Cytosolic and mitochondrial phosphoenolpyruvate carboxykinase of the bullfrog,Rana catesbeiana,liver

The mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase(transphosphorylating), EC 4.1.1.32) occurring in the bullfrog (Rana catesbeiana) liver were studied. The enzymes in the two intracellular compartments of both tadpole and adult frog liver were immunologically identical. Both radioactively-labelled forms of the mitochondrial and cytosolic phosphoenolpyruvate carboxykinase from bullfrog liver were imported at the same rate into intact mitochondria in vitro. The mitochondrial and cytosolic enzyme activities did not respond to the administration of glucagon, glucocorticoid, quinolinate and d-mannoheptulose which are known as enhancers of phosphoenolpyruvate carboxykinase, but were found to increase during natural metamorphosis. The former activity was markedly increased in the tadpoles treated with 3,5,3′-triiodothyronine. It was supposed that in the bullfrog liver the phosphoenolpyruvate carboxykinase localized in the mitochondria is of central importance in phosphoenolpyruvate synthesis from oxaloacetate     

Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons ofCucurbita pepo L.

3-Mercaptopicolinic acid, a non-competitive inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.19) was used to study the control of gluconeogenesis by this enzyme in germinating marrow (Cucurbita pepo) cotyledons. In vitro, phosphoenolpyruvate carboxykinase was inhibited by 3-mercaptopicolinic acid, with aKi of 5.9 M. At 25°C the inhibitor caused an increase in the label incorporated from [2-¹⁴C]acetate into CO₂, and a decrease in the label incorporated into the insoluble and neutral fractions. Phosphoenolpyruvate carboxykinase had a flux control coefficient for gluconeogenesis (C _PEPCK ^J ) of between 0.7 and 1.0. 3-Mercaptopicolinic acid was a less effective inhibitor of phosphoenolpyruvate carboxykinase at lower temperatures (Ki = 8.6 M at 17°C, 13.3 M at 10°C) and had similar effects on the metabolism of [2-¹⁴C]acetate by marrow cotyledons when the temperature was reduced to 17°C and 10°C. The control coefficient for this enzyme did not change with temperature, indicating that phosphoenolpyruvate carboxykinase exerts a high degree of control over gluconeogenesis at all temperatures examined.Abbreviations PEP Phosphoenolpyruvate - PEPCK PEP carboxykinase The authors thank Dr. Ian Woodrow (University of Melbourne, Australia) for helpful discussions. This work was supported by a grant from the Science and Engineering Research Council, U.K. (GR/F 50978).     

Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver

Hepatic steatosis, the accumulation of lipids in the liver, is widely believed to result in insulin resistance. To test the causal relationship between hepatic steatosis and insulin resistance, we generated mice that overexpress acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step of triacylglycerol (TG) biosynthesis, in the liver (Liv-DGAT2 mice). Liv-DGAT2 mice developed hepatic steatosis, with increased amounts of TG, diacylglycerol, ceramides, and unsaturated long-chain fatty acyl-CoAs in the liver. However, they had no abnormalities in plasma glucose and insulin levels, glucose and insulin tolerance, rates of glucose infusion and hepatic glucose production during hyperinsulinemic-euglycemic clamp studies, or activities of insulin-stimulated signaling proteins in the liver. DGAT1 overexpression in the liver also failed to induce glucose or insulin intolerance. Our results indicate that DGAT-mediated lipid accumulation in the liver is insufficient to cause insulin resistance and show that hepatic steatosis can occur independently of insulin resistance.     

Broad expression of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase provide evidence for gluconeogenesis in human tissues other than liver and kidney

The importance of renal and hepatic gluconeogenesis in glucose homeostasis is well established, but the cellular localization of the key gluconeogenic enzymes liver fructose-1,6-bisphosphatase (FBPase) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in these organs and the potential contribution of other tissues in this process has not been investigated in detail. Therefore, we analyzed the human tissue localization and cellular distribution of FBPase and PEPCK immunohistochemically. The localization analysis demonstrated that FBPase was expressed in many tissues that had not been previously reported to contain FBPase activity (e.g., prostate, ovary, suprarenal cortex, stomach, and heart). In some multicellular tissues, this enzyme was detected in specialized areas such as epithelial cells of the small intestine and prostate or lung pneumocytes II. Interestingly, FBPase was also present in pancreas and cortex cells of the adrenal gland, organs that are involved in the control of carbohydrate and lipid metabolism. Although similar results were obtained for PEPCK localization, different expression of this enzyme was observed in pancreas, adrenal gland, and pneumocytes type I. These results show that co-expression of FBPase and PEPCK occurs not only in kidney and liver, but also in a variety of organs such as the small intestine, stomach, adrenal gland, testis, and prostate which might also contribute to gluconeogenesis. Our results are consistent with published data on the expression of glucose-6-phosphatase in the human small intestine, providing evidence that this organ may play an important role in the human glucose homeostasis.     

Genetic control of serine dehydratase and phosphoenolpyruvate carboxykinase in mice

Inbred strains of mice were found to differ with regard to their endogenous activities of the liver enzymes serine dehydratase (SD) and phosphoenolpyruvate carboxykinase (PEPCK). The strain distribution patterns for the activity of each enzyme were identical. On feeding of high-protein diets or on fasting, the activities of both enzymes were induced in a concordant fashion which suggested the control of both enzymes by a single gene. Genetic analysis established that the induction of both enzymes on feeding of high-protein diets was controlled by a single gene (Sdr-1), whereas the induction of SD, but not of PEPCK, on fasting was controlled by different single gene (Sdr-2). The lack of segregation of the backcross generations with respect to PEPCK activities obtained on fasting precluded the establishment of any association of the response of PEPCK to fasting with either the Sdr-1 or Sdr-2 locus. The strain of mice (BALB/cJ) that had the ability to maximally induce both gluconeogenic enzymes under both dietary treatments failed to survive a fast as long as those strains with less ability to induce. This suggests that the ability to induce key enzymes in gluconeogenesis when food is unavailable is of little consequence with regard to their ability to produce essential nutrients necessary for survival.     

Inflammation inhibits the expression of phosphoenolpyruvate carboxykinase in liver and adipose tissue

Inhibition of adipocyte triglyceride biosynthesis is required for fatty acid mobilization during inflammation. Triglyceride biosynthesis requires glycerol 3-phosphate and phosphoenolpyruvate carboxykinase (PEPCK) plays a key role. We demonstrate that LPS, zymosan, and TNF-α decrease PEPCK in liver and fat. Turpentine decreases PEPCK in liver, but not in fat. The LPS-induced decrease in PEPCK does not occur in TLR4 deficient animals, indicating that this receptor is required. The LPS-induced decrease in hepatic PEPCK does not occur in TNF receptor/IL-1 receptor knockout mice, but occurs in fat, indicating that TNF-α/IL-1 is essential for the decrease in liver but not fat. In 3T3-L1 adipocytes TNF-α, IL-1, IL-6, and IFNγ inhibit PEPCK indicating that there are multiple pathways by which PEPCK is decreased in adipocytes. The binding of PPARγ and RXRα to the PPARγ response element in the PEPCK promoter is markedly decreased in adipose tissue nuclear extracts from LPS treated animals. Lipopolysaccharide and zymosan reduce PPARγ and RXRα expression in fat, suggesting that a decrease in PPARγ and RXRα accounts for the decrease in PEPCK. Thus, there are multiple cytokine pathways by which inflammation inhibits PEPCK expression in adipose tissue which could contribute to the increased mobilization of fatty acids during inflammation.     

Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase

Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity. One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme. When the modification is carried out in the presence of GDPMn, the enzyme retains 97% of its activity with almost no incorporation of label. The specificity of the reaction is further supported by the detection of a unique fluorescent peptide from the trypsin-treated modified enzyme. Fluorescence emission of enzyme-bound AEDANS shows a broad band centered at 470 nm and presents a monoexponential decay with a lifetime of 19 ns. These data indicate that the probe-binding site is considerably less polar than water and similar in polarity to ethanol. Anisotropy determinations give evidence for restricted rotational freedom for AEDANS bound to the rat carboxykinase, while acrylamide quenching studies reveal limited accessibility to the probe site. The results are consistent with specific labeling of rat liver phosphoenolpyruvate carboxykinase at or near the GDP site. The characteristics of the nucleotide-binding sites of rat liver and yeast (ATP) phosphoenolpyruvate carboxykinase are compared.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.     

Phosphorus-31 nuclear relaxation rate studies of the nucleotides on phosphoenolpyruvate carboxykinase

The interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd less than 2 microM) than for GDP (Kd = 15 microM) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the alpha- and beta-31P of IDP to Mn2+ are identical (3.5 A). The Mn2+ distance to the beta- and gamma-31P of ITP is also identical (3.7 A) and is 0.2 A further from the alpha-phosphorus. In the presence of P-enolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a second sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the phosphorus atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 X 10(4) s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)