for the attenuation of the age-related decline with training, however, remain to be determined.

Effects of Short-Chain Acyl-CoA Dehydrogenase Deficiency on Developmental Expression of Metabolic Enzyme Genes in the Mouse

Patients with an acyl-CoA dehydrogenase deficiency share the disease features of hypoglycemia, hyperammonemia, tissue fatty change, hypoketonemia, carnitine deficiency, and organic acidemia due to apparent disruption of normal fatty acid, glucose, and urea metabolism. Most of the acute clinical episodes occur in young children. These episodes are precipitated by fasting and are often fatal, with thein vivomechanisms essentially unknown. Since the genes of the rate controlling enzymes of these pathways are tissue and developmentally regulated at the transcriptional level, we measured, throughout neonatal development, the steady-state mRNA levels of long-chain, medium-chain, and short-chain (SCAD) acyl-CoA dehydrogenases, pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), carbamyl phosphate synthetase I (CPS), ornithine transcarbamylase (OTC), and argininosuccinate synthetase (AS) in fed or fasted SCAD-deficient BALB/cByJ mice compared to BALB/cBy controls. Overall, our results showed no major effects on expression of acyl-CoA dehydrogenases due to SCAD deficiency, regardless of age or fasting. In SCAD-deficient mice we found depressed mRNA expression and enzyme activity for the urea cycle enzymes CPS and AS at 6 days of age, and found no apparent effects on expression of gluconeogenic enzymes PC or PEPCK. There was a period of overall lower gene expression for most genes at 6 and 15 days, which appears to be in parallel with the developmental period when children with these diseases are most severely affected.

Glucokinase and cytosolic phosphoenolpyruvate carboxykinase (GTP) in the human liver. Regulation of gene expression in cultured hepatocytes.

Glucokinase and phosphoenolpyruvate carboxykinase are key enzymes of glucose metabolism in the rat liver. The former is considered to be instrumental in regulating glucose hepatic release/uptake according to the glycaemia level, and cytosolic phosphoenolpyruvate carboxykinase is a major flux-generating enzyme for gluconeogenesis. The level of expression of both enzymes and the regulation of their mRNAs in the human liver cell were investigated. Surgical biopsies of liver from patients undergoing partial hepatectomies and parenchymal hepatocytes derived from the biopsies were used to assay glucokinase, hexokinase and phosphoenolpyruvate carboxykinase activities. Hepatocytes were placed in culture and the actions of insulin, glucagon and cAMP on glucokinase and phosphoenolpyruvate carboxykinase mRNAs were studied. The main results are: (a) glucokinase accounts for 95% of the glucose phosphorylation activity of human hepatocytes, although this fact is masked in assays of total liver tissue; (b) glucokinase activity is set at a lower level in human hepatocytes than in rat hepatocytes, and vice-versa for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase; and (c) as previously shown in rat liver, glucokinase and phosphoenolpyruvate carboxykinase mRNAs are regulated in a reciprocal fashion in human hepatocytes, insulin inducing the first enzyme and repressing the latter, whereas glucagon has opposite effects. These data have interesting implications with respect to metabolic regulation and intracellular hormone signaling in the human liver.

Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: opposite effects of dexamethasone and isoprenaline on transcription

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in gluconeogenesis in liver and in glyceroneogenesis in adipose tissue. These processes, and PEPCK, are regulated by a number of hormones, some of which have different effects on the enzyme in liver and adipose tissue. To explore this phenomenon, PEPCK gene expression was studied in 3T3-F442A adipocytes maintained in a serum-free medium. The beta-adrenergic agonist isoprenaline (isoproterenol) and a cyclic AMP analogue (8-CPT-cAMP) increased PEPCK mRNA. A maximal 3-fold induction occurred in 2 h. Dexamethasone decreased PEPCK mRNA by 80% in 4 h. Dexamethasone also counteracted the inductive effects of isoprenaline and 8-CPT-cAMP. Run-on transcription experiments showed that the isoprenaline and dexamethasone actions were, at least in part, exerted at the level of PEPCK gene transcription. These effects were further analysed by using transient and stable transfection of adipocytes with a plasmid containing bp -2100 to 69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene. In such cells isoprenaline stimulated CAT expression, an effect that was prevented if the cells were also exposed to dexamethasone.

Inhibition by recombinant human interleukin-6 of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and of the insulin-dependent induction of glucokinase gene expression in cultured rat hepatocytes: regulation of gene transcription and messenger RNA degradation.

The influence of recombinant human interleukin-6, the major mediator of the inflammatory response in liver, on the glucagon- and insulin-dependent induction of the phosphoenolpyruvate carboxykinase and glucokinase gene, respectively, was monitored on the level of gene transcription, mRNA abundance and enzyme activity in cultured rat hepatocytes. As control markers of the interleukin-6-induced acute-phase response the mRNA levels of the acute phase proteins alpha 2-macroglobulin and beta-fibrinogen were determined. In cultured rat hepatocytes, recombinant human interleukin-6, added simultaneously with glucagon and insulin, lowered the maximal increase in glucagon-induced phosphoenolpyruvate carboxykinase mRNA levels after 2 hr and the maximal increase in glucokinase mRNA levels after 3 hr to about 30%, respectively. It inhibited the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene transcription and phosphoenolpyruvate carboxykinase enzyme activity, as well as the insulin-induced increases in glucokinase gene transcription and glucokinase enzyme activity. Recombinant human interleukin-6 increased the mRNA levels of the acute-phase proteins alpha 2-macroglobulin and beta-fibrinogen gradually over 4 to 6 hr. Recombinant human interleukin-6, added 2 hr after glucagon or 3 hr after insulin at the maximum of the hormone-induced enzyme mRNA levels, almost doubled the decay rate of phosphoenolpyruvate carboxykinase mRNA and glucokinase mRNA. The results show that interleukin-6 induced the expression of inflammatory proteins and simultaneously inhibited the hormone-induced expression of enzymes of intermediary metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of prolonged hyperglycemia on metabolic alterations in the subtotally pancreatectomized rat.

A delayed onset of diabetes is characteristic of subtotally pancreatectomized patients in whom persistent hyperglycemia per se is documented to lead to the development of insulin resistance. This study was conducted to elucidate the metabolic alterations seen during transition of the acute to chronic phase after subtotal pancreatectomy (SP). Eight male Sprague-Dawley rats were studied 2 weeks after surgery in the acute phase, and the other eight at 4 weeks in the chronic phase. Phosphoenolpyruvate carboxykinase (PEPck) for gluconeogenesis and the malic enzyme for de novo fatty acid synthesis in the liver showed a reciprocal change, the former activity being increased, while the latter was suppressed. Both alterations were more pronounced in the chronic phase. In the acute phase, lipoprotein lipase (LPL) for triglyceride clearance decreased in the adipose tissue, while that in the cardiac and skeletal muscle became significantly elevated. The latter elevations were decreased in the chronic phase. Sustained hyperglycemia in the SP rats not only increased the changes in PEPck and malic enzyme activities but reversed the tissue-specific muscle LPL elevations. These changes might help to explain the wasting condition seen in surgically induced diabetic patients.

Inhibition by PGE 2 of glucagon-induced increase in phosphoenolpyruvate carboxykinase mRNA and acceleration of mRNA degradation in cultured rat hepatocytes

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E 2 has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE 2 to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE 2 was added concomitantly with glucagon. This inhibition by PGE 2 of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE 2 accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE 2 is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE 2 may reduce the hepatic gluconeogenic capacity via a G i-linked signal chain.

Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene.

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Ascaris suum: Cloning of a cDNA Encoding Phosphoenolpyruvate Carboxykinase

A cDNA encoding phosphoenolpyruvate carboxykinase (PEPCK) from Ascaris suum was cloned by complementation of a strain of Escherichia coli deficient in PEPCK and malic enzyme. The product of this cDNA was enzymatically similar to a recombinant PEPCK obtained from Haemonchus contortus by the same method. Comparison of the predicted amino acid sequence of A. suum PEPCK with other PEPCKs showed that this enzyme is most closely related to the H. contortus enzyme. The two nematode enzymes share considerable homology in regions thought to be functionally involved in substrate binding and catalysis, some of which distinguish the nematode enzymes from PEPCKs from other organisms. This analysis suggests a structural explanation for the kinetic differences seen between nematode and vertebrate PEPCKs and supports the hypothesis that nematode PEPCK is a target for selective inhibition.

Effects of okadaic acid on expression of phospho enol pyruvate carboxykinase in cultured rat hepatocytes

In normal rat hepatocytes in primary culture the level of mRNA encoding the key gluconeogenic enzyme phospho enol pyruvate carboxykinase (PEPCK) is increased by the cyclic AMP analogue, chlorophenylthio cyclic AMP (cpt cAMP), and this response is reversed by insulin. The protein-phosphatase inhibitor okadaic acid diminished the stimulatory effects of cpt cAMP on PEPCK mRNA. Protein kinase A remained fully active in the presence of okadaic acid, therefore, the insulin-mimetic actions of okadaic acid were localised to a site subsequent to initial protein kinase A activation. Insulin produced a decrease in PEPCK mRNA expression which was similar to that of okadaic acid both in extent and mechanism (i.e., lack of change in protein kinase A activation). The effects of okadaic acid on PEPCK mRNA amount were not additive with those of insulin and the effects of insulin were not abolished by okadaic acid. These data suggest that okadaic acid and insulin may interact with the cAMP regulation of the PEPCK gene expression at a common site. The mechanisms by which this may be attained are discussed in relation to what is known about the control of specific protein kinases and protein phosphatases by insulin and okadaic acid and of the importance of protein phosphorylation state to regulation of gene-transcriptional processes.

A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely.

A cytosolic cell-free system prepared from rat liver was used to study the effect of bivalent cations on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Steady-state concentrations of oxaloacetate in the range 5-50 microM were generated from increasing concentrations of malate+fumarate (10:1); 2 mM ITP and 3 mM Mg2+ were added as cofactors. Micromolar concentrations of Mn2+, Fe2+ and, to a lesser extent, of Zn2+ and Co2+ were shown to stimulate PEPCK activity. Vmax. (mumol/min per g of liver) increased from 0.67 to 1.68 on addition of 5 microM Fe2+ and to 2.34 with 2 microM Mn2+, whereas no significant effect on the Km for oxaloacetate was observed. The apparent K(a) values (total) were 0.62 microM for Mn2+, 1.48 microM for Zn2+, 1.92 microM for Co2+ and 3.37 microM for Fe2+, being 2-8-fold lower than the corresponding published values. Variations of the free Mn2+ concentration were obtained (a) by increasing the Mn2+ concentration (i.e. activation curve) and (b) by simultaneous addition of Mn2+ and increasing concentrations of the chelating agent EGTA (i.e. inactivation curve). Different results were obtained for the activation and inactivation curves. The inactivation curve showed that PEPCK activity was almost unaffected by variations of the free Mn2+ concentration over the range 0.05-0.15 microM. Under comparable experimental conditions, rat liver arginase (another Mn(2+)-dependent enzyme) was completely inactivated. From kinetic evidence, the existence of two distinct molecular forms of cytosolic rat liver PEPCK with different Mn2+ affinities is postulated. Considering the high affinity of PEPCK for Mn2+ and its relative insensitivity to changes in the free Mn2+ concentration, it seems rather unlikely that changes in the free cation concentration play a major role in regulating PEPCK activity in vivo.

Metabolic enzymes in coelomic cells (eleocytes) of the polychaete Nereis virens: sex specific changes during sexual maturation

The activities of some enzymes of the intermediary metabolism and the content of soluble protein and carbohydrate (glycogen plus free glucose) were measured in one type of coelomic cells (eleocytes) of the polychaete Nereis virens. Specimens used in this study were collected between 1989 and 1991 in Oosterscheldt Bay, The Netherlands, and divided into six different stages of sexual maturation as determined by the mean oocyte volume. In both sexes, the soluble protein content in eleocytes of immature individuals (11 mg ml−1 cell vol) increased three-fold. In prespawning N. virens the soluble protein content decreased to less than 2 mg protein ml−1 cell vol in females but not in males. In both sexes, the carbohydrate content decreased continuously from immature [300 μmol glucose equivalent (equiv) ml−1 cell vol] to prespawning individuals (< 40 μmol glucose equiv ml−1 cell vol). During the time course of maturation, the specific activities (expressed as units mg−1 protein) of pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, alanine aminotransferase and glutamate dehydrogenase decreased in both sexes. A transient increase in the specific activities was found for glycogen phosphorylase and aspartate aminotransferase. No major changes were found for hexokinase, lactase dehydrogenase, glucose-6-phosphate dehydrogenase and malic enzyme. Sex specific differences were found for the activities of citrate synthase and isocitrate dehydrogenase, which were higher in males. the specific activities of the latter enzyme increased more than ten-fold in males, but only four-fold in female eleocytes during maturation. In eleocytes of prespawning females, the activities of most enzymes showed extremely high variations not found in prespawning males. For two enzymes of fatty acid catabolism, β-hydroxyacyl-CoA dehydrogenase and β-hydroxybutyrate dehydrogenase, only traces of activities were detected, suggesting the absence of significant fatty acid catabolism in the eleocytes. Compared to the eleocytes, the body wall tissue showed ten-fold higher activities of phosphofructokinase, whereas the eleocytes displayed higher activities of the amino acid interconverting enzymes glutamate dehydrogenase and alanine aminotransferase and the glyconeogenic enzyme phosphoenolpyruvate carboxykinase. Citrate synthase activities were similar for both tissues. In the coelomic fluid of N. virens, glucose (< 0.1 to 3.5 mM) and d-lactate (0.1 to 4 mM) were present and represent exogenous substrates for the eleocyte metabolism.

The atp2 operon of the green bacterium Chlorobium limicola

The operon (atp2) encoding the β and ϵ subunits of F-ATPase from Chlorobium limicola was cloned and sequenced. In contrast with purple bacteria these genes are arranged in a separate operon similar to the cyanobacteria. The operon terminates with a pronounced stem-loop structure. About 0.8 kb upstream of the β subunit a gene encoding the enzyme phospho enolpyruvate carboxykinase was identified. This gene is transcribed in the opposite direction of the atp2 operon and also ends with a stem-loop structure. These genes of green bacteria are among the first to be sequenced, and therefore the genetic distance between these genes and corresponding genes from other bacteria and eukaryotes was studied. Even though the operon structure resembles that of cyanobacteria, the evolutionary tree compiled from these data places the chlorobium gene close to purple bacteria. Chlorobium limicola β and ϵ subunits complemented Escherichia coli mutants defective in the corresponding subunits, indicating that the hybrid enzyme formed from subunits of the two bacteria is active in ATP synthesis.

Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and the glycolytic intermediates.

The beta cells of the pancreatic islets of Langerhans respond to changes in glucose concentration by varying the rate of insulin synthesis and secretion. Beta cells sense glucose concentration by the levels of the products of glucose catabolism. Distinctive beta-cell proteins glucose transporter 2 and glucokinase catalyze the first two steps in beta-cell glucose catabolism. To test whether either protein controls the sensitivity of the beta cell to glucose by controlling the rate of glucose catabolism, we used gene-transfer techniques to express the isoenzymes glucose transporter 1 and hexokinase I in beta cells and measured the response to glucose of the insulin gene promoter. Cells expressing glucose transporter 1 do not differ significantly from control cells, but in cells expressing hexokinase I, insulin promoter activity increases, reaches a maximum by 1 mM glucose, and does not respond to changes in glucose concentration within the physiologic range. We conclude that glucokinase catalyzes the rate-limiting step of glucose catabolism in beta cells and, therefore, acts as the glucose sensor. Pyruvate, the end product of anaerobic glycolysis, is readily oxidized by mitochondria in normal beta cells but cannot substitute for glucose as a stimulator of insulin synthesis and secretion. We found that pyruvate can stimulate the insulin promoter in cells expressing the bacterial gluconeogenic enzyme phosphoenolpyruvate carboxykinase, which allows the conversion of pyruvate to phosphoenolpyruvate and the earlier intermediates of glycolysis. We conclude that the intermediates of anaerobic glycolysis between fructose 1,6-diphosphate and phosphoenolpyruvate are essential for beta-cell glucose sensing.

Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice.

The decreased synthesis of hepatic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of gluconeogenesis, that occurs during endotoxemia was shown previously in rats to occur at the transcriptional level. In the current study, the exogenous administration of human recombinant tumor necrosis factor (TNF), a proximal mediator of endotoxic shock, reduced the PEPCK transcription rate, mRNAPEPCK levels, and PEPCK enzyme activity in a time- and dose-dependent manner in CD-1 mice. Comparable amounts of circulating TNF were measured in mice 2 h after injection of human recombinant TNF (10(5) U) or a 50% lethal dose of Escherichia coli endotoxin (20 mg/kg). Direct action of TNF to decrease the PEPCK transcription rate was confirmed in vitro with H-4-II-E Reuber hepatoma cells, in which a dose-dependent inhibition of PEPCK transcription was observed with 1 to 100 U of TNF per ml. A role for TNF-elicited changes in PEPCK gene expression during endotoxemia was confirmed by the protective effect of rabbit polyclonal antibodies to recombinant murine TNF. C57BL/6 mice passively immunized with anti-TNF 4 h prior to endotoxin challenge exhibited normal PEPCK enzyme activity. Neutralization of circulating TNF with anti-TNF failed, however, to prevent the hypoglycemia commonly observed during endotoxemia, suggesting the participation of other mediators. Anti-TNF treatment reduced circulating interleukins 1 and 6 at 3 and 6 h after endotoxin treatment, respectively. These results suggest that during endotoxemia, the development of hypoglycemia is multifaceted and that several cytokines are most likely involved. The findings from the Reuber hepatoma cell model afford an opportunity in future work to map putative cytokine response elements in the PEPCK promoter responsible for perturbed hormonal regulation of the gene during endotoxemia.