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J Biol Chem. 2006 Jul 14;281(28):19000-8. 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. From the full text article: Introduction 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 beta-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-gamma (PPAR-gamma) coactivator-1alpha (PGC-1alpha) is a highly inducible transcriptional coactivator that integrates multiple interconnected metabolic pathways in liver (1). PGC-1alpha controls transcription of genes involved in hepatic gluconeogenesis, fatty acid catabolism, oxidative phosphorylation (OXPHOS), and mitochondrial biogenesis (1–3). Although PGC-1alpha was originally identified in a yeast two-hybrid screen of PPAR-gamma-interacting factors in a brown adipocyte cDNA library (4), it is now known to coactivate myriad nuclear receptor and non-nuclear receptor transcription factors in a variety of cell types (1). Expression is enriched in tissues with a high capacity for mitochondrial OXPHOS, including heart, skeletal muscle, and brown adipose tissue (1, 4). Although hepatic PGC-1alpha levels are relatively low in normal, ad libitum-fed mice, its expression is robustly induced by acute food deprivation or diabetes mellitus (5, 6), states when rates of fatty acid oxidation and gluconeogenesis are increased. Overexpression of PGC-1alpha in liver transcriptionally activates genes involved in hepatic gluconeogenesis, fatty acid catabolism, and OXPHOS (2, 3, 6, 7), whereas acute loss of function (adenovirus-driven RNA interference) markedly down-regulates expression of genes involved in each of these processes (8). Similarly, liver-specific PGC-1alpha gene deletion in mice impairs the expression of gluconeogenic genes in response to acute food deprivation (9). Surprisingly, recent studies of two independently derived strains of mice in which the PGC-1alpha gene was constitutively disrupted in a whole-animal fashion (PGC-1alpha–/– mice) have shown that the expression of many known PGC-1alpha target genes was unaltered (10) or only modestly altered in liver (11). Despite this, rates of fatty acid beta-oxidation (10), mitochondrial respiration (10), and oxygen consumption (11) were significantly diminished in hepatocytes from PGC-1alpha–/– mice. Moreover, one PGC-1alpha–/– mouse line exhibited significant hepatic steatosis following a 24-h fast, likely due to diminished capacity for FAO (10). Although previous studies have provided significant evidence implicating PGC-1alpha 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-1alpha–/– mice provide a unique opportunity to address this issue. Accordingly, we studied the isolated perfused liver of PGC-1alpha–/– 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-1alpha–/– mice, gluconeogenic gene expression was normal under fed and fasted conditions. Rates of fatty acid beta-oxidation and tricarboxylic acid cycle flux were also defective in PGC-1alpha–/– 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-1alpha–/– mice are secondary to deficits in mitochondrial oxidative metabolism and tricarboxylic acid cycle activity but not gluconeogenic enzyme expression. … Results PGC-1alpha-deficient Livers Have Diminished Gluconeogenic Flux - Livers from 24-h-fasted PGC-1alpha–/– mice and their littermate controls were isolated and perfused with a non-recirculating perfusion medium for 60 min. The PGC-1alpha–/– 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 (18) in PGC-1alpha–/– livers compared with WT controls (Fig. 1c). There was no difference in the (H5-H6s)/H2 ratio (15) between groups, indicating that the fraction of glucose production due to gluconeogenesis from glycerol was unchanged. However, the H6s/H2 (19) ratio was significantly decreased in PGC-1alpha–/– 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-1alpha–/– mice versus fasted WT mice (Fig. 1d). These data suggest that hepatic glycogen cycling is altered in PGC-1alpha–/– 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-1alpha–/– 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-1alpha–/– livers, there was no difference in absolute rates of glycogenolysis between PGC-1alpha–/– 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-1alpha–/– livers. However, absolute flux through GNGPEP was dramatically decreased in PGC-1alpha–/– 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-1alpha–/– and control livers. Absolute flux through the pathway mal/OAA > pyr/PEP (Fig. 2a, v6) was halved in PGC-1alpha–/– livers compared with WT livers, indicating that PEPCK flux was remarkably impaired (Fig. 2c). Fig. 2c also shows that PGC-1alpha–/– 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-1alpha–/– livers. PGC-1alpha 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-1alpha–/– mice, we examined the fasting-induced expression of genes encoding PEPCK, glucose-6-phosphatase (Glc-6-P), and PC in PGC-1alpha–/– livers. Surprisingly, the hepatic expression of PEPCK, Glc-6-P, and PC was equally and robustly induced by fasting in WT and PGC-1alpha–/– 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-1alpha and gluconeogenic gene expression. PGC-1alpha 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-1alpha is not required for the activation of gluconeogenic gene expression in response to acute fasting or gluconeogenic stimuli and suggest the existence of PGC-1alpha-independent regulatory mechanisms. PGC-1alpha-deficient Livers Have Diminished Energy Production - Because PGC-1alpha is a well recognized transcriptional regulator of fatty acid catabolism, OXPHOS, and mitochondrial biogenesis (2, 3), flux through biochemical pathways important for hepatic energy homeostasis was also measured. We found that hepatic oxygen consumption in the isolated perfused PGC-1alpha–/– 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-1alpha–/– 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-1alpha–/– livers versus WT controls (Fig. 5b). beta-oxidation of octanoate (Fig. 2a, v9) was also decreased by 25% in the PGC-1alpha–/– liver (Fig. 5b), which is consistent with our previous report using [3H]palmitate in isolated hepatocytes (10). These findings collectively indicate a significant energetic disadvantage due to loss of PGC-1alpha function. Decreased flux through beta-oxidation and the reactions of the tricarboxylic acid cycle suggested decreased mitochondrial NADH production. To investigate whether the mitochondrial redox state was impacted, liver tissue was extracted after perfusion and assayed for the redox pair AcAc and BHB. Surprisingly, PGC-1alpha–/– livers had a 2-fold higher BHB/AcAc ratio compared with control livers, indicating an increase in the NADH/NAD+ ratio (Fig. 5c) (25). Highly reduced mitochondria are usually associated with hepatic physiology in which energy production overmatches energy utilization, which contrasts our findings of impaired tricarboxylic acid cycle and beta-oxidation activity in the PGC-1alpha–/– liver. To determine whether this imbalance might impact downstream energy metabolism, total ATP, ADP, and AMP were assayed by HPLC of the liver extract. The absolute concentrations of ATP and ADP were decreased by ~40% in the PGC-1alpha–/– liver but did not reach statistical significance, and we found no difference in the calculated adenosine energy charge (Fig. 5d). PGC-1alpha Controls the Expression of Tricarboxylic Acid Cycle and OXPHOS Enzymes in Liver - We previously demonstrated that, despite reduced rates of FAO in hepatocytes isolated from PGC-1alpha–/– mice, the expression of several genes involved in FAO (carnitine palmitoyltransferase 1alpha, very long chain acyl-CoA dehydrogenase, and medium chain acyl-CoA dehydrogenase) was unaffected by PGC-1alpha deficiency (10), which is notable given the significant defect in beta-oxidation flux. Therefore, we examined the expression of genes encoding tricarboxylic acid cycle and OXPHOS enzymes. We found that the expression of the tricarboxylic acid cycle enzymes, citrate synthase (CS), isocitrate dehydrogenase 3alpha (IDH), and succinate dehydrogenase (SDH) subunit A was significantly diminished in liver of PGC-1alpha knock-out mice (Fig. 6a), whereas the expression of malate dehydrogenase (MDH) 2 was not altered. As has been reported in other tissues of PGC-1alpha–/– mice, the expression of several genes encoding enzymes involved in OXPHOS, including cytochrome c (CytC), CytC oxidase 2 (COX2), COX4, and the beta-subunit of ATP synthase was also diminished (Fig. 6b). Given the apparent crucial role of tricarboxylic acid cycle deficiency in the control of hepatic energy and glucose homeostasis by PGC-1alpha, we examined whether PGC-1alpha activation was sufficient to induce the expression of tricarboxylic acid cycle genes. Wild-type mice were injected intravenously with an adenovirus driving the expression of murine PGC-1alpha and/or GFP (vector control) and hepatic gene expression examined 5 days post-infection. As predicted from the loss-of-function studies, the expression of CS, SDH, IDH, and MDH was robustly activated by PGC-1alpha overexpression in the livers of mice (Fig. 7a). The expression of CytC, COX2, and COX4 was also strongly induced by PGC-1alpha (Fig. 7b). Collectively, these combined gain-of-function and loss-of-function studies identify multiple enzymes in the tricarboxylic acid cycle as target genes of PGC-1alpha in liver. Discussion A flurry of recent studies has shown that the PGC-1 family of coactivators transcriptionally regulates enzymes involved in mitochondrial OXPHOS, FAO, and glucose homeostasis (1–3). Consistent with this, we demonstrate that chronic PGC-1alpha deficiency leads to significant impairments in hepatic beta-oxidation, tricarboxylic acid cycle, and gluconeogenic flux. Altered metabolic flux through these pathways correlated to decrements in the expression of multiple enzymes in the tricarboxylic acid cycle and OXPHOS pathways. However, deficits in hepatic gluconeogenic and mitochondrial fatty acid beta-oxidation flux observed in PGC-1alpha–/– mice did not correlate with altered expression of key enzymes involved in gluconeogenesis or fatty acid catabolism. Based on these findings, we postulate that the gluconeogenic and beta-oxidation defects in PGC-1alpha–/– mice are secondary to tricarboxylic acid cycle, OXPHOS, or generalized mitochondrial dysfunction and suggest that these findings elucidate novel mechanisms by which diminished PGC-1alpha activity impacts glucose and fatty acid homeostasis. The hepatic PGC-1alpha system is activated in both types 1 and 2 models of diabetes mellitus (5, 6). Because PGC-1alpha transcriptionally activates the expression of genes encoding gluconeogenic enzymes, PGC-1alpha overactivity is thought to contribute to uncontrolled hepatic glucose production in the diabetic state. In support of this, RNA interference-mediated knockdown of hepatic PGC-1alpha improved glucose homeostasis in a rodent model of diabetes (8). In contrast to the strong activation observed in liver, the expression of PGC-1alpha and several downstream target genes involved in OXPHOS is actually diminished in the skeletal muscle of diabetic patients (26). Whether skeletal muscle PGC-1alpha system inactivity plays a causative role in the development of diabetes or is a secondary consequence of metabolic perturbations of the disease is still unclear. However, it has been postulated that PGC-1alpha system deficiencies may exacerbate lipid accumulation and drive the development of skeletal muscle insulin resistance. Interestingly, PGC-1alpha–/– mice exhibit enhanced insulin sensitivity on standard chow and are protected against high fat diet-induced insulin resistance (10, 11). These findings prompted us to examine the effects of PGC-1alpha deficiency on hepatic metabolic flux, particularly because PGC-1alpha influences hepatic glucose production, a principal constituent of whole-body glucose homeostasis. The marked impairment we observed in hepatic glucose production may explain, in part, the enhanced insulin sensitivity of PGC-1alpha–/– mice. The hepatic metabolic phenotype of PGC-1alpha-deficient mice is reminiscent of mice nullizygous for PPARalpha, a liver-enriched transcription factor partner of PGC-1alpha, which also exhibit defects in hepatic fatty acid oxidation and glucose production (27) and are insulin sensitive (28–30). PPARalpha–/– mice exhibit normal hepatic PEPCK and Glc-6-P expression under fasting conditions (27, 31) but are severely hypoglycemic (32). We postulate that defects in mitochondrial metabolism underlie the observed defects in hepatic gluconeogenesis in both PGC-1alpha–/– and PPARalpha–/– mice. This notion is supported by other genetic models of altered mitochondrial energy metabolism. For example, ablation of beta-oxidation enzymes leads to hypoglycemia during fasting (33, 34), whereas children with inborn errors in mitochondrial FAO or OXPHOS often present with hypoglycemia secondary to defects in gluconeogenesis (35–37). The precise lesion (i.e. beta-oxidation, tricarboxylic acid cycle, or OXPHOS) that leads to this metabolic bottleneck in this and other models is unclear and will require further study. Conversely, in mice with a liver-specific knockout of PEPCK, tricarboxylic acid cycle flux is impaired (14), despite up-regulation of some tricarboxylic acid cycle enzymes (38). Collectively, these studies indicate that cataplerosis related to GNGPEP and tricarboxylic acid cycle flux are exquisitely interdependent (14, 39, 40) and support the existence of bidirectional cross-talk between hepatic energy generation and gluconeogenic pathways. We propose that, in the PGC-1alpha–/– liver, impaired hepatic energy production necessarily inhibits the energetically costly process of gluconeogenesis. Interestingly, GNGglycerol (the conversion of glycerol to glucose), which occurs in the cytosol and results in net production of ATP, was unaffected in PGC-1alpha–/– livers. Given the strong activation of gluconeogenic enzymes following PGC-1alpha overexpression (6), our finding that PGC-1alpha is not required for full expression of these enzymes, especially during fasting when PGC-1alpha is induced, is surprising. Previous work has demonstrated that liver-specific PGC-1alpha deficiency attenuates the fasting-induced activation of PEPCK and Glc-6-P (9). Acute RNA interference-mediated knockdown of PGC-1alpha in liver also causes a profound down-regulation of gluconeogenic enzyme gene expression (8). In contrast, the expression of gluconeogenic enzymes in the two models of constitutive whole-animal PGC-1alpha deficiency was unaltered (current study) or actually increased (11). However, it should be noted that gluconeogenic gene expression in response to dexamethasone and forskolin was defective in the other constitutive PGC-1alpha–/– mouse strain (11). It is likely that the differences in gluconeogenic gene expression among the various models of PGC-1alpha deficiency are explained by whole-animal versus liver-specific deficiency or are related to the developmental timing of PGC-1alpha deactivation. The data obtained from the two chronic, whole-animal PGC-1alpha-deficient models suggest compensatory adaptations by other transactivators. Two related proteins with regions of homology to PGC-1alpha (PGC-1beta and PGC-related coactivator) have been identified as part of the PGC-1 family. Although PGC-1beta functionally overlaps with PGC-1alpha in its effects on mitochondrial FAO and OXPHOS, the beta isoform has distinct effects on gluconeogenic and lipogenic gene expression (7, 41). Overexpression of PGC-1beta fails to drive a gluconeogenic response and interacts poorly with HNF4alpha and FOXO1, transcription factors controlling PEPCK and Glc-6-P expression (7). To our knowledge, the effects of the PGC-related coactivator on gluconeogenesis have not yet been characterized. Additionally, TORC2, a transcriptional coactivator outside of the PGC-1alpha family, has recently been shown to stimulate gluconeogenesis in fasted liver (42). In the context of the constitutive PGC-1alpha-deficient liver, other transcriptional coactivators likely compensate for PGC-1alpha to transactivate the expression of gluconeogenic genes. The finding that tricarboxylic acid cycle enzymes are direct targets of PGC-1alpha is not surprising, given that PGC-1alpha controls many other aspects of mitochondrial oxidative metabolism. As was recently demonstrated in skeletal muscle (43), PGC-1alpha activation coordinately induces multiple pathways (beta-oxidation, tricarboxylic acid cycle, and OXPHOS) to synchronize the capacity of the entire ATP synthesis pathway of the mitochondrion. Our findings suggest that PGC-1alpha deficiency leads to a coordinate deactivation of each of these metabolic pathways and reaffirm the critical role that PGC-1alpha plays in controlling energy homeostasis in the liver. Summary - In summary, PGC-1alpha loss of function caused decreased hepatic expression of enzymes in the tricarboxylic acid cycle and the electron transport chain. However, PGC-1alpha deficiency did not impact the expression of known PGC-1alpha target genes involved in fatty acid beta-oxidation or gluconeogenesis, suggesting compensatory changes in the transcriptional control of these pathways in response to chronic PGC-1alpha deficiency. Nevertheless, primary defects of the tricarboxylic acid cycle and OXPHOS pathways caused impaired flux through fatty acid beta-oxidation, the tricarboxylic acid cycle, anaplerotic pathways, and GNGPEP. These studies unveil novel mechanisms of PGC-1alpha action and identify biochemical pathways that may be most impacted by altered PGC-1alpha activity in pathologic states, including obesity-related insulin resistance and diabetes. Categories: 2006, PGC-1alpha, PPARalpha, Diabetes, Energy metabolism, Fatty acid metabolism, Fatty acid oxidation, Gluconeogenesis, Glycogenolysis, Hypoglycemia, Ketosis, Krebs cycle, Mitochondria, Oxidative phosphorylation, PEPCK, Pyruvate carboxylase, Cataplerosis, Anaplerosis |