Proposed involvement of adipocyte glyceroneogenesis and phosphoenolpyruvate carboxykinase in the metabolic syndrome

Elevated concentration of plasma non-esterified fatty acids (NEFA) is now recognized as a key factor in the onset of insulin-resistance and type 2 diabetes mellitus. During fasting, circulating NEFAs arise from white adipose tissue (WAT) as a consequence of lipolysis from stored triacylglycerols. However, a significant part of these FAs (30-70%) is re-esterified within the adipocyte, so that a recycling occurs and net FA output is much less than < true > lipolysis. Indeed, a balance between two antagonistic processes, lipolysis and FA re-esterification, controls the rate of net FA release from WAT. During fasting, re-esterification requires glyceroneogenesis defined as the de novo synthesis of glycerol-3-P from pyruvate, lactate or certain amino acids. The key enzyme in this process is the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C; EC 4.1.1.32). Recent advance has stressed the role of glyceroneogenesis and of PEPCK-C in FA release from WAT. Results indicate that glyceroneogenesis is indeed important to lipid homeostasis and that a disregulation in this pathway may have profound pathophysiological effects. The present review focuses on the regulation of glyceroneogenesis and of PEPCK-C gene expression and activity by FAs, retinoic acids, glucocorticoids and the hypolipidemic class of drugs, thiazolidinediones.     

Thiazolidinediones block fatty acid release by inducing glyceroneogenesis in fat cells

Thiazolidinediones are used to treat type 2 diabetes mellitus because they decrease plasma glucose, insulin, triglyceride, and fatty acid levels. Thiazolidinediones are agonists for peroxisome proliferator-activated receptor gamma, a nuclear receptor that is highly expressed in fat tissue. We identify glyceroneogenesis as a target of thiazolidinediones in cultured adipocytes and fat tissues of Wistar rats. The activation of glyceroneogenesis by thiazolidinediones occurs mainly in visceral fat, the same fat depot that is specifically implicated in the progression of obesity to type 2 diabetes. The increase in glyceroneogenesis is a result of the induction of its key enzyme, phosphoenolpyruvate carboxykinase, whose gene expression is peroxisome proliferator-activated receptor gamma-dependent in adipocytes. The main role of this metabolic pathway is to allow the re-esterification of fatty acids via a futile cycle in adipocytes, thus lowering fatty acid release into the plasma. The importance of such a fatty acid re-esterification process in the control of lipid homeostasis is highlighted by the existence of a second thiazolidinedione-induced pathway involving glycerol kinase. We show that glyceroneogenesis accounts for at least 75% of the whole thiazolidinedione effect. Because elevated plasma fatty acids promote insulin resistance, these results suggest that the glyceroneogenesis-dependent fatty acid-lowering effect of thiazolidinediones could be an essential aspect of the antidiabetic action of these drugs.     

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.     

Inactivation of phosphoenolpyruvate carboxykinase by acetaldehyde.

Preincubation with acetaldehyde at 37°C inactivates rat liver phosphoenolpyruvate carboxykinase. The inactivation is dependent upon the acetaldehyde concentration and the pH and duration of preincubation, and is prevented but not reversed by glutathione. The binding of the substrate ITP appears to be affected in the inactivation process. This effect of acetaldehyde might contribute to inhibition of gluconeogenesis resulting from ethanol metabolism.     

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.     

Fasciola hepatica: inhibition of phosphoenolpyruvate carboxykinase, and end-product formation by quinolinic acid and 3-mercaptopicolinic acid

Quinolinic acid and 3-mercaptopicolinic acid act as inhibitors of Fasciola hepatica phosphoenolpyruvate carboxykinase. Low concentrations of these compounds (0.1 mM quinolinate and 0.01 mM 3-mercaptopicolinate) resulted in noncompetitive inhibition, which became mixed inhibition at higher concentrations (1.5 and 0.15 mM, respectively). 3-mercaptopicolinic acid proved to be a much more potent effector than quinolinic acid. Both quinolinic acid and 3-mercaptopicolinic acid caused a significant reduction in the total amount of end product excreted, again 3-mercaptopicolinate being more effective than quinolinate. When glucose was present in the medium, both propionate and acetate levels fell significantly with both inhibitors; however, only 3-mercaptopicolinic acid caused an effect in the absence of glucose.     

Activation and inactivation of phosphoenolpyruvate carboxykinase by ferrous ions.

Phosphoenolpyruvate carboxykinase from rat liver cytosol is activated by Fe2+ ions in either direction of catalysis. Preincubation of the purified enzyme with Fe2+ ions causes a time-dependent irreversible loss of activity; this is not seen with unpurified enzyme. Purified enzyme can be protected from inactivation by Fe2+ ions by partially purified protein fractions from liver (ferroactivator fractions). The possible role of ferroactivator and Fe2+ ions in regulating phosphoenolpyruvate carboxykinase is discussed.     

Inactivation by glucose of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase showed high activity in Saccharomyces cerevisiae grown on gluconeogenic carbon sources. Addition of glucose to such cultures caused a rapid loss of the phosphoenolpyruvate carboxykinase activity. Fructose or mannose had the same effect as glucose, while 2-deoxyglucose or galactose were without effect. The inactivation was an irreversible process, since the regain of the activity was dependent of de novo protein synthesis. Cycloheximide did not prevent inactivation. All strains of the genus Saccharomyces tested showed inactivation of their phosphoenolpyruvate carboxykinase upon addition of glucose; this behaviour was not restricted to this genus.Non-Standard Abbreviations FbPase fructose bisphosphatase [EC 3.1.3.11 fructose-1,6-bisphosphate hydrolase] - PEPCK phosphoenolpyruvate carboxykinase [EC 4.1.49 ATP: oxalacetate carboxylase (transphosphorylating)] - YPE yeast-peptone-ethanol A preliminary account of these results was presented at the Fourth International Symposium on Yeasts, Vienna, Austria, July 1974     

Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn²⁺ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990–994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3–6 orders of magnitude lower values of V _max/K _m, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased K _m values for Mn²⁺ and PEP as compared with wild-type enzyme, suggesting a role of His²²⁵ in Mn²⁺ and PEP binding. From 1.5–1.6 Kcal/mol lower affinity for the 3(2)-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His²²⁵ and Asp²⁶³ in nucleotide binding.     

The role of phosphoenolpyruvate carboxykinase in amino acid metabolism in muscle.

Starvation or feeding rats on a high-protein diet, valine or isoleucine, but not leucine, increases the activity of muscle phosphoenolpyruvate carboxykinase, but has no effect on NADP+-linked malate dehydrogenase. This suggests that muscle phosphoenolpyruvate carboxykinase is involved in oxidation or conversion of some amino acids to alanine.     

Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli.

Phosphoenolpyruvate and oxaloacetate are key intermediates at the junction between catabolism and biosynthesis. Alteration of carbon flow at these branch points will affect the growth yield and the formation of products. We attempted to modulate the metabolic flow between phosphoenolpyruvate and oxaloacetate by overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from a multicopy plasmid under the control of the tac promoter. It was found that overexpression of phosphoenolpyruvate carboxylase decreased the rates of glucose consumption and organic acid excretion, but the growth and respiration rates remained unchanged. Consequently, the growth yield on glucose was improved. This result indicates that the wild-type level of phosphoenolpyruvate carboxylase is not optimal for the most efficient glucose utilization in batch cultures. On the other hand, overexpression of phosphoenolpyruvate carboxykinase increased glucose consumption and decreased oxygen consumption relative to those levels required for growth. Therefore, the growth yield on glucose was reduced because of a higher rate of fermentation product excretion. These data provide useful insights into the regulation of central metabolism and facilitate further manipulation of pathways for metabolite production.