Diminished hepatic gluconeogenesis via defects in tricarboxylic acid cycle flux in peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)-deficient mice

Abstract

The peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha) 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-1alpha 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-1alpha(-/-) mice using isotopomer-based NMR with complementary gene expression analyses. Hepatic glucose production was diminished in PGC-1alpha(-/-) livers coincident with reduced gluconeogenic flux from phosphoenolpyruvate. Surprisingly, the expression of PGC-1alpha target genes involved in gluconeogenesis was unaltered in PGC-1alpha(-/-) compared with WT mice under fed and fasted conditions. Flux through tricarboxylic acid cycle and mitochondrial fatty acid beta-oxidation pathways was also diminished in PGC-1alpha(-/-) livers. The expression of multiple genes encoding tricarboxylic acid cycle and oxidative phosphorylation enzymes was significantly depressed in PGC-1alpha(-/-) mice and was activated by PGC-1alpha overexpression in the livers of WT mice. Collectively, these findings suggest that chronic whole-animal PGC-1alpha deficiency results in defects in hepatic glucose production that are secondary to diminished fatty acid beta-oxidation and tricarboxylic acid cycle flux rather than abnormalities in gluconeogenic enzyme gene expression per se.

FIGURE 1. Sources of substrate used for glucose production in the PGC-1α^−/− mice

a, the graph depicts mean (±S.E.) glucose production by isolated perfused livers from WT and PGC-1α^−/− mice determined by glucose assay of the perfusate. b, deuterium NMR spectra of MAG derived from glucose produced by the isolated perfused liver. The peak area of H2, H5, and H6s are used to determine the relative contributions of glycogenolysis, gluconeogenesis from glycerol, and gluconeogenesis from PEP. c, the NMR data indicates that PGC-1α^−/− livers form a greater fraction of their glucose production from glycogen and less from PEP compared with control livers. d, the graph depicts mean (±S.E.) hepatic glycogen levels in WT or PGC-1α^−/− mice given ad libitum access to food or after a 24-h fast. *, p < 0.05 versus WT fasted mice. PPM, parts/million.

FIGURE 2. Defects in hepatic gluconeogenesis and altered flux through gluconeogenic pathways in PGC-1α^−/− mice

a, the diagram illustrates the metabolic pathways under investigation by the combination of deuterium and ¹³C tracers. The three major sources of glucose production in liver are denoted v2 (glycogenolysis), v3 (gluconeogenesis from glycerol, GNG_glycerol), and v4 (gluconeogenesis from phosphoenolpyruvate, GNG_pep). PEPCK flux is a major constituent of total efflux from the hepatic tricarboxylic acid (TCA) cycle, which is estimated as total anaplerosis by the ¹³C NMR spectra of perfusate glucose and represented by v6. Pyruvate cycling (v5) denotes pathways such as pyruvate kinase or the malic enzyme, which regenerate pyruvate rather than contributing to gluconeogenesis. b and c, the graphs depict mean (±S.E.) absolute flux through the pathways shown in a. b, the graph shows the absolute flux through pathways leading to glucose production: glycogenolysis (hexose units), GNG_glycerol (triose units), or GNG_PEP (triose units) as determined by deuterium NMR of perfusate glucose. c, fluxes contributing to GNG_PEP determined from ¹³C NMR. Mice were fasted 24 h before the liver perfusion experiment. *, p < 0.05 versus WT mice.

FIGURE 3. Fasting-induced activation of genes involved in gluconeogenesis is normal in PGC-1α^−/− liver

Graphs depict mean (±S.E.) levels of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (Glc-6-P), and pyruvate carboxylase (PC) mRNA collected from the livers of fed and fasted WT and PGC-1α^−/− mice determined by SYBR green RT-PCR (n = 7). Values are normalized to fed WT mice (= 1.0) and corrected to 36B4 RNA levels. *, p < 0.05 versus fed mice. AU, arbitrary units.

FIGURE 4. Normal 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.

FIGURE 5. Defective tricarboxylic acid cycle activity and β-oxidation in isolated perfused livers from fasted PGC-1α^−/− mice

Graphs depict mean (±S.E.) values in isolated perfused livers from 24-h fasted mice. a, perfusate was assayed to measure hepatic oxygen consumption and ketogenesis. The rate of oxygen consumption was measured by oxygen electrode, and ketogenesis was determined from the sum of effluent acetoacetate and β-hydroxybutyrate. b, tricarboxylic acid (TCA) cycle flux or citrate synthase (CS) flux was measured using a combination of ²H and ¹³C NMR data from perfusate glucose. Total β-oxidation was determined from ketone production and tricarboxylic acid cycle flux. c, perfused livers were extracted and assayed for acetoacetate (AcAc) and β-hydroxybutyrate (BHB) as an indication of mitochondrial redox state. d, total high energy nucleotides in liver extracts were determined by HPLC. *, p < 0.05 versus WT liver; **, p < 0.1 versus WT liver. AEC, adenosine energy charge.

FIGURE 6. Diminished expression of genes encoding enzymes in tricarboxylic acid cycle enzymes and genes involved in mitochondrial OXPHOS in PGC-1α^−/− mice

Graphs depict mean (±S.E.) levels of citrate synthase (CS), isocitrate dehydrogenase (IDH) succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) (a) or cytochrome c (CytC), CytC oxidase (COX2), COX4, and the β-subunit of ATP synthase mRNA (b) collected from liver of WT and PGC-1α^−/− mice determined by SYBR green RT-PCR (n = 7). Values are normalized to WT mice (= 1.0) and corrected to 36B4 RNA levels. *, p < 0.05 versus WT mice. AU, arbitrary units.

FIGURE 7. PGC-1α overexpression drives transcriptional activation of tricarboxylic acid cycle enzymes

Graphs depict mean (±S.E.) levels of CS, IDH, SDH, and MDH (a) or CytC, COX2, and COX4 mRNA (b) collected from the livers of WT mice infected with virus driving the expression of GFP or PGC-1α as determined by SYBR green RT-PCR (n = 7). Values are normalized to GFP-infected mice (= 1.0) and corrected to 36B4 RNA levels. *, p < 0.05 versus GFP mice. AU, arbitrary units.