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• W2036407890 abstract "Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator involved in several aspects of energy metabolism. It is induced or activated under different stimuli in a highly tissue-specific manner and subsequently partners with certain transcription factors in those tissues to execute various biological programs. In the fasted liver, PGC-1α is induced and interacts with hepatocyte nuclear factor 4α (HNF4α) and other transcription factors to activate gluconeogenesis and increase hepatic glucose output. Given the broad spectrum of liver genes responsive to HNF4α, we sought to determine those that were specifically targeted by the combination of PGC-1α and HNF4α. Coexpression of these two molecules in murine stem cells reveals a high induction of mRNA for apolipoproteins A-IV and C-II. Forced expression of PGC-1α in mouse and human hepatoma cells increases the mRNA of a subset of apolipoproteins implicated in very low density lipoprotein and triglyceride metabolism, including apolipoproteins A-IV, C-II, and C-III. Coactivation of the apoC-III/A-IV promoter region by PGC-1α occurs through a highly conserved HNF4α response element, the loss of which completely abolishes activation by PGC-1α and HNF4α. Adenoviral infusion of PGC-1α into live mice increases hepatic expression of apolipoproteins A-IV, C-II, and C-III and increases serum and very low density lipoprotein triglyceride levels. Conversely, knock down of PGC-1α in vivo causes a decrease in both apolipoprotein expression and serum triglyceride levels. These data point to a crucial role for the PGC-1α/HNF4α partnership in hepatic lipoprotein metabolism. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator involved in several aspects of energy metabolism. It is induced or activated under different stimuli in a highly tissue-specific manner and subsequently partners with certain transcription factors in those tissues to execute various biological programs. In the fasted liver, PGC-1α is induced and interacts with hepatocyte nuclear factor 4α (HNF4α) and other transcription factors to activate gluconeogenesis and increase hepatic glucose output. Given the broad spectrum of liver genes responsive to HNF4α, we sought to determine those that were specifically targeted by the combination of PGC-1α and HNF4α. Coexpression of these two molecules in murine stem cells reveals a high induction of mRNA for apolipoproteins A-IV and C-II. Forced expression of PGC-1α in mouse and human hepatoma cells increases the mRNA of a subset of apolipoproteins implicated in very low density lipoprotein and triglyceride metabolism, including apolipoproteins A-IV, C-II, and C-III. Coactivation of the apoC-III/A-IV promoter region by PGC-1α occurs through a highly conserved HNF4α response element, the loss of which completely abolishes activation by PGC-1α and HNF4α. Adenoviral infusion of PGC-1α into live mice increases hepatic expression of apolipoproteins A-IV, C-II, and C-III and increases serum and very low density lipoprotein triglyceride levels. Conversely, knock down of PGC-1α in vivo causes a decrease in both apolipoprotein expression and serum triglyceride levels. These data point to a crucial role for the PGC-1α/HNF4α partnership in hepatic lipoprotein metabolism. The liver is central to systemic nutrient metabolism. It regulates both catabolic and anabolic processes that maintain proper blood levels of protein, carbohydrate, and lipid. The liver responds to blood-borne hormones and neurotransmitters, executing biological programs to ensure that the energy demands of peripheral tissues are satisfied. Many of these programs exert metabolic changes through the activation or induction of specific transcription factors, which in turn increase the expression of key enzymes or other downstream regulators.The peroxisome proliferator-activated receptor γ (PPARγ) 4The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PGC-1α, PPARγ coactivator-1 α; HNF4α, hepatocyte nuclear factor 4 α; VLDL, very low density lipoprotein; RNAi, RNA interference; GFP, green fluorescent protein; DR-1, direct repeat separated by one nucleotide. 4The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PGC-1α, PPARγ coactivator-1 α; HNF4α, hepatocyte nuclear factor 4 α; VLDL, very low density lipoprotein; RNAi, RNA interference; GFP, green fluorescent protein; DR-1, direct repeat separated by one nucleotide. coactivator-1α (PGC-1α) (1Puigserver 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 (3030) Google Scholar) has emerged as an important regulator of several liver functions. It positively regulates hepatic heme biosynthesis and links fasting to acute attacks of porphyria (2Handschin 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 (280) Google Scholar). PGC-1α induces CYP7A1, the rate-limiting enzyme of bile acid synthesis (3De Fabiani E. Mitro N. Gilardi F. Caruso D. Galli G. Crestani M. J. Biol. Chem. 2003; 278: 39124-39132Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 4Shin D.J. Campos J.A. Gil G. Osborne T.F. J. Biol. Chem. 2003; 278: 50047-50052Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 5Bhalla S. Ozalp C. Fang S. Xiang L. Kemper J.K. J. Biol. Chem. 2004; 279: 45139-45147Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). It also partners with liver X receptor α and farnesoid X receptor in modulating cholesterol and triglyceride metabolism (6Zhang Y. Castellani L.W. Sinal C.J. Gonzalez F.J. Edwards P.A. Genes Dev. 2004; 18: 157-169Crossref PubMed Scopus (291) Google Scholar, 7Oberkofler H. Schraml E. Krempler F. Patsch W. Biochem. J. 2003; 371: 89-96Crossref PubMed Scopus (75) Google Scholar). In the hepatic response to fasting, PGC-1α coordinates the induction of genes involved in gluconeogenesis, fatty acid oxidation, and ketogenesis (8Rhee 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 (464) Google Scholar, 9Spiegelman B.M. Heinrich R. Cell. 2004; 119: 157-167Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Its coactivation of the liver-enriched transcription factors HNF4α and FOXO1, as well as the glucocorticoid receptor, results in the induction of gluconeogenic enzymes and increased hepatic glucose output (10Yoon 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 (1495) Google Scholar, 11Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1154) Google Scholar). The functional loss or genetic knock out of either HNF4α or FOXO1 completely abrogates the ability of PGC-1α to activate hepatic gluconeogenesis. PGC-1α interacts tightly with HNF4α in vitro and strongly coactivates this transcription factor on the phosphoenolpyruvate carboxykinase and glucose-6-phosphatase promoters (8Rhee 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 (464) Google Scholar, 10Yoon 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 (1495) Google Scholar).HNF4α has been recognized as a broad regulator of liver function (12Sladek F.M. Receptor. 1994; 4: 64PubMed Google Scholar, 13Spath G.F. Weiss M.C. Mol. Cell. Biol. 1997; 17: 1913-1922Crossref PubMed Scopus (104) Google Scholar). Ectopic expression of HNF4α in de-differentiated hepatoma cells or fibroblasts induces both a morphology and genetic markers characteristic of hepatic epithelium (14Spath G.F. Weiss M.C. J. Cell Biol. 1998; 140: 935-946Crossref PubMed Scopus (133) Google Scholar, 15Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Nat. Genet. 2003; 34: 292-296Crossref PubMed Scopus (453) Google Scholar). Although the total knock out of HNF4α is embryonic lethal due to defects in gastrulation (16Chen W.S. Manova K. Weinstein D.C. Duncan S.A. Plump A.S. Prezioso V.R. Bachvarova R.F. Darnell Jr., J.E. Genes Dev. 1994; 8: 2466-2477Crossref PubMed Scopus (479) Google Scholar), embryos lacking HNF4α can be rescued in midgestation through tetraploid complementation (17Duncan S.A. Nagy A. Chan W. Development. 1997; 124: 279-287Crossref PubMed Google Scholar). Analysis of E12.0 fetuses null for HNF4α shows that a lack of HNF4α impairs differentiation into a fully functional hepatic parenchyma that expresses its normal complement of serum and metabolic factors, such as apolipoproteins and fatty acid-binding proteins (18Li J. Ning G. Duncan S.A. Genes Dev. 2000; 14: 464-474PubMed Google Scholar). Conditional knock out of the gene in the liver through a tissue-specific cre-lox system produces viable offspring that suffer from severe defects in lipid homeostasis; they accumulate lipid in the liver and exhibit reduced serum triglyceride levels (19Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (849) Google Scholar). It is clear that the transcriptional targets of HNF4α are numerous and varied. In fact, using chromatin immunoprecipitation in human hepatocytes combined with promoter microarrays, HNF4α localizes to 12% of 13,000 promoters examined (20Odom D.T. Zizlsperger N. Gordon D.B. Bell G.W. Rinaldi N.J. Murray H.L. Volkert T.L. Schreiber J. Rolfe P.A. Gifford D.K. Fraenkel E. Bell G.I. Young R.A. Science. 2004; 303: 1378-1381Crossref PubMed Scopus (1065) Google Scholar).Given the important functional interaction of PGC-1α with HNF4α and the broad roles of these two molecules in hepatic function, it is quite likely that their partnership influences multiple aspects of liver metabolism beyond that which has been described. Through microarray analysis, we now identify a subset of apolipoproteins as transcriptional targets that are particularly dependent on the cooperative action of PGC-1α and HNF4α. Adenoviral infusion of PGC-1α into live mice results in the hepatic induction of these apolipoproteins and a concomitant increase in serum and VLDL triglyceride. Knock down of PGC-1α via adenoviral RNAi decreases apolipoprotein mRNA and serum triglyceride levels. These data indicate that the PGC-1α/HNF4α partnership plays a critical role in hepatic lipoprotein synthesis and export.EXPERIMENTAL PROCEDURESMicroarray Analysis—Affymetrix array hybridization and scanning were performed by the Core Facility at Dana-Farber Cancer Institute using Murine 430 2.0 chips. Array data were analyzed with d-CHIP software.RNA Isolation and Analysis—Total RNA was isolated from liver or cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For quantitative real-time PCR analysis, 2μg of total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamer primers (Roche Applied Science). Relative mRNA abundance normalized to 18 S rRNA levels was determined with the ΔΔCt method after amplification using an iCycler iQ real-time PCR detection system (Bio-Rad) and SYBR Green (Bio-Rad). For Northern hybridizations, 20 μg of each sample of RNA was resolved on a formaldehyde gel, transferred to nylon membrane using a TurboBlotter (Schleicher & Schuell), and hybridized with 32P-labeled gene-specific probes. Hybridization to ribosomal protein 36B4 was included as a loading control.Animal Experiments—All animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee. Animals were fed standard rodent chow and housed in a controlled environment with 12-h light and dark cycles. Fasts were started at the beginning of a dark cycle, and mice were deprived of food for the indicated amount of time before being sacrificed.Adenoviral Infection—Adenoviral GFP and PGC-1α have been described previously (54Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D.M. Kelly D.P. J. Clin. Investig. 2000; 106: 847-856Crossref PubMed Scopus (997) Google Scholar). An adenoviral vector encoding HNF4α was generated by cloning an HNF4α cDNA into AdTrack-CMV (Invitrogen). Cultured cells were infected with the indicated adenovirus at a multiplicity of infection of 50 at 90% confluence. 36 h after infection, cells were harvested and their RNA isolated. Female, 8-week-old C57 BL/6 mice were transduced with CsCl-purified adenovirus via tail vein injection. Mice were each tail vein injected with 0.2 OD of adenovirus (∼2 × 1011 viral particles/mouse). Four days later, they were subjected to a 24-h fast. The mice were sacrificed on the fifth day and their livers and plasma harvested.Cell Culture, Transfections, and Reporter Gene Assays—F9 teratocarcinoma cells were cultured on gelatinized plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. H2.35 mouse hepatoma cells were kept in Dulbecco's modified Eagle's medium supplemented with 4% fetal bovine serum and 1 μm dexamethasone. HepG2 cells were cultured in 10% fetal bovine serum in Dulbecco's modified Eagle's medium. Cells were transfected in 6-well dishes at 70% confluence using Superfect (Qiagen) according to the manufacturer's protocol. Each transfection involved a 1:1:10 ratio of reporter gene:transcription factor(s):coactivator for a total of 3.6 μg of DNA. Equal amounts of DNA were used for all transfection combinations by using appropriate vector DNA. For reporter gene assays, cells were harvested 24 h after transfection. Luciferase levels were determined and normalized to β-galactosidase expression. All transfection experiments were repeated at least three times in triplicate.Site-directed Mutagenesis—Site-directed mutagenesis of the apoC-III/A-IV luciferase reporter (28Carrier J.C. Deblois G. Champigny C. Levy E. Giguere V. J. Biol. Chem. 2004; 279: 52052-52058Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) was performed using overlapping primers and the Expand Long Template PCR system (Roche Applied Science). HNF4α-binding Site 1 was mutated from 5′-TGGGCAAAGGTCA-3′ to 5′-TGGGCAAtGcgCA-3′, which introduced an FspI site. Site 2 was mutated from 5′-TGGACCTTGTTCT-3′ to 5′-TGGACCTTcTaga-3′, which introduced an XbaI site.Chromatin Immunoprecipitation—Experiments were performed using a chromatin immunoprecipitation kit (EZ ChIP) following the manufacturer's protocol (Upstate Biotechnology). H2.35 cells were transfected in 15-cm plates at 70% confluence with plasmids expressing GFP, FLAG-tagged PGC-1α, or FLAG-tagged PGC-1α plus HNF4α. Two days later cells were harvested, DNA-protein complexes cross-linked in formaldehyde, and immunoprecipitation reactions performed using M2 anti-FLAG resin (Sigma). After reverse cross-linking, DNA was purified by spin columns and subsequently analyzed by PCR.Triglyceride Measurements and Lipoprotein Fractionation—Total serum triglyceride was measured using a colorimetric assay kit (337; Sigma). For fractionation experiments, serum was diluted 1:1 (v/v) with phosphate-buffered saline and centrifuged to remove any debris. 100 μl of diluted sample was injected into a Superose 6 HR 10/30 gel filtration column for analysis of plasma lipoproteins, performed using a fast protein liquid chromatography system (Pharmacia ÄKTA) at 4 °C (55Ha Y.C. Barter P.J. J. Chromatogr. 1985; 341: 154-159Crossref PubMed Scopus (51) Google Scholar). Samples were eluted at a flow rate of 0.4 ml/min in buffer containing 0.05 m phosphate and 0.15 m NaCl, pH 7.0. Fractions of 0.3 ml were collected for analysis. Triglyceride levels in each fraction were measured enzymatically using an Infinity Triglyceride (GPO-Trinder) kit (Sigma).RESULTSCombined Expression of PGC-1α and HNF4α Induces Apolipoproteins in Cultured Cells—To determine the genes that are dependent upon cooperation between PGC-1α and HNF4α, we exogenously expressed these two molecules in F9 teratocarcinoma stem cells using adenoviral vectors. These cells have the ability to produce all three germ layers and have been used heavily as a model system for differentiation (21Alonso A. Breuer B. Steuer B. Fischer J. Int. J. Dev. Biol. 1991; 35: 389-397PubMed Google Scholar). Table 1 is a partial list of the genes most highly induced by the combined ectopic expression of PGC-1α and HNF4α as determined by microarray analysis. Apolipoproteins C-II and A-IV are highly induced by the combination of HNF4α and PGC-1α, whereas either molecule alone does not induce them to nearly the same extent. Other apolipoproteins detected by the microarray are not significantly increased by these two molecules (supplemental Table S1).TABLE 1Highest scoring targets of AdPGC1α + AdHNF4α co-expressionFold expression over GFPGFP alonePGC-1α aloneHNF4α alonePGC-1α + HNF4αApolipoprotein C-II1118.51810.56 Aminoacylase 1131.2182.36674.17 RIKEN cDNA 4932432N11 gene136.61270.45 Purinergic receptor P2X, ligand-gated ion channel, 311201.21229.43Apolipoprotein A-IV111156.09 Retinol dehydrogenase 7112.35118.08 C-type lectin-related f14.022.11105.13 PGC-1α143.08NA94.08 G0/G1 switch gene 21NA5.8571.01 HNF4α14.7567.170.52 Open table in a new tab To confirm the results of the microarray analysis, we analyzed the RNA from the F9 cells with quantitative PCR. As shown in Fig. 1, apoC-II and apoA-IV are highly induced by PGC-1α and HNF4α, much more so than by either protein alone. Interestingly, apoA-I and apoC-III are also significantly induced by PGC-1α and HNF4α in this context, despite their relatively low expression in the arrays. HNF4α activates the apoB promoter (22Metzger S. Halaas J.L. Breslow J.L. Sladek F.M. J. Biol. Chem. 1993; 268: 16831-16838Abstract Full Text PDF PubMed Google Scholar), and although there is a 20-fold induction of apoB by HNF4α alone, PGC-1α only further augments this by a modest 2-fold. The expression of apoE, another target of HNF4α (23Dang Q. Walker D. Taylor S. Allan C. Chin P. Fan J. Taylor J. J. Biol. Chem. 1995; 270: 22577-22585Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), is not increased in the presence of ectopic PGC-1α and HNF4α. Likewise, although the epithelial marker E-cadherin is expressed in response to forced HNF4α expression in fibroblasts (14Spath G.F. Weiss M.C. J. Cell Biol. 1998; 140: 935-946Crossref PubMed Scopus (133) Google Scholar), it fails to be activated in these cells by the co-expression of PGC-1α and HNF4α. In summary, PGC-1α robustly coactivates a subset, but not all, of the downstream targets of HNF4α.Apolipoprotein biosynthesis is most abundant in the liver in vivo. To examine the effects of PGC-1α and HNF4α in cells derived from liver, we exogenously expressed the two molecules in H2.35 mouse hepatoma cells through adenoviral transduction and subsequently harvested RNA. Northern analysis in Fig. 2A shows that the mRNAs for apolipoproteins A-IV, C-II, and C-III are induced to detectable levels only in the presence of both PGC-1α and HNF4α. ApoA-IV and apoC-II are expressed 11-fold over their levels with HNF4α alone, whereas apoC-III is increased 14-fold as measured by quantitative PCR (supplemental Table S2). Unlike its effect in F9 cells, ectopic HNF4α in H2.35 cells is, alone, sufficient to increase apoE levels. However, the addition of PGC-1α does not significantly enhance this induction. Likewise, HNF4α alone is able to induce apoB by 18-fold, and PGC-1α does not further increase its levels (supplemental Table S2). PGC-1β, a closely related homolog of PGC-1α that has been shown to regulate hepatic lipid synthesis and export (24Lin J. Yang R. Tarr P.T. Wu P.H. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. Newgard C.B. Spiegelman B.M. Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), fails to induce these same genes. This is most likely due, at least in part, to the fact that PGC-1β coactivates HNF4α poorly (25Lin J. Tarr P.T. Yang R. Rhee J. Puigserver P. Newgard C.B. Spiegelman B.M. J. Biol. Chem. 2003; 278: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar).FIGURE 2The ectopic combination of PGC-1α and HNF4α induces apolipoproteins in hepatoma cells. A, H2.35 mouse hepatoma cells were infected with the indicated adenoviruses, and their RNA was analyzed by Northern blotting with radiolabeled mouse cDNA probes. 36B4 was blotted as a loading control. B, HepG2 human hepatoma cells were infected with the indicated adenoviruses, and their RNA was analyzed by Northern blotting with radiolabeled human cDNA probes. 36B4 was blotted as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether PGC-1α-mediated induction of apolipoproteins occurs within human cells as well as those from mouse, we expressed PGC-1α in HepG2 hepatoma cells. As in the H2.35 cells, PGC-1α is able to induce the same subset of apolipoproteins (Fig. 2B). However, in contrast to its expression in H2.35 cells, ectopic PGC-1α in HepG2 cells is alone sufficient to activate expression of these genes. In fact, adenoviral PGC-1α is much more effective in inducing these genes than is adenoviral HNF4α. For example, the level of apoA-IV is more than 30 times higher in the setting of adenoviral PGC-1α than in the presence of adenoviral HNF4α (supplemental Table S3). This is likely the result of a high endogenous level of HNF4α (Fig. 2B) through which the exogenously expressed PGC-1α may act.PGC-1α Coactivates HNF4α on the apoC-III/A-IV Promoter—The apoA-IV and apoC-III genes are separated in the mouse and human genomes by an ∼6-kb intergenic region (26Omori K. Vergnes L. Zakin M.M. Ochoa A. Gene. 1995; 159: 231-234Crossref PubMed Scopus (12) Google Scholar). Because apoA-IV and apoC-III are transcribed in opposite directions, this intergenic region serves as a common 5′-flanking sequence for both genes (27Vergnes L. Taniguchi T. Omori K. Zakin M.M. Ochoa A. Biochim. Biophys. Acta. 1997; 1348: 299-310Crossref PubMed Scopus (35) Google Scholar). To test whether this region is responsive to PGC-1α coactivation of HNF4α, we transfected plasmids encoding these two factors along with an apoC-III/A-IV promoter luciferase construct into H2.35 hepatoma cells. Recent studies in intestinal epithelial cells report that PGC-1α coactivates estrogen-related receptor α on this promoter construct (28Carrier J.C. Deblois G. Champigny C. Levy E. Giguere V. J. Biol. Chem. 2004; 279: 52052-52058Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Fig. 3A shows that PGC-1α robustly coactivates HNF4α on this reporter to nearly 400-fold over base line. No significant activation occurs when PGC-1α is co-expressed with other liver transcription factors, including a constitutively active allele of FOXO1, glucocorticoid receptor (GR), PPARα, and PPARγ (Fig. 3A). Previous reports have shown that PGC-1α coactivates these factors in other contexts (1Puigserver 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 (3030) Google Scholar, 11Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1154) Google Scholar, 29Puigserver P. Adelmant G. Wu Z. Fan M. Xu J. O'Malley B. Spiegelman B.M. Science. 1999; 286: 1368-1371Crossref PubMed Scopus (492) Google Scholar).FIGURE 3PGC-1α coactivates and is recruited to the apoC-III/A-IV promoter region. A, H2.35 cells were transfected with an apoC-III/A-IV luciferase reporter and plasmids encoding the transcription factors estrogen-related receptor α (ERRα), FOXO1, GR, HNF4α, PPARα, or PPARγ. Reporter activity was measured for each of these transcription factors in the absence and presence of PGC-1α and depicted as –fold change over the first lane (vector alone). Transfections were repeated at least three times in triplicate. B, a diagram of the apoC-III/A-IV intergenic region. The full-length mouse promoter is 6 kb long, and two previously identified HNF4α response elements are depicted. Conservation of these sites between human and mouse genomes is also shown. C, H2.35 cells were transfected with plasmids expressing GFP, FLAG epitope-tagged PGC-1α, or a combination of HNF4α and tagged PGC-1α. Following formaldehyde cross-linking, chromatin immunoprecipitation assays were performed using anti-FLAG resin. Co-immunoprecipitated DNA and 5% of input DNA were used as template for PCR primers flanking site 1, site 2, and a control region on chromosome 9 downstream of the apoA-IV gene. All PCR reactions were carried out to 23 cycles. D, H2.35 cells were transfected with PGC-1α, HNF4α, and either a wild-type or mutant apoC-III/A-IV luciferase reporter. HNF4α-binding sites 1 and 2 were mutated as described under “Experimental Procedures.” PGC-1α coactivation of HNF4α was measured on each of the different reporters and depicted as –fold change over the first lane (PGC-1α alone). Transfections were repeated at least three times in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next identified the HNF4α response elements through which PGC-1α exerts its effects. Fig. 3B shows two previously characterized HNF4α-binding sites, labeled sites 1 and 2, located in the proximal promoters of apoC-III and apoA-IV, respectively (30Lavrentiadou S.N. Hadzopoulou-Cladaras M. Kardassis D. Zannis V.I. Biochemistry. 1999; 38: 964-975Crossref PubMed Scopus (48) Google Scholar, 31Ktistaki E. Lacorte J.M. Katrakili N. Zannis V.I. Talianidis I. Nucleic Acids Res. 1994; 22: 4689-4696Crossref PubMed Scopus (67) Google Scholar). These two direct repeats (DR-1) were also identified by NUBIScan, a computer algorithm that predicts DNA recognition sites for nuclear receptors (32Podvinec M. Kaufmann M.R. Handschin C. Meyer U.A. Mol. Endocrinol. 2002; 16: 1269-1279Crossref PubMed Scopus (152) Google Scholar). Site 1 is a DR-1 that is 100% conserved between mouse and human. Chromatin immunoprecipitations were performed with antibodies against FLAG epitope-tagged PGC-1α, expressed alone or with HNF4α in H2.35 hepatoma cells. Only the immunoprecipitates containing cross-linked complexes of PGC-1α and HNF4α with genomic DNA are enriched with fragments encompassing site 1 and site 2, indicating that PGC-1α is recruited to both of these sites through HNF4α (Fig. 3C). Primers that amplify a control region on chromosome 9 downstream from the apoC-III/apoA-IV gene cluster show no PGC-1α-specific enrichment.To test the functional significance of these two putative HNF4α-response elements, we disrupted one of the two half-sites within each DR-1 through site-directed mutagenesis. Whereas mutation of site 2 leaves PGC-1α coactivation completely intact, mutation of site 1 utterly abolishes the PGC-1α effect (Fig. 3D). The absolute loss of response to PGC-1α when site 1 is mutated is observed in both the full-length reporter as well as in a truncated version containing 700 bp of apoC-III proximal promoter (data not shown). Taken together, these data show that PGC-1α directly coactivates HNF4α on the apolipoprotein C-III/A-IV intergenic region through an HNF4α-responsive element.Apolipoproteins A-IV and C-II Are Induced in Fasted Liver—We next investigated the physiological significance of PGC-1α-mediated induction of apolipoproteins. Because PGC-1α is an integral part of the hepatic fasting response, we looked at the expression of apolipoproteins in fasted mice. Previous reports demonstrate mild increases in apoA-I during fasting and more robust increases in apoA-IV (33LeBoeuf R.C. Caldwell M. Kirk E. J. Lipid Res. 1994; 35: 121-133Abstract Full Text PDF PubMed Google Scholar). Northern blotting in Fig. 4 shows that the genes for PGC-1α and apolipoproteins A-IV and C-II are indeed induced under 6- and 24-h food deprivation. Whereas apoC-II is increased roughly 2-fold at the 24-h time point, apoA-IV is induced over 70-fold as assessed by quantitative PCR (supplemental Fig. S1). ApoA-IV has been closely linked to triglyceride metabolism, and apoC-II is a requisite cofactor for lipoprotein lipase activity and uptake of free fatty acids into extrahepatic tissues. Given the importance of apolipoproteins in lipid metabolism and their regulation by PGC-1α in vitro, we next investigated the role of PGC-1α in the regulation of triglyceride levels in vivo.FIGURE 4Fasting induces hepatic mRNA for PGC-1α, apoA-IV, and apoC-II. Mice were fasted for 6 and 24 h and sacrificed or refed for another 24 h and then sacrificed. Liver mRNA was analyzed for PGC-1α and apolipoprotein transcript levels via Northern blotting using radiolabeled mouse cDNA probes. 36B4 was blotted as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hepatic PGC-1α Levels Influence Serum and VLDL Triglyceri" @default.
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