Title: Diminished Hepatic Gluconeogenesis via Defects in Tricarboxylic Acid Cycle Flux in Peroxisome Proliferator-activated Receptor γ Coactivator-1α (PGC-1α)-deficient Mice
Abstract: The peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) 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-1α 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-1α–/– mice using isotopomer-based NMR with complementary gene expression analyses. Hepatic glucose production was diminished in PGC-1α–/– livers coincident with reduced gluconeogenic flux from phosphoenolpyruvate. Surprisingly, the expression of PGC-1α target genes involved in gluconeogenesis was unaltered in PGC-1α–/– compared with WT mice under fed and fasted conditions. Flux through tricarboxylic acid cycle and mitochondrial fatty acid β-oxidation pathways was also diminished in PGC-1α–/– livers. The expression of multiple genes encoding tricarboxylic acid cycle and oxidative phosphorylation enzymes was significantly depressed in PGC-1α–/– mice and was activated by PGC-1α overexpression in the livers of WT mice. Collectively, these findings suggest that chronic whole-animal PGC-1α deficiency results in defects in hepatic glucose production that are secondary to diminished fatty acid β-oxidation and tricarboxylic acid cycle flux rather than abnormalities in gluconeogenic enzyme gene expression per se. The peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) 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-1α 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-1α–/– mice using isotopomer-based NMR with complementary gene expression analyses. Hepatic glucose production was diminished in PGC-1α–/– livers coincident with reduced gluconeogenic flux from phosphoenolpyruvate. Surprisingly, the expression of PGC-1α target genes involved in gluconeogenesis was unaltered in PGC-1α–/– compared with WT mice under fed and fasted conditions. Flux through tricarboxylic acid cycle and mitochondrial fatty acid β-oxidation pathways was also diminished in PGC-1α–/– livers. The expression of multiple genes encoding tricarboxylic acid cycle and oxidative phosphorylation enzymes was significantly depressed in PGC-1α–/– mice and was activated by PGC-1α overexpression in the livers of WT mice. Collectively, these findings suggest that chronic whole-animal PGC-1α deficiency results in defects in hepatic glucose production that are secondary to diminished fatty acid β-oxidation and tricarboxylic acid cycle flux rather than abnormalities in gluconeogenic enzyme gene expression per se. Flux through hepatic gluconeogenesis, fatty acid oxidation (FAO), 3The abbreviations used are: FAO, fatty acid oxidation; WT, wild-type; OXPHOS, oxidative phosphorylation; PC, pyruvate carboxylase; PPAR, peroxisome proliferator-activated receptor; PEPCK, phosphoenolpyruvate carboxykinase; GFP, green fluorescent protein; MAG, monoacetone glucose; GNGglycerol, gluconeogenesis from glycerol; PEP, P-enolpyruvate; CS, citrate synthase; AcAc, acetoacetate; BHB, β-hydroxybutyrate; HPLC, high pressure liquid chromatography; RT, reverse transcription; Glc-6-P, glucose-6-phosphatase; IDH, isocitrate dehydrogenase; SDH, succinate dehydrogenase; MDH, malate dehydrogenase; CytC, cytochrome c; COX2, CytC oxidase 2. 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 β-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 proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) is a highly inducible transcriptional coactivator that integrates multiple interconnected metabolic pathways in liver (1Lin J. Handschin C. Spiegelman B.M. Cell Metab. 2005; 1: 361-370Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar). PGC-1α controls transcription of genes involved in hepatic gluconeogenesis, fatty acid catabolism, oxidative phosphorylation (OXPHOS), and mitochondrial biogenesis (1Lin J. Handschin C. Spiegelman B.M. Cell Metab. 2005; 1: 361-370Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar, 2Kelly D.P. Scarpulla R.C. Genes Dev. 2004; 18: 357-368Crossref PubMed Scopus (1020) Google Scholar, 3Spiegelman B.M. Heinrich R. Cell. 2004; 119: 157-167Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Although PGC-1α was originally identified in a yeast two-hybrid screen of PPARγ-interacting factors in a brown adipocyte cDNA library (4Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3154) Google Scholar), it is now known to coactivate myriad nuclear receptor and non-nuclear receptor transcription factors in a variety of cell types (1Lin J. Handschin C. Spiegelman B.M. Cell Metab. 2005; 1: 361-370Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar). Expression is enriched in tissues with a high capacity for mitochondrial OXPHOS, including heart, skeletal muscle, and brown adipose tissue (1Lin J. Handschin C. Spiegelman B.M. Cell Metab. 2005; 1: 361-370Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar, 4Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3154) Google Scholar). Although hepatic PGC-1α levels are relatively low in normal, ad libitum-fed mice, its expression is robustly induced by acute food deprivation or diabetes mellitus (5Herzig S. Long F. Jhala U.S. Hedrick S. Quinn R. Bauer A. Rudolph D. Schutz G. Yoon C. Puigserver P. Spiegelman B. Montminy M. Nature. 2001; 413: 179-183Crossref PubMed Scopus (1164) Google Scholar, 6Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1551) Google Scholar), states when rates of fatty acid oxidation and gluconeogenesis are increased. Overexpression of PGC-1α in liver transcriptionally activates genes involved in hepatic gluconeogenesis, fatty acid catabolism, and OXPHOS (2Kelly D.P. Scarpulla R.C. Genes Dev. 2004; 18: 357-368Crossref PubMed Scopus (1020) Google Scholar, 3Spiegelman B.M. Heinrich R. Cell. 2004; 119: 157-167Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 6Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1551) Google Scholar, 7Rhee J. Inoue Y. Yoon J.C. Puigserver P. Fan M. Gonzalez F.J. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4012-4017Crossref PubMed Scopus (480) Google Scholar), whereas acute loss of function (adenovirus-driven RNA interference) markedly down-regulates expression of genes involved in each of these processes (8Koo S.H. Satoh H. Herzig S. Lee C.H. Hedrick S. Kulkarni R. Evans R.M. Olefsky J. Montminy M. Nat. Med. 2004; 10: 530-534Crossref PubMed Scopus (482) Google Scholar). Similarly, liver-specific PGC-1α gene deletion in mice impairs the expression of gluconeogenic genes in response to acute food deprivation (9Handschin C. Lin J. Rhee J. Peyer A.K. Chin S. Wu P.H. Meyer U.A. Spiegelman B.M. Cell. 2005; 122: 505-515Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Surprisingly, recent studies of two independently derived strains of mice in which the PGC-1α gene was constitutively disrupted in a whole-animal fashion (PGC-1α–/– mice) have shown that the expression of many known PGC-1α target genes was unaltered (10Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: e101Crossref PubMed Scopus (792) Google Scholar) or only modestly altered in liver (11Lin J. Wu P.H. Tarr P.T. Lindenberg K.S. St-Pierre J. Zhang C.Y. Mootha V.K. Jager S. Vianna C.R. Reznick R.M. Cui L. Manieri M. Donovan M.X. Wu Z. Cooper M.P. Fan M.C. Rohas L.M. Zavacki A.M. Cinti S. Shulman G.I. Lowell B.B. Krainc D. Spiegelman B.M. Cell. 2004; 119: 121-135Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar). Despite this, rates of fatty acid β-oxidation (10Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: e101Crossref PubMed Scopus (792) Google Scholar), mitochondrial respiration (10Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: e101Crossref PubMed Scopus (792) Google Scholar), and oxygen consumption (11Lin J. Wu P.H. Tarr P.T. Lindenberg K.S. St-Pierre J. Zhang C.Y. Mootha V.K. Jager S. Vianna C.R. Reznick R.M. Cui L. Manieri M. Donovan M.X. Wu Z. Cooper M.P. Fan M.C. Rohas L.M. Zavacki A.M. Cinti S. Shulman G.I. Lowell B.B. Krainc D. Spiegelman B.M. Cell. 2004; 119: 121-135Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar) were significantly diminished in hepatocytes from PGC-1α–/– mice. Moreover, one PGC-1α–/– mouse line exhibited significant hepatic steatosis following a 24-h fast, likely due to diminished capacity for FAO (10Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: e101Crossref PubMed Scopus (792) Google Scholar). Although previous studies have provided significant evidence implicating PGC-1α in the transcriptional control of genes encoding enzymes involved in gluconeogenesis, FAO, and the tricarboxylic acid cycle, less is known about the impact of this coactivator on metabolic flux through these key pathways in intact liver. PGC-1α–/– mice provide a unique opportunity to address this issue. Accordingly, we studied the isolated perfused liver of PGC-1α–/– mice by deuterium and 13C NMR spectroscopy isotopomer analysis. These studies were complemented with gene expression analyses examining multiple genes encoding enzymes in the relevant hepatic metabolic pathways. Surprisingly, we found that, despite marked deficits in rates of gluconeogenic flux in liver of fasted PGC-1α–/– mice, gluconeogenic gene expression was normal under fed and fasted conditions. Rates of fatty acid β-oxidation and tricarboxylic acid cycle flux were also defective in PGC-1α–/– mice, which correlated with diminished expression of tricarboxylic acid cycle enzymes and genes involved in OXPHOS. Taken together, these data suggest that gluconeogenic defects in PGC-1α–/– mice are secondary to deficits in mitochondrial oxidative metabolism and tricarboxylic acid cycle activity but not gluconeogenic enzyme expression. Animal Studies—The generation and general characterization of PGC-1α–/– mice has been recently described (10Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: e101Crossref PubMed Scopus (792) Google Scholar). Six-week-old PGC-1α–/– mice with age- and sex-matched wild-type (WT) control mice were employed. Short term fasting studies were performed with individually housed mice that were either food-deprived for 24 h or given ad libitum access to normal mouse chow. For adenoviral injection, C57BL/6 mice were injected intravenously with adenovirus driving the expression of GFP or PGC-1α as previously described (12Bernal-Mizrachi C. Weng S. Feng C. Finck B.N. Knutsen R.H. Leone T.C. Coleman T. Mecham R.P. Kelly D.P. Semenkovich C.F. Nat. Med. 2003; 9: 1069-1075Crossref PubMed Scopus (179) Google Scholar) and sacrificed 5 days later for tissue collection. Liver Glycogen—Hepatic glycogen content was determined as described by Passonneau and Lauderdale (13Passonneau J.V. Lauderdale V.R. Anal. Biochem. 1974; 60: 405-412Crossref PubMed Scopus (630) Google Scholar) using freeze-clamped liver tissue from WT or PGC-1α–/– mice fasted for 24 h. Liver Perfusion Experiments—Livers were isolated and perfused from 24-h-fasted mice as previously described (14Burgess S.C. Hausler N. Merritt M. Jeffrey F.M.H. Storey C. Milde A. Koshy S. Lindner J. Magnuson M.A. Malloy C.R. Sherry A.D. J. Biol. Chem. 2004; 279: 48941-48949Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Briefly, a midline laparotomy was performed to expose the liver and portal circulatory system. The liver was heparinized, and the portal vein was cannulated. The hepatic vein and inferior vena cava were dissected, and the perfusate flow through the portal vein was started simultaneously with a peristaltic pump at 8 ml/min in a non-recirculation circuit. The liver was suspended in a beaker containing effluent perfusate at 37 °C. Perfusate was siphoned off and stored on ice. The perfusate was composed of Krebs-Henseleit bicarbonate buffer containing 1.5 mm lactate, 0.15 mm pyruvate, 0.25 mm glycerol, 0.2 mm octanoate, 0.2 mm [U-13C3]propionate and 3% v/v D2O. Oxygen consumption was measured by oxygen electrode. Fractions (2 ml) of perfusate were collected at 10-min intervals and stored at –80 °C until assay for glucose. Liver perfusions were performed for 60 min, and the last 30 min of perfusate was combined for NMR analysis (n = 5WT and 6 PGC-1α–/–). Acetoacetate and β-hydroxybutyrate production was measured in a separate group of animals under the exact same conditions (n = 5 WT and 6 PGC-1α–/–). Upon completion, the liver was freeze-clamped and stored at –80 °C until further analysis. Sample Preparation and NMR Analyses—Glucose was isolated from the effluent perfusate and then converted to its monoacetone glucose (MAG) derivative as previously described (14Burgess S.C. Hausler N. Merritt M. Jeffrey F.M.H. Storey C. Milde A. Koshy S. Lindner J. Magnuson M.A. Malloy C.R. Sherry A.D. J. Biol. Chem. 2004; 279: 48941-48949Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). MAG was then analyzed by 2H (15Jones J.G. Solomon M.A. Cole S.M. Sherry A.D. Malloy C.R. Am. J. Physiol. 2001; 281: E848-E856Crossref PubMed Google Scholar, 16Burgess S.C. Nuss M. Chandramouli V. Hardin D.S. Rice M. Landau B.R. Malloy C.R. Sherry A.D. Anal. Biochem. 2003; 318: 321-324Crossref PubMed Scopus (38) Google Scholar) and 13C (15Jones J.G. Solomon M.A. Cole S.M. Sherry A.D. Malloy C.R. Am. J. Physiol. 2001; 281: E848-E856Crossref PubMed Google Scholar) NMR spectroscopy at 14.1% tesla using a broadband probe tuned to 92 and 150 MHz, respectively. Peak areas in the resulting spectra were measured using the peak fitting routine in the spectral analysis program NUTS (Acorn NMR Inc., Freemont, CA). Metabolic Profile—Metabolic fluxes were calculated from the NMR peak areas and biochemical assay of glucose as previously described (14Burgess S.C. Hausler N. Merritt M. Jeffrey F.M.H. Storey C. Milde A. Koshy S. Lindner J. Magnuson M.A. Malloy C.R. Sherry A.D. J. Biol. Chem. 2004; 279: 48941-48949Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 17Jin E.S. Jones J.G. Merritt M.E. Burgess S.C. Malloy C.R. Sherry A.D. Anal. Biochem. 2004; 327: 149-155Crossref PubMed Scopus (91) Google Scholar). Deuterium NMR spectra of MAG were used to determine the relative 2H enrichments of glucose at the H2, H5, and H6s positions. In turn, these enrichments were used to calculate the relative fractions of glucose production from glycogenolysis, gluconeogenesis from glycerol (GNGglycerol), and gluconeogenesis from P-enolpyruvate (PEP) originating from lactate or amino acids via the tricarboxylic acid cycle (GNGPEP) (15Jones J.G. Solomon M.A. Cole S.M. Sherry A.D. Malloy C.R. Am. J. Physiol. 2001; 281: E848-E856Crossref PubMed Google Scholar, 18Chandramouli V. Ekberg K. Schumann W.C. Kalhan S.C. Wahren J. Landau B.R. Am. J. Physiol. 1997; 273: E1209-E1215PubMed Google Scholar, 19Landau B.R. Wahren J. Chandramouli V. Schumann W.C. Ekberg K. Kalhan S.C. J. Clin. Investig. 1995; 95: 172-178Crossref PubMed Scopus (184) Google Scholar). Absolute fluxes were determined by multiplying the relative fluxes by total glucose production (14Burgess S.C. Hausler N. Merritt M. Jeffrey F.M.H. Storey C. Milde A. Koshy S. Lindner J. Magnuson M.A. Malloy C.R. Sherry A.D. J. Biol. Chem. 2004; 279: 48941-48949Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 17Jin E.S. Jones J.G. Merritt M.E. Burgess S.C. Malloy C.R. Sherry A.D. Anal. Biochem. 2004; 327: 149-155Crossref PubMed Scopus (91) Google Scholar). Anaplerosis and pyruvate cycling fluxes were determined from the C2 multiplets in the 13C NMR spectra of MAG using previously reported equations (14Burgess S.C. Hausler N. Merritt M. Jeffrey F.M.H. Storey C. Milde A. Koshy S. Lindner J. Magnuson M.A. Malloy C.R. Sherry A.D. J. Biol. Chem. 2004; 279: 48941-48949Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar,17Jin E.S. Jones J.G. Merritt M.E. Burgess S.C. Malloy C.R. Sherry A.D. Anal. Biochem. 2004; 327: 149-155Crossref PubMed Scopus (91) Google Scholar,20Jones J.G. Naidoo R. Sherry A.D. Jeffrey F.M.H. Cottam G.L. Malloy C.R. FEBS Lett. 1997; 412: 131-137Crossref PubMed Scopus (86) Google Scholar). Hepatic anaplerosis must be balanced by disposal pathways which, under typical conditions in the liver, is dominated by flux through PEPCK but may also have minor contributions, for instance, from the malic enzyme. For simplicity, we refer to this measurement as PEPCK, although it represents the total disposal fluxes whose sum must equal anaplerosis and thus is a maximal estimate of PEPCK. The portion of anaplerosis contributed by pyruvate cycling could be, again, from the malic enzyme (Pyr → OAA → Mal → Pyr) or from pyruvate kinase (Pyr → OAA → PEP → Pyr), and it should be pointed out that these two pathways cannot be distinguished from each other using the tracer technique employed here. The difference between anaplerosis and pyruvate cycling is equal to gluconeogenesis from PEP, which allows the absolute fluxes determined by glucose production and the deuterium NMR data to be extended to the fluxes intersecting the tricarboxylic acid cycle. β-oxidation (octanoate units) was calculated from ketogenesis and citrate synthase (CS) flux. Rates of fatty acid β-oxidation were calculated under the assumption that citrate synthase flux and ketogenesis represent the only fate of β-oxidation-derived acetyl-CoA (see the following): β-oxidation = (CS + 2 × ketogenesis)/4, where CS is in 2 carbon units of acetyl-CoA, ketogenesis is in 4 carbon units (2 acetyl-CoA), and β-oxidation is in 8 carbon (octanyl) units. The sum of CS and 2 × ketogenesis is divided by 4, because there are 4 acetyl-CoAs generated per octanoate. Analytical Measurements—Perfusate fractions designated for analytical analysis (2-ml fractions) were thawed and extracted with perchloric acid prior to assay. Glucose was assayed by standard enzyme-coupled reactions (21Kunst A. Draeger B. Ziegenhorn J. Bergmeyer H.U. Methods of Enzymatic Analysis. 3rd Ed. VCH Verlagsgesllschaft, Weinheim, Germany1984: 163-172Google Scholar), and these data were used to determine the rate of hepatic glucose production. Acetoacetate (AcAc) and β-hydroxybutyrate (BHB) were measured by the method of Williamson et al. (22Williamson D.H. Mellanby J. Krebs H.A. Biochem. J. 1962; 82: 90-96Crossref PubMed Scopus (1053) Google Scholar). The rate of AcAc and BHB production were summed to represent the rate of ketogenesis. Frozen liver tissue was divided and perchloric acid extracted for analysis of AcAc, BHB (100 mg), HPLC analysis of adenylate nucleotides (100 mg), and 13C NMR analysis (1 g). Ketone concentrations in the tissue extracts were determined by enzyme-linked assays (22Williamson D.H. Mellanby J. Krebs H.A. Biochem. J. 1962; 82: 90-96Crossref PubMed Scopus (1053) Google Scholar). The HPLC assay for ATP, ADP, and AMP was performed as described by Stochii et al. (23Stocchi V. Cucchiarini L. Canestrari F. Piacentini M.P. Fornaini G. Anal. Biochem. 1987; 167: 181-190Crossref PubMed Scopus (195) Google Scholar) on a Dionex (Palo Alto, Ca) HPLC system equipped with UV light detector and Supelco C18 reverse phase column. Hepatocyte Isolation and Metabolic Analyses—Primary cultures of mouse hepatocytes were obtained from WT and PGC-1α–/– mice as described previously (24Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). For gene expression analyses with isolated hepatocytes, cells were stimulated for 6 h with vehicle or dexamethasone (1 μm) and 8-bromo-cyclic AMP (1 mm). Quantitative Real-time RT-PCR—First-strand cDNA was generated by reverse transcription (RT) using total hepatic RNA. Real-time RT-PCR was performed using the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA) and the SYBR green kit. Primer sets were designed to span exon splice borders and are shown in supplemental Table 1. Arbitrary units of target mRNA were corrected by measuring the levels of 36B4 RNA. Statistical Analyses—For quantitative data, statistical comparisons were made using analysis of variance coupled to Scheffe's test or Student's t test assuming unequal variances. All data are presented as means ± S.E. with a statistically significant difference defined as p < 0.05. PGC-1α-deficient Livers Have Diminished Gluconeogenic Flux—Livers from 24-h-fasted PGC-1α–/– mice and their littermate controls were isolated and perfused with a non-recirculating perfusion medium for 60 min. The PGC-1α–/– livers produced 60% less glucose over the last 45 min of the perfusion (Fig. 1a). To determine the source of the glucose produced in these experiments, deuterated water was included in the perfusion medium and the effluent glucose was analyzed by 2H NMR (Fig. 1b). A lower H5/H2 ratio suggests a lower fractional contribution of gluconeogenesis and a higher contribution of glycogenolysis to glucose production (18Chandramouli V. Ekberg K. Schumann W.C. Kalhan S.C. Wahren J. Landau B.R. Am. J. Physiol. 1997; 273: E1209-E1215PubMed Google Scholar) in PGC-1α–/– livers compared with WT controls (Fig. 1c). There was no difference in the (H5-H6s)/H2 ratio (15Jones J.G. Solomon M.A. Cole S.M. Sherry A.D. Malloy C.R. Am. J. Physiol. 2001; 281: E848-E856Crossref PubMed Google Scholar) between groups, indicating that the fraction of glucose production due to gluconeogenesis from glycerol was unchanged. However, the H6s/H2 (19Landau B.R. Wahren J. Chandramouli V. Schumann W.C. Ekberg K. Kalhan S.C. J. Clin. Investig. 1995; 95: 172-178Crossref PubMed Scopus (184) Google Scholar) ratio was significantly decreased in PGC-1α–/– livers, indicating a decrease in gluconeogenesis from PEP as a fraction of glucose production (Fig. 1c). The finding that glycogenolysis was a significant source of glucose after a 24-h fast was surprising but agreed with a 2-fold elevation in liver glycogen content in fasted PGC-1α–/– mice versus fasted WT mice (Fig. 1d). These data suggest that hepatic glycogen cycling is altered in PGC-1α–/– mice perhaps to allow for significant glycogenolysis to compensate for a relative reduction in gluconeogenic flux following prolonged fasting. Nevertheless, absolute glycogen levels were still low compared with the fed state. Therefore, to determine which pathways contributed to decreased glucose production (Fig. 2a, v1) in the PGC-1α–/– liver, the absolute flux through glycogenolysis, GNGglycerol, and GNGPEP (v2, v3, and v4, respectively, in Fig. 2a) pathways was quantified. Despite the increased fraction of glucose derived from glycogen in the PGC-1α–/– livers, there was no difference in absolute rates of glycogenolysis between PGC-1α–/– and WT livers (Fig. 2b) due to the overall decrease in glucose production. In addition, the flux from glycerol to glucose (GNGglycerol) was not significantly different between the WT and PGC-1α–/– livers. However, absolute flux through GNGPEP was dramatically decreased in PGC-1α–/– livers (Fig. 2b), indicating that the primary defect in glucose output was at the level of gluconeogenesis from substrates that pass through the tricarboxylic acid cycle (e.g. lactate, pyruvate, or amino acids) via the combined activity of PC and PEPCK. To investigate flux through the PEP pathways, we measured tricarboxylic acid cycle anaplerosis and pyruvate cycling (as illustrated in Fig. 2a) by 13C NMR isotopomer analysis of the effluent glucose from PGC-1α–/– and control livers. Absolute flux through the pathway mal/OAA → pyr/PEP (Fig. 2a, v6) was halved in PGC-1α–/– livers compared with WT livers, indicating that PEPCK flux was remarkably impaired (Fig. 2c). Fig. 2c also shows that PGC-1α–/– livers had decreased flux through pyruvate kinase or malic enzyme-catalyzed pyruvate cycling (v5) (Fig. 2c) as determined by 13C NMR isotopomer analysis. This may be a compensatory response to decreased PEPCK flux to augment GNGPEP by sparing PEP from this "futile cycle." Without the attenuated pyruvate cycling, gluconeogenesis would be close to zero in the PGC-1α–/– livers. PGC-1α Is Not Required for Basal or Fasting-induced Expression of Gluconeogenic Enzymes—Because NMR isotopomer analyses indicated that gluconeogenesis, especially via the PEPCK pathway, is defective in PGC-1α–/– mice, we examined the fasting-induced expression of genes encoding PEPCK, glucose-6-phosphatase (Glc-6-P), and PC in PGC-1α–/– livers. Surprisingly, the hepatic expression of PEPCK, Glc-6-P, and PC was equally and robustly induced by fasting in WT and PGC-1α–/– mice (Fig. 3). The expression of gluconeogenic enzymes was also evaluated in isolated hepatocytes stimulated ex vivo with 8-bromo-cyclic AMP and dexamethasone, which is known to induce PGC-1α and gluconeogenic gene expression. PGC-1α deficiency again did not affect the activation of PEPCK or Glc-6-P gene expression in response to this stimulus (Fig. 4). Collectively, these data indicate that PGC-1α is not required for the activation of gluconeogenic gene expression in response to acute fasting or gluconeogenic stimuli and suggest the existence of PGC-1α-independent regulatory mechanisms.FIGURE 4Normal induction of gluconeogenic enzymes in PGC-1α–/– hepatocytes. Graphs depict mean levels of PEPCK and Glc-6-P mRNA collected from hepatocytes isolated from WT and PGC-1α–/– mice determined by SYBR green RT-PCR (n = 6). Hepatocytes were treated in culture with vehicle or dexamethasone (dex)(1 μm) and 8-bromo-cyclic AMP (8 Br cAMP)(1 mm) for 6 h prior to RNA collection. Values are normalized to vehicle-treated WT hepatocytes (=1.0) and corrected to 36B4 RNA levels in the same sample. *, p < 0.05 versus vehicle-treated hepatocytes. AU, arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PGC-1α-deficient Livers Have Diminished Energy Production—Because PGC-1α is a well recognized transcriptional regulator of fatty acid catabolism, OXPHOS, and mitochondrial biogenesis (2Kelly D.P. Scarpulla R.C. Genes Dev. 2004; 18: 357-368Crossref PubMed Scopus (1020) Google Scholar, 3Spiegelman B.M. Heinrich R. Cell. 2004; 119: 157-167Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), flux through biochemical pathways important for hepatic energy homeostasis was also measured. We found that hepatic oxygen consumption in the isolated perfused PGC-1α–/– livers was reduced by 25% compared with control livers (Fig. 5a). In addition, total ketone (AcAc and BHB) production (Fig. 2a, v8) was decreased 30% in the PGC-1α–/– liver compared with controls (Fig. 5a). Carbon-13 isotopomer analysis of the effluent glucose revealed a 2-fold impairment of tricarboxylic acid cycle citrate synthase flux (Fig. 2a, v7) in PGC-1α–/– livers versus WT controls (Fig. 5b). β-oxidation of octanoate (Fig. 2a, v9) was also decreased by 25% in the PGC-1α–/– liver (Fig. 5b), which is consistent with our pre