Phosphoenolpyruvate carboxykinase and the critical role of cataplerosis in the control of hepatic metabolism

100

The peroxisome proliferator-activated receptor ␥ (PPAR␥) coactivator 1␣ (PGC-1␣) is a highly inducible transcriptional coactivator implicated in the coordinate regulation of genes encoding enzymes involved in hepatic fatty acid oxidation, oxidative phosphorylation, and gluconeogenesis. The present study sought to assess the effects of chronic PGC-1␣ deficiency on metabolic flux through the hepatic gluconeogenic, fatty acid oxidation, and tricarboxylic acid cycle pathways. To this end, hepatic metabolism was assessed in wild-type (WT) and PGC-1␣ ؊/؊ mice using isotopomer-based NMR with complementary gene expression analyses. Hepatic glucose production was diminished in PGC-1␣ ؊/؊ livers coincident with reduced gluconeogenic flux from phosphoenolpyruvate. Surprisingly, the expression of PGC-1␣ target genes involved in gluconeogenesis was unaltered in PGC-1␣ ؊/؊ compared with WT mice under fed and fasted conditions. Flux through tricarboxylic acid cycle and mitochondrial fatty acid ␤-oxidation pathways was also diminished in PGC-1␣ ؊/؊ livers. The expression of multiple genes encoding tricarboxylic acid cycle and oxidative phosphorylation enzymes was significantly depressed in PGC-1␣ ؊/؊ mice and was activated by PGC-1␣ overexpression in the livers of WT mice. Collectively, these findings suggest that chronic wholeanimal PGC-1␣ deficiency results in defects in hepatic glucose production that are secondary to diminished fatty acid ␤-oxidation and tricarboxylic acid cycle flux rather than abnormalities in gluconeogenic enzyme gene expression per se.Flux through hepatic gluconeogenesis, fatty acid oxidation (FAO), 3 tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation (OXPHOS) pathways can be modulated at multiple regulatory levels. Substrate availability, post-translational modification, and transcriptional regulation of genes encoding enzymes at various points can influence the capacity for, and the rate of flux through, each of these pathways. Moreover, flux through one pathway has an inevitable impact on the flux of the others. For instance, mitochondrial FAO is the principal source of energy in the hepatocyte, impacting the amount of chemical work that can be performed by the liver. Furthermore, the tricarboxylic acid cycle not only oxidizes acetyl-CoA generated by ␤-oxidation and produces reducing equivalents for ATP synthesis but also supplies carbons necessary for gluconeogenesis through pyruvate carboxylase (PC) and P-enolpyruvate carboxykinase (PEPCK). Thus, the tricarboxylic acid cycle is a critical hub linking FAO with gluconeogenesis and OXPHOS pathways.Recent work has shown that the peroxisome proliferatoractivated receptor ␥ (PPAR␥) coactivator-1␣ (PGC-1␣) is a highly inducible transcriptional coactivator that integrates multiple interconnected metabolic pathways in liver (1). PGC-1␣ controls transcription of genes involved in hepatic gluconeogenesis, fatty acid catabolism, oxidative phosphorylation (OXPHOS), and mitochondrial biogenesis (1-3). Although PGC-1␣ was originally identified ...

Section: Discussionmentioning

confidence: 70%

Diminished Hepatic Gluconeogenesis via Defects in Tricarboxylic Acid Cycle Flux in Peroxisome Proliferator-activated Receptor γ Coactivator-1α (PGC-1α)-deficient Mice

Burgess

Leone

Wende

et al. 2006

100

“…1 and 2). Accumulation of these intermediates was observed in the liver of mice in which liver PEPCK was inactivated by homologous recombination (38). Although these mice die very young, another type of liver PEPCK knock-out mice has a normal life span and does not suffer from hypoglycemia, presumably because renal GNG compensates for the absence of liver GNG from amino acids and lactate (30).…”

Section: Resultsmentioning

confidence: 99%

Metabolomic and Mass Isotopomer Analysis of Liver Gluconeogenesis and Citric Acid Cycle

Yang

Kombu

Kasumov

et al. 2008

We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. Rat livers were perfused with lactate or pyruvate ؎ aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH 13 CO 3 . Other livers were perfused with dimethyl [1,4-13 C 2 ]succinate ؎ mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic carbon leaves the liver as citric acid cycle intermediates, mostly ␣-ketoglutarate. The efflux of gluconeogenic carbon ranges from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the condensation, in the liver, of (i) aminooxyacetate with pyruvate, ␣-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.Although hypothesis-based research is the gold standard of biological investigation, it is necessary from time to time to expand its scope by a period of data-based discovery research. The current omic revolution is fulfilling this need. By widening the knowledge base, omics and in particular metabolomics (1-5) identify unknown correlations, allowing the formulation and testing of new hypotheses. We hypothesized that the scope of metabolomics could be enhanced by associating it with mass isotopomer analysis, a powerful technique developed over the past 16 years (reviewed in Refs. 6, 7). This association would allow the identification of unexpected labeling patterns of metabolites, pointing to unknown reactions and/or regulatory mechanisms (8). We used this association of techniques to expand our previous studies on the influence of metabolic zonation of the liver on the labeling patterns of metabolites and on metabolic rates calculated from these labeling patterns.Over the past 12 years, questions were raised on the potential influence of the metabolic zonation of the liver (9) on the 13 Clabeling pattern of glucose released by gluconeogenesis (GNG), 2 glycogenolysis, or both. The zonated structure of the liver results in translobular gradients of enzyme activities and of blood substrate concentrations (glycerol, acetate, and NH 4 ϩ ) (10 -12). Also, the computation of the mass isotopomer distribution (MID) of (i) glucose labeled from [ (10,13,14). This results in underestimations of fractional rates of glucose, fatty acid, and sterol synthesis calculated from the MID of these compounds synthesized from a 13 C-labeled substrate. In this study, we hypothesized that the metabolic zonation of the liver might result in incompatible labeling of intermediates extracted from the whole liver. Such incompatibilities were suggested in 1970 by Veneziale et...

“…in the conversion of oxalacetate to P-enolpyruvate), this enzyme links cataplerosis with a major biosynthetic pathway: glucose synthesis. Deletion of the gene for PEPCK-C in mouse liver leads to ablated gluconeogenesis in that tissue (8,9), whereas a total body deletion of the enzyme results in profound hypoglycemia and death. It is well established that changes in the rate of transcription of the gene for PEPCK-C is a critical step in establishing the overall activity of the enzyme in rodent liver and kidney cortex, tissues that synthesize glucose (7).…”

Section: Gluconeogenesismentioning

confidence: 99%

What Is the Metabolic Role of Phosphoenolpyruvate Carboxykinase?

Yang

Kalhan

Hanson

2009

Self Cite

227

204

The enzyme phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) (PEPCK) 2 has the unusual distinction of being very well studied but metabolically misunderstood. As we will document in this minireview, the enzyme has been almost exclusively linked to gluconeogenesis to the point that changes in the levels of PEPCK mRNA or its activity are associated with the control of hepatic glucose output and, more recently, with alterations in life span. That a tissue such as brown adipose tissue, which does not make glucose, has more PEPCK activity on a protein basis than is present in the liver is largely ignored. In addition, all eukaryotes have a gene for both a mitochondrial (PEPCK-M) and cytosolic (PEPCK-C) form of the enzyme. In the livers of most mammals studied to date (including humans), 50% of the total PEPCK activity is PEPCK-M. However, for reasons to be discussed, only PEPCK-C has been studied in any detail in mammals. Thus, the "strange case of PEPCK-M" deserves our attention. This minireview is an attempt to broaden our prospective on the metabolic role of this enzyme by reviewing the body of literature that has accumulated demonstrating that PEPCK plays a key role in a several metabolic processes associated with cataplerosis. Some Facts about PEPCKPEPCK catalyzes the following reaction.Although the reaction is reversible physiologically, the K m(oxalacetate) (12 M) and K m(GTP) (13 M) are within the concentration range of these substrates in mammalian tissues, suggesting that PEPCK normally synthesizes P-enolpyruvate from oxalacetate. This is further supported by the fact that pyruvate carboxylase, a mitochondrial enzyme that can also synthesize oxalacetate, is present in many tissues and has a generally higher V max (12 units/g in rat liver) than PEPCK (6 units/g) (1). PEPCK uses GTP or ITP, but not ATP, as a phosphate donor to form P-enolpyruvate. Most bacteria and yeast studied to date contain an ATP-linked enzyme, which has little sequence similarity to mammalian PEPCK. However, Mukhopadhyay et al.(2) have purified a GTP-linked PEPCK from Mycobacterium smegmatis, which has a molecular mass of 72 kDa; this agrees well with the molecular mass of mammalian (3) and avian (4) PEPCK. In addition, the M. smegmatis enzyme has a 47-50% sequence identity to human and chicken PEPCK-C and a 46% similarity to chicken PEPCK-M (2), suggesting that the genes for the two isoforms of the enzyme diverged from a common bacterial enzyme, perhaps by duplication and gene rearrangement. The genes for human PEPCK-M and human and rat PEPCK-C contain the same organization: 10 exons and 9 introns (3, 5). The gene for human PEPCK-M is 9839 bp in length, whereas the genes for rat (6138 bp) and human (5345 bp) PEPCK-C are smaller; the size of the introns in PEPCK-M largely accounts for this difference.