Modulation of PGC-1 Coactivator Pathways in Brown Fat Differentiation through LRP130
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m805431200
ISSN1083-351X
AutoresMarcus P. Cooper, Marc Uldry, Shingo Kajimura, Zoltàn Arany, Bruce M. Spiegelman,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe PGC-1 coactivators are important regulators of oxidative metabolism. We previously demonstrated that LRP130 is a binding partner of PGC-1α, required for hepatic gluconeogenesis. LRP130 is the gene mutated in Leigh syndrome French Canadian variant, a rare neurodegenerative disease. The importance of LRP130 in other, non-hepatocyte biology remains obscure. To better understand PGC-1 coactivator function in brown fat development, we explored the metabolic role of LRP130 in brown adipocyte differentiation. We show that LRP130 is preferentially enriched in brown fat compared with white, and induced in a PGC-1-dependent manner during differentiation. Despite intact PGC-1 coactivator expression, brown fat cells deficient for LRP130 exhibit attenuated expression of several genes characteristic of brown fat, including uncoupling protein 1. Oxygen consumption studies support a specific defect in proton leak due to attenuated uncoupling protein 1 expression. Notably, brown fat cell development common to both PGC-1 coactivators is governed by LRP130. Conversely, the cAMP response controlled by PGC-1α is not regulated by LRP130. These data implicate LRP130 in brown fat cell development and differentiation. The PGC-1 coactivators are important regulators of oxidative metabolism. We previously demonstrated that LRP130 is a binding partner of PGC-1α, required for hepatic gluconeogenesis. LRP130 is the gene mutated in Leigh syndrome French Canadian variant, a rare neurodegenerative disease. The importance of LRP130 in other, non-hepatocyte biology remains obscure. To better understand PGC-1 coactivator function in brown fat development, we explored the metabolic role of LRP130 in brown adipocyte differentiation. We show that LRP130 is preferentially enriched in brown fat compared with white, and induced in a PGC-1-dependent manner during differentiation. Despite intact PGC-1 coactivator expression, brown fat cells deficient for LRP130 exhibit attenuated expression of several genes characteristic of brown fat, including uncoupling protein 1. Oxygen consumption studies support a specific defect in proton leak due to attenuated uncoupling protein 1 expression. Notably, brown fat cell development common to both PGC-1 coactivators is governed by LRP130. Conversely, the cAMP response controlled by PGC-1α is not regulated by LRP130. These data implicate LRP130 in brown fat cell development and differentiation. Obesity is a global health concern, predisposing to insulin resistance, hypertension, dyslipidemia, type 2 diabetes, and certain cancers. Its pathogenesis is multifactorial, involving neurohormonal, behavioral, genetic, and adipocyte-specific elements. Integration of these factors establishes whole body energy balance.Adipocytes constitute important regulators of systemic energy balance, and play a central role in obesity due to their inherent capacity to store or consume excess calories. The duality of the adipocyte lineage is illustrated in two different types of fat cells. White adipocytes store fat as triacylglycerol during caloric excess and release free fatty acids during fasting. In contrast, brown adipocytes oxidize triacylglycerol to generate heat (thermogenesis) (1Hansen J.B. Kristiansen K. Biochem. J. 2006; 398: 153-168Crossref PubMed Scopus (137) Google Scholar, 2Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4423) Google Scholar). Besides muscular shivering (2Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4423) Google Scholar, 3Hull D. Segall M.M. J. Physiol. 1965; 181: 468-477Crossref PubMed Scopus (23) Google Scholar, 4Simon H.E. Am. J. 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J. 2006; 398: 153-168Crossref PubMed Scopus (137) Google Scholar, 2Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4423) Google Scholar). Transcription factors involved in brown fat cell regulation include PPARγ2 (17Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1627) Google Scholar, 18Tai T.A. Jennermann C. Brown K.K. Oliver B.B. MacGinnitie M.A. Wilkison W.O. Brown H.R. Lehmann J.M. Kliewer S.A. Morris D.C. Graves R.A. J. Biol. Chem. 1996; 271: 29909-29914Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), PPARα (19Barbera M.J. Schluter A. Pedraza N. Iglesias R. Villarroya F. Giralt M. J. Biol. Chem. 2001; 276: 1486-1493Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), thyroid receptor, ATF-2, FoxC2 (20Cederberg A. Gronning L.M. Ahren B. Tasken K. Carlsson P. Enerback S. 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The PGC-1 family of coactivators regulate Ucp1 gene expression and several brown fat selective genes during brown fat development, and following cold exposure or treatment with cAMP mimetic. PGC-1 (PPARγ coactivator-1α) is the founding member of the PGC-1 family and was isolated from a brown fat library to identify PPARγ-interacting proteins (24Puigserver 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). PGC-1β is the closest homolog of PGC-1α (25Lin J. Puigserver P. Donovan J. Tarr P. Spiegelman B.M. J. Biol. Chem. 2002; 277: 1645-1648Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) with PRC being more distally related (26Andersson U. Scarpulla R.C. Mol. Cell. Biol. 2001; 21: 3738-3749Crossref PubMed Scopus (292) Google Scholar). PGC-1α and PGC-1β exhibit complementary function during brown fat development (27Uldry M. Yang W. St Pierre J. Lin J. Seale P. Spiegelman B.M. Cell Metab. 2006; 3: 333-341Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). Complementation is shared at the level of several brown fat specific genes, including Ucp1, as well as, mitochondrial biogenesis. Either member of the family is singly sufficient to establish basal Ucp1 gene expression in cells and animals (27Uldry M. Yang W. St Pierre J. Lin J. Seale P. Spiegelman B.M. Cell Metab. 2006; 3: 333-341Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 28Lin 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 (985) Google Scholar, 29Leone 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. 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Identification of such a factor would help delineate developmental control from the environmental control, and shed insight on how the PGC-1s preferentially influence one program over another.We previously demonstrated that LRP130 (leucine-rich protein 130), also known as LRPPRC (leucine-rich protein pentatricopeptide repeat-containing motif), is a component of the PGC-1α holo-complex (32Cooper M.P. Qu L. Rohas L.M. Lin J. Yang W. Erdjument-Bromage H. Tempst P. Spiegelman B.M. Genes Dev. 2006; 20: 2996-3009Crossref PubMed Scopus (85) Google Scholar). LRP130 is mutated in a rare neurological disorder called Leigh Syndrome French Canadian variant (33Mootha V.K. Lepage P. Miller K. Bunkenborg J. Reich M. Hjerrild M. Delmonte T. Villeneuve A. Sladek R. Xu F. Mitchell G.A. Morin C. Mann M. Hudson T.J. Robinson B. Rioux J.D. Lander E.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 605-610Crossref PubMed Scopus (466) Google Scholar). Patients suffer a devastating neurological disease as well as liver dysfunction (34Morin C. Mitchell G. Larochelle J. Lambert M. Ogier H. Robinson B.H. De Braekeleer M. Am. J. Hum. Genet. 1993; 53: 488-496PubMed Google Scholar). LRP130 localizes to the nucleus and mitochondria, and has been shown to influence gene expression in both compartments (32Cooper M.P. Qu L. Rohas L.M. Lin J. Yang W. Erdjument-Bromage H. Tempst P. Spiegelman B.M. Genes Dev. 2006; 20: 2996-3009Crossref PubMed Scopus (85) Google Scholar, 35Xu F. Morin C. Mitchell G. Ackerley C. Robinson B.H. Biochem. J. 2004; 382: 331-336Crossref PubMed Scopus (138) Google Scholar, 36Tsuchiya N. Fukuda H. Nakashima K. Nagao M. Sugimura T. Nakagama H. Biochem. Biophys. Res. Commun. 2004; 317: 736-743Crossref PubMed Scopus (34) Google Scholar). We recently demonstrated that LRP130 is an important coregulator of PGC-1α-dependent gluconeogenesis. Although LRP130 was shown to interact with PGC-1β (32Cooper M.P. Qu L. Rohas L.M. Lin J. Yang W. Erdjument-Bromage H. Tempst P. Spiegelman B.M. Genes Dev. 2006; 20: 2996-3009Crossref PubMed Scopus (85) Google Scholar), the functional importance of interaction with this PGC-1 member was not studied. We show here that LRP130 is a novel and important regulator of brown fat development. Notably it regulates the developmental program mediated by the PGC-1 coactivators, but does not influence the cAMP response. These results suggest that LRP130 is critical for the complementary actions of the PGC-1s during brown fat development.EXPERIMENTAL PROCEDURESCell Culture and Adipocyte Differentiation—We digested brown fat from wild-type and PGC-1α KO newborn mice (28Lin 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 (985) Google Scholar) with collagenase to isolate brown fat preadipocytes (37Fasshauer M. Klein J. Kriauciunas K.M. Ueki K. Benito M. Kahn C.R. Mol. Cell. Biol. 2001; 21: 319-329Crossref PubMed Scopus (148) Google Scholar, 38Klein J. Fasshauer M. Klein H.H. Benito M. Kahn C.R. Bioessays. 2002; 24: 382-388Crossref PubMed Scopus (102) Google Scholar, 39Tseng Y.H. Kriauciunas K.M. Kokkotou E. Kahn C.R. Mol. Cell. Biol. 2004; 24: 1918-1929Crossref PubMed Scopus (149) Google Scholar). The cells were immortalized by pBABE SV40 T retroviral infection, and selected in 2 μg/ml puromycin. Maintenance media contained 20% FBS, Dulbecco's modified Eagle's medium, 20 mm HEPES pH 7.55 (Invitrogen). Differentiation day 0: cells grown to 100% confluence were treated with 20 nm insulin, 1 nm T3, 500 nm dexamethasone, 125 μm indomethacin, 500 μm isobutylmethylxanthine in 10% FBS. Differentiation day 2: medium was changed to 20 nm insulin, 1 nm T3 in 10% FBS, and replenished every 48 h. Full differentiation happened between days 5 and 8. Treatment with 10 μm forskolin activated the thermogenic program. Stimulation for 4 h induced Ucp1 and PGC-1α, mitochondrial-encoded genes required 24 h.We induced 3T3 L1 adipocyte differentiation as previously described (40Green H. Meuth M. Cell. 1974; 3: 127-133Abstract Full Text PDF PubMed Scopus (809) Google Scholar, 41Spiegelman B.M. Frank M. Green H. J. Biol. Chem. 1983; 258: 10083-10089Abstract Full Text PDF PubMed Google Scholar). Differentiation day 0: cells grown to 100% confluence were treated with 20 nm insulin, 1000 nm dexamethasone, 250 μm isobutylmethylxanthine in 10% FBS. Differentiation day 2: medium was changed to 20 nm insulin, 10% FBS, and replenished every 48 h. Full differentiation happened between days 5 and 7.siLRP130 and LRP130 Stable Cell Lines—We stably expressed shRNA constructs in cell lines referred to as siCtrl and siLRP130. Briefly, we cloned LRP130 RNAi sequence, 5′-GAAGCTAGATGACCTGTTT-3′ (Dharmacon), into pSuperRetro GFP (42Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3945) Google Scholar), and produced retrovirus in phoenix cells. Infected brown preadipocytes were selected by MoFlo cell sorting. We previously demonstrated the specificity of this LRP130 RNAi in several cell types and by comparing it to a separate LRP130 RNAi sequence, 5′-GCAGTTAGGTGTCGTATAT-3′ (32Cooper M.P. Qu L. Rohas L.M. Lin J. Yang W. Erdjument-Bromage H. Tempst P. Spiegelman B.M. Genes Dev. 2006; 20: 2996-3009Crossref PubMed Scopus (85) Google Scholar). A control shRNA cell line was generated for comparison and the sequence has been previously published (43Fan M. Rhee J. St Pierre J. Handschin C. Puigserver P. Lin J. Jaeger S. Erdjument-Bromage H. Tempst P. Spiegelman B.M. Genes Dev. 2004; 18: 278-289Crossref PubMed Scopus (246) Google Scholar). Uldry et al. (27Uldry M. Yang W. St Pierre J. Lin J. Seale P. Spiegelman B.M. Cell Metab. 2006; 3: 333-341Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar) previously described generation of PGC-1 double deficient brown fat cells.3T3-F442A cells were transduced with pMSCV human LRP130 retrovirus. After digestion with BamHI/PmeI, human LRP130-FLAG was cloned into the BglII/HpaI site of pMSCV puromycin. To generate stable human LRP130-FLAG cells, we infected cells with the retrovirus and selected in 2 μg/ml puromycin. Empty pMSCV retrovirus of similar titer was used to create a control cell line. Culture conditions and differentiation were identical to brown fat cells. Retroviral infection occasionally reduced adipogenesis in 3T3-F442A cells; therefore, 1 μm rosiglitazone (Sigma-Aldrich) was used in a few experiments to promote full differentiation. Results were comparable to treatments without rosiglitazone.Animal Experiments—All animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee at the Dana-Farber Cancer Institute. Mice were maintained on standard rodent chow with 12-h light-dark cycles. For acute cold exposure studies, we housed male C57BL/6 mice, ages 3-4 weeks, at 4 °C for 4 h. Brown adipose tissue (BAT) was harvested and mRNA isolated.Immunodetection of Proteins—Whole cell protein lysates were prepared by 3 freeze/thaw cycles in FLAG lysis buffer: 50 mm Tris-Cl, pH 7.8, 137 mm NaCl, 10 mm NaF, 1 mm EDTA, 1% Triton X-100, 10% glycerol, and 0.2% sarkosyl (N-lauryl sarcosine). Samples were clarified at 14,000 × g for 10 min at 4 °C, and quantified using Bradford reagent (44Bearden Jr., J.C. Biochim. Biophys. Acta. 1978; 533: 525-529Crossref PubMed Scopus (393) Google Scholar). Rabbit polyclonal antibody raised against the C terminus of LRP130 was generated by PrimmBiotech Inc. We detected PGC-1β with affinity purified rabbit polyclonal antibody. Antibodies against, C-V-α and NDUFA9, were purchased from Mitosciences. FLAG M2 monoclonal antibody and Ucp1 antibody are available from Sigma-Aldrich, and tubulin antibody from Abcam. We probed with FLAG antibody per the manufacturer's protocol. All other antibodies were used at 1:1000 in TBS/0.1% Tween-20, 5% nonfat dry milk.Electron Microscopy and Stereological Measurements—Samples were fixed, sectioned (∼60 nm), and visualized on a JEOL 1200EX (27Uldry M. Yang W. St Pierre J. Lin J. Seale P. Spiegelman B.M. Cell Metab. 2006; 3: 333-341Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). We laid a grid onto randomly selected micrographs (n > 30), and counted points falling onto mitochondria or cell area. The fraction of total points attributable to mitochondria was expressed as mitochondrial density (45St Pierre J. Lin J. Krauss S. Tarr P.T. Yang R. Newgard C.B. Spiegelman B.M. J. Biol. Chem. 2003; 278: 26597-26603Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar).Reporter Assays—After stable transduction with pMSCV control or hLRP130, 3T3-F442A cells were transiently transfected in 6-well dishes using Fugene (Roche Applied Sciences). We used 500 ng of the -4kbto1+ bp Ucp1 promoter fused to a luciferase reporter (46Cao W. Daniel K.W. Robidoux J. Puigserver P. Medvedev A.V. Bai X. Floering L.M. Spiegelman B.M. Collins S. Mol. Cell. Biol. 2004; 24: 3057-3067Crossref PubMed Scopus (421) Google Scholar), and 1500 ng of RSV-β-galactosidase to normalize each transfection. To exclude nonspecific activation, we used empty reporter vector. Reporter activity was measured on days 2, 4, and 8 of differentiation.Chromatin Immunoprecipitation—Mature brown fat cells grown in a 15-cm dish were used for 4 chromatin immunoprecipitation reactions. The reactions were processed using the EZ ChIP kit from Upstate. PPARγ (H-100 X), FLAG M2, mouse, and rabbit IgG were purchased from Sigma. For each reaction, we used either 6 μg of IgG, 3 μg of PPARγ, or 3 μg of FLAG antibody. ChIP experiments were performed three times; one representative experiment is shown. The primers for the promoters were: Ucp1 enhancer, forward 5′-TGAGGCTGATATCCCCAGAGA-3′, reverse 5′-TCTGTGTGTCCTCTGGGCATAA-3′; Ucp1-200 bp promoter, forward 5′-AATCAGGAACTGGTGCCAAATC-3′, reverse 5′-AGGTCTCCAAAGAGCTGCTAGTG-3′; aP2 (FABP4) DR-1 sites -4 kb promoter forward 5′-TTCCCAGCAGGAATCAGGTAG-3′, reverse 5′-CTGGGAACTCCATTTGCTCTC-3′; 18S gene, forward 5′-AGTCCCTGCCCTTTGTACACA-3′, reverse 5′-CGATCCGAGGGCCTCACTA-3′.Oxygen Consumption—Cells in suspension were washed twice in 10% FBS, Dulbecco's modified Eagle's medium, and resuspended in respiration mediuim: DPBS, 2 mm glucose, 1 mm pyruvate, 2% bovine serum albumin. Total respiration and proton leak ("uncouple respiration") were measured as previously described (45St Pierre J. Lin J. Krauss S. Tarr P.T. Yang R. Newgard C.B. Spiegelman B.M. J. Biol. Chem. 2003; 278: 26597-26603Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 47Mootha V.K. Handschin C. Arlow D. Xie X. St Pierre J. Sihag S. Yang W. Altshuler D. Puigserver P. Patterson N. Willy P.J. Schulman I.G. Heyman R.A. Lander E.S. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6570-6575Crossref PubMed Scopus (547) Google Scholar). We normalized respiration rates to an aliquot of total cell protein lysate.RNA Analysis—For Northern blots, total RNA was isolated using Trizol (Invitrogen). 10-20 μg was resolved on a formaldehyde gel, transferred to nylon membrane, and hybridized with gene-specific probes. We used a 1430-2523 bp probe for LRP130, 1-810 bp for PGC-1α, and 1500-2400 bp for PGC-1β.For quantitative PCR, total RNA was isolated with Trizol, and further purified with RNAesay columns (Qiagen). We reverse-transcribed RNA with a kit (Applied Biosystems), and measured signal intensity using SyberGreen (Applied Biosystems). Samples were normalized to TBP using the ΔΔCt method, and expressed as relative gene expression. Primer sequences are shown in supplemental Fig. S4.Statistical Analysis—For bar graph analyses, we used two-way analysis of variance with Bonferroni post-test. One asterisk denotes p < 0.05, two: p < 0.01, three: p < 0.001.RESULTSLRP130 Is Expressed in a Brown Fat-selective Manner—Certain regulators of brown fat development are selectively expressed in brown fat, and their expression may be further driven by differentiation or cold exposure. LRP130 mRNA is enriched in brown fat, compared with white. Its distribution across tissues is similar to PGC-1α and PGC-1β (Fig. 1A). LRP130 gene expression increases ∼5-fold during differentiation of immortalized brown fat cells, but does not change during differentiation of immortalized 3T3-L1 white fat cells (Fig. 1B). Upon cold exposure, however, LRP130 gene expression remains unchanged in brown fat (Fig. 1C). Notably Ucp1, PGC-1α and deiodinase 2 are all induced as expected (Fig. 1D). In parallel, stimulation of mature brown fat cells with forskolin does not induce LRP130 gene expression (data not shown).Effect of LRP130 Depletion on Adipogenesis and the Brown Fat Program—To assess the role of LRP130 in brown fat cell differentiation, we generated brown fat cells deficient for LRP130, using retroviral delivery of shRNA (Fig. 2A). To confirm that the shRNA against LRP130 is functional, we assessed protein level (Fig. 2B), and a target gene, Cox3 (Fig. 2A). Two genes, FABP4 (aP2) and Glut4, are induced during differentiation of brown or white fat cells. Expression of these genes were unaffected by depletion of LRP130, suggesting that adipogenesis is intact. Additionally, microscopic examination revealed no gross morphological defects (Fig. 2C), indicating LRP130 is not required for lipid accumulation.FIGURE 2Adipogenic and morphological features of brown fat cells deficient for LRP130.A, gene expression of select genes at day 7 of differentiation (n = 3). B, protein expression of LRP130 in stably depleted brown fat cells at day 7 differentiation. A detailed gene expression profile for RNAi no. 2 is in supplemental Fig. S1. C, phase contrast of cells stably transduced with control shRNA (siControl) or shRNA against LRP130 (siLRP130). Error bars represent mean (±S.E.). *. p < 0.05; **, p < 0.01; ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next we evaluated the capacity of LRP130-deficient cells to regulate the brown adipocyte phenotype. As shown in Fig. 3A, several brown fat genes regulated by the PGC-1 coactivators are attenuated in LRP130-deficient cells, Ucp1, Cidea, Cox7a1, but not Elovl3. This is very similar to the pattern observed in PGC-1 double-deficient cells (Fig. 3B). Although moderately blunted in PGC-1 double-deficient cells, mRNA expression of mitochondrial transcription factor A (TFAM) is unaffected in LRP130-deficient cells (Fig. 3C). Similarly, the gene expression of cytochrome c (cycs), a component of the electron transport chain and marker of mitochondrial biogenesis, was unaffected by LRP130 depletion. Unlike cyctochrome c, Cox7a1 is a component of the electron transport chain that is also a brown fat-specific gene (21Seale P. Kajimura S. Yang W. Chin S. Rohas L.M. Uldry M. Tavernier G. Langin D. Spiegelman B.M. Cell Metab. 2007; 6: 38-54Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar). Its gene expression is attenuated by LRP130 depletion, highlighting the importance of LRP130 in the brown fat program. Similar genetic findings using a second RNAi against LRP130 were observed (supplemental Fig. S1).FIGURE 3LRP130 phenocopies certain features of PGC-1 double-deficient brown fat cells. Designated αKO + siβ, PGC-1-deficient cells are PGC-1α-null cells with stable shPGC-1β knock-down. Designated siCtrl, the control cells contain a control shRNA. Gene expression profile of brown fat genes and mitochondrial genes in LRP130-deficient (A and C) versus PGC-1 double-deficient mature brown fat cells (B and D) (n = 3). In comparison to siCtrl cell, PGC-1α-null cells stably expressing a control shRNA showed no difference (data not shown). Error bars represent mean (±S.E.). *, p < 0.05; **, p < 0.01; ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Prior studies have demonstrated that cAMP treatment or cold exposure induces PGC-1α, which then drives the expression of Ucp1 and deiodinase 2. To determine the effect of LRP130 on this program, we stimulated mature brown fat cells deficient for LRP130 with forskolin, an adenylate cyclase agonist. As shown in Fig. 4A, absolute expression of Ucp1 is reduced ∼70% in untreated and cAMP-treated LRP130-deficient cells; however, the fold change from baseline is identical to control cells, about 20-25-fold. Fold change and absolute level of PGC-1α was similar between both groups. Longer treatments with cyclic adenylate agonist induce several mitochondrial genes. As anticipated, cytochrome c (cycs), a nuclear encoded mitochondrial gene, is unaffected, but the mitochondrial-encoded genes are attenuated in LRP130-deficient cells (Fig. 4B). Although not required for Ucp1 induction following cAMP stimulation, LPR130 is required for the induction of the mitochondrial-encoded genes.FIGURE 4Depletion of LRP130 does not alter cAMP mediated induction of Ucp1.A, gene expression of Ucp1 and PGC-1α in mature brown fat cells treated with forskolin for 4 h. Untreated siCtrl cells were statistically compared with untreated siLRP130 cells. cAMP-treated siCtrl cells were statistically compared with cAMP-treated siLRP130 cells (n = 4). B, gene expression of several mitochondrial-encoded genes, and nuclear-encoded cytochrome c (Cycs). 24 h of cAMP treatment proved optimal for inducing these genes (n = 4). Untreated siCtrl cells were statisticall
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