NT-PGC-1α deficiency decreases mitochondrial FA oxidation in brown adipose tissue and alters substrate utilization in vivo
2018; Elsevier BV; Volume: 59; Issue: 9 Linguagem: Inglês
10.1194/jlr.m085647
ISSN1539-7262
AutoresJi-Hyun Kim, Min Sung Park, Kyoung‐Soo Ha, Chulhong Park, Jisu Lee, Randall L. Mynatt, Ji Suk Chang,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoTranscriptional coactivator PPAR γ coactivator (PGC)-1α and its splice variant N-terminal (NT)-PGC-1α mediate transcriptional regulation of brown adipose tissue (BAT) thermogenesis in response to changes in ambient temperature. PGC-1α is dispensable for cold-induced BAT thermogenesis as long as NT-PGC-1α is present. However, the functional significance of NT-PGC-1α in BAT has not been determined. In the present study, we generated NT-PGC-1α−/− mice to investigate the effect of NT-PGC-1α deficiency on adaptive BAT thermogenesis. At thermoneutrality, NT-PGC-1α−/− mice exhibited abnormal BAT phenotype with increased accumulation of large lipid droplets concomitant with marked downregulation of FA oxidation (FAO)-related genes. Consistent with transcriptional changes, mitochondrial FAO was significantly diminished in NT-PGC-1α−/− BAT. This alteration, in turn, enhanced glucose utilization within the NT-PGC-1α−/− BAT mitochondria. In line with this, NT-PGC-1α−/− mice had higher reliance on carbohydrates. In response to cold or β3-adrenergic receptor agonist, NT-PGC-1α−/− mice transiently exhibited lower thermogenesis but reached similar thermogenic capacities as their WT littermates. Collectively, these findings demonstrate that NT-PGC-1α is an important contributor to the maintenance of FAO capacity in BAT at thermoneutrality and provide deeper insights into the relative contributions of PGC-1α and NT-PGC-1α to temperature-regulated BAT remodeling. Transcriptional coactivator PPAR γ coactivator (PGC)-1α and its splice variant N-terminal (NT)-PGC-1α mediate transcriptional regulation of brown adipose tissue (BAT) thermogenesis in response to changes in ambient temperature. PGC-1α is dispensable for cold-induced BAT thermogenesis as long as NT-PGC-1α is present. However, the functional significance of NT-PGC-1α in BAT has not been determined. In the present study, we generated NT-PGC-1α−/− mice to investigate the effect of NT-PGC-1α deficiency on adaptive BAT thermogenesis. At thermoneutrality, NT-PGC-1α−/− mice exhibited abnormal BAT phenotype with increased accumulation of large lipid droplets concomitant with marked downregulation of FA oxidation (FAO)-related genes. Consistent with transcriptional changes, mitochondrial FAO was significantly diminished in NT-PGC-1α−/− BAT. This alteration, in turn, enhanced glucose utilization within the NT-PGC-1α−/− BAT mitochondria. In line with this, NT-PGC-1α−/− mice had higher reliance on carbohydrates. In response to cold or β3-adrenergic receptor agonist, NT-PGC-1α−/− mice transiently exhibited lower thermogenesis but reached similar thermogenic capacities as their WT littermates. Collectively, these findings demonstrate that NT-PGC-1α is an important contributor to the maintenance of FAO capacity in BAT at thermoneutrality and provide deeper insights into the relative contributions of PGC-1α and NT-PGC-1α to temperature-regulated BAT remodeling. Brown adipose tissue (BAT) is specialized in dissipating energy in the form of heat. This process, known as nonshivering thermogenesis, requires an abundant fuel supply, a high number of mitochondria, and high levels of uncoupling protein 1 (UCP1), a BAT-specific transport protein located in the inner mitochondrial membrane (IMM) (1.Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4521) Google Scholar). UCP1 uncouples fuel-driven proton gradient from ATP synthesis and promotes proton leak across the IMM, thus resulting in heat production (2.Nedergaard J. Golozoubova V. Matthias A. Asadi A. Jacobsson A. Cannon B. UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency.Biochim. Biophys. Acta. 2001; 1504: 82-106Crossref PubMed Scopus (465) Google Scholar, 3.Golozoubova V. Hohtola E. Matthias A. Jacobsson A. Cannon B. Nedergaard J. Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold.FASEB J. 2001; 15: 2048-2050Crossref PubMed Scopus (358) Google Scholar). BAT not only allows rodents to maintain body temperature at temperatures below the thermoneutral zone, but also plays a role in whole-body energy balance (4.Rothwell N.J. Stock M.J. A role for brown adipose tissue in diet-induced thermogenesis.Nature. 1979; 281: 31-35Crossref PubMed Scopus (1197) Google Scholar, 5.Himms-Hagen J. Brown adipose tissue thermogenesis: interdisciplinary studies.FASEB J. 1990; 4: 2890-2898Crossref PubMed Scopus (415) Google Scholar). Adult humans also have functional BAT depots that contribute to metabolic homeostasis (6.Ouellet V. Labbe S.M. 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Lewin D.A. Forrest W. Adams S.H. Cold elicits the simultaneous induction of fatty acid synthesis and beta-oxidation in murine brown adipose tissue: prediction from differential gene expression and confirmation in vivo.FASEB J. 2002; 16: 155-168Crossref PubMed Scopus (159) Google Scholar, 9.Mottillo E.P. Balasubramanian P. Lee Y.H. Weng C. Kershaw E.E. Granneman J.G. Coupling of lipolysis and de novo lipogenesis in brown, beige, and white adipose tissues during chronic beta3-adrenergic receptor activation.J. Lipid Res. 2014; 55: 2276-2286Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10.Sanchez-Gurmaches J. Tang Y. Jespersen N.Z. Wallace M. Martinez Calejman C. Gujja S. Li H. Edwards Y.J.K. Wolfrum C. 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At thermoneutral temperature (28–32°C), where heat-producing demands are minimal, the aforementioned BAT thermogenic program is inactivated (1.Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4521) Google Scholar). Temperature-sensitive transcriptional coactivator PPAR γ coactivator 1-α (PGC-1α) is a master regulator of adaptive BAT thermogenesis (17.Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3070) Google Scholar, 18.Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. et al.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3176) Google Scholar). PGC-1α is upregulated by cold/β3-AR stimulation and activates a number of transcription factors, leading to increased expression of UCP1 and many mitochondrial genes involved in mitochondrial biogenesis and oxidative metabolism (17.Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3070) Google Scholar, 18.Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. et al.Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3176) Google Scholar). Previously, we showed that alternative splicing of the PGC-1α gene produces a shorter but functional isoform of PGC-1α [N-terminal (NT)-PGC-1α] in rodents and humans (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Mice lacking both PGC-1α and NT-PGC-1α are unable to produce heat from BAT due to impaired induction of UCP1 and mitochondrial gene expression in response to cold (20.Lin 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. et al.Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice.Cell. 2004; 119: 121-135Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar). In contrast, mice selectively deficient in full-length PGC-1α can maintain normal BAT function and activate cold-induced thermogenesis because NT-PGC-1α upregulates UCP1 and many mitochondrial genes involved in FA transport, β-oxidation, the TCA cycle, and electron transport in the absence of PGC-1α (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 21.Kim J. Fernand V.E. Henagan T.M. Shin J. Huypens P. Newman S. Gettys T.W. Chang J.S. Regulation of brown and white adipocyte transcriptome by the transcriptional coactivator NT-PGC-1alpha.PLoS One. 2016; 11: e0159990PubMed Google Scholar, 22.Jun H.J. Joshi Y. Patil Y. Noland R.C. Chang J.S. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism.Diabetes. 2014; 63: 3615-3625Crossref PubMed Scopus (28) Google Scholar, 23.Chang J.S. Fernand V. Zhang Y. Shin J. Jun H.J. Joshi Y. Gettys T.W. NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis.J. Biol. Chem. 2012; 287: 9100-9111Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The present study aimed at elucidating the functional significance of NT-PGC-1α in adaptive BAT thermogenesis using newly generated NT-PGC-1α−/− mice. By assessing the thermogenic response to warm (28°C) or cold (4°C)/β3-AR agonist, we here demonstrate that NT-PGC-1α is dispensable for mitochondrial biogenesis and respiration, but specifically important for mitochondrial FAO in quiescent BAT and in fully activated BAT where the maximum FA-oxidizing capacity is needed. Taken together with our previous studies, our findings underscore the relative contributions of PGC-1α and NT-PGC-1α to temperature-regulated BAT remodeling. The animal protocol for the study was approved by the Institutional Animal Care and Use Committee at the Pennington Biomedical Research Center, and the procedures were carried out in accordance with the approved guidelines. Mice were housed at a room temperature of 22°C under a 12 h light/12 h dark cycle with ad libitum feeding (standard chow 5001, LabDiet, St. Louis, MO). For cohort 1, 4 week old female mice were kept at 22°C or housed at near thermoneutrality (28°C) for 3 weeks. For cohort 2, female mice housed at 28°C for 3 weeks were singly housed and exposed to 4°C. Core rectal temperature was measured at baseline and every 1 h over the 5 h period of cold exposure. For cohort 3, male mice housed at 28°C were weighed, and their body composition was measured using a Bruker Mouse Minispec (Bruker Optics, Billerica, MA). Mice were then placed in indirect calorimetry chambers (Columbus Instruments, Columbus, OH) and monitored for VO2 and VCO2 for 4 h prior to and after CL316243 injection (1 mg/kg body weight). For the cohort 4, male mice housed at 28°C were determined for body weight and composition and placed in indirect calorimetry chambers. After 3 days in the chambers, mice were intraperitoneally injected with CL316243 (1 mg/kg body weight/day) for 4 days and continuously monitored for VO2 and VCO2. Locomotor activity was measured while the mice were in the chambers. Energy expenditure (EE) was calculated as VO2 × [3.815 + (1.232 × RQ)] × 4.187 and expressed as kilojoules per hour. After removing from indirect calorimetry chambers, mice were injected with CL316243 for an additional 3 days. At the end of experiments (cohorts 1–4), BAT and white adipose tissue (WAT) were collected in the fed state. Gene targeting and chimeric mouse production were performed by the Transgenic Core at Pennington Biomedical Research Center. DNA derived from a C57BL6 genomic BAC library clone (RP24-399J12) was used to generate a targeting vector containing fused exons 6 and 7 and the loxP-neo-loxP. Targeted albino B6 embryonic stem cells carrying the mutant allele were injected into C57BL6 blastocytes, and chimeric mice were mated to C57BL6 mice to obtain heterozygous offspring containing a germline mutant allele. These heterozygotes were subsequently mated to Zp3-Cre transgenic mice (Jackson Laboratory catalog no. 003651) that express Cre recombinase under the control of ZP3 promoter, which is activated in growing oocytes (24.Lewandoski M. Wassarman K.M. Martin G.R. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line.Curr. Biol. 1997; 7: 148-151Abstract Full Text Full Text PDF PubMed Google Scholar). The Cre recombinase-mediated deletion of the neo cassette was confirmed by PCR. Heterozygous NT-PGC-1α+/− mice were mated to obtain homozygous NT-PGC-1α−/− mice. Genotyping was performed by PCR using ear punch DNA. Total RNA was reverse-transcribed for quantitative real-time PCR (qPCR) analysis as described previously (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23.Chang J.S. Fernand V. Zhang Y. Shin J. Jun H.J. Joshi Y. Gettys T.W. NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis.J. Biol. Chem. 2012; 287: 9100-9111Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 25.Chang J.S. Huypens P. Zhang Y. Black C. Kralli A. Gettys T.W. Regulation of NT-PGC-1alpha subcellular localization and function by protein kinase A-dependent modulation of nuclear export by CRM1.J. Biol. Chem. 2010; 285: 18039-18050Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Relative expression levels of genes were determined after normalization to cyclophilin by the 2-ΔΔCt method. For quantitative analysis of mitochondrial biogenesis, mitochondrial and nuclear DNA were isolated, and their respective copy numbers were measured using qPCR with primers for ND1 and LPL as described previously (23.Chang J.S. Fernand V. Zhang Y. Shin J. Jun H.J. Joshi Y. Gettys T.W. NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis.J. Biol. Chem. 2012; 287: 9100-9111Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Palmitate oxidation was measured as previously described (22.Jun H.J. Joshi Y. Patil Y. Noland R.C. Chang J.S. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism.Diabetes. 2014; 63: 3615-3625Crossref PubMed Scopus (28) Google Scholar, 26.Huynh F.K. Green M.F. Koves T.R. Hirschey M.D. Measurement of fatty acid oxidation rates in animal tissues and cell lines.Methods Enzymol. 2014; 542: 391-405Crossref PubMed Scopus (94) Google Scholar). In brief, freshly isolated adipose explants were homogenized in SET buffer containing 250 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4). Tissue homogenates were incubated with reaction mixture containing 62.5 mM sucrose, 12.5 mM potassium phosphate, 100 mM potassium chloride, 1.25 mM magnesium chloride, 0.5 mM EDTA, 1.25 mM l-carnitine, 0.125 mM malic acid, 1.25 mM DTT, 62.5 µM NAD+, 2 mM ATP, 62.5 µM CoA, 10 mM Tris-HCl (pH 7.4), 200 µM palmitic acid coupled to BSA, and [1-14C]palmitate (0.65 µCi/ml). After 30 min of incubation at 37°C, 70% perchloric acid was added to stop the reaction. The 14CO2 produced during the incubation was trapped in 1 M sodium hydroxide, and the acidified portion of the incubation mixture was collected for liquid scintillation counting. Total palmitate oxidation was determined and normalized to total protein content in each sample. Interscapular BAT was freshly isolated, cut into small pieces (∼20 mg), and incubated at 37°C under agitation in Krebs–Henseleit buffer (KHB) containing 5 mM glucose and 2% BSA with or without isoproterenol (1 µM). After 6 h of incubation, an aliquot (25 µl) of the supernatants was incubated with 125 µl of glycerol assay buffer (pH 9.5) containing 1 mg/ml β-NAD, 1.8 mM ATP, GAPDH, and glycerokinase. Glycerol release was determined by measuring the absorbance at 320 nm. For inguinal and visceral adipose tissue, fresh fat explants were digested at 37°C with collagenase in KHB containing 5 mM glucose and 2% BSA, filtered through nylon mesh, and centrifuged. Isolated adipocytes were then incubated with or without isoproterenol (0.1 and 1 µM) at 37°C in KHB containing 5 mM glucose and 2% BSA. Glycerol was measured from infranatants after 2 h of incubation. Glycerol levels were normalized to total genomic DNA content in each sample. Oxygen consumption rates (OCRs) of BAT were measured as described previously (22.Jun H.J. Joshi Y. Patil Y. Noland R.C. Chang J.S. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism.Diabetes. 2014; 63: 3615-3625Crossref PubMed Scopus (28) Google Scholar). Briefly, fresh BAT explants were cut into small pieces (∼5 mg) and placed in a respirometric chamber of the OROBOROS Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria) containing the respiration buffer. Oxygen consumption was measured in the presence of malate/pyruvate, rotenone/succinate, or malate/palmitoyl carnitine, followed by injection of antimycin A. Mitochondrial respiration was determined by subtracting antimycin A-independent nonmitochondrial respiration and normalized by tissue weight. Tissue samples were fixed in 10% neutral-buffered formalin, paraffin embedded, and sectioned (5 µm) by the Cell Biology and Bioimaging Core at Pennington Biomedical Research Center. H&E-stained paraffin sections were scanned using a Hamamatsu NanoZoomer slide scanner (Hamamatsu, Japan). Tissue samples (40–60 mg) were homogenized in the Standard Diluent Assay Reagent provided by a Triglyceride Colorimetric Assay kit (Cayman Chemical, Ann Arbor, MI), and TAG concentrations were measured as described in the manufacturer's instructions. Whole-cell extracts were prepared from tissues by homogenization in RIPA buffer containing protease and phosphatase inhibitor cocktail (25.Chang J.S. Huypens P. Zhang Y. Black C. Kralli A. Gettys T.W. Regulation of NT-PGC-1alpha subcellular localization and function by protein kinase A-dependent modulation of nuclear export by CRM1.J. Biol. Chem. 2010; 285: 18039-18050Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and subjected to Western blot analysis using following antibodies: anti-phospho-HSL (S563), anti-HSL, anti-ATGL (Cell Signaling), and anti-actin (Sigma). For immunoprecipitation, the whole-cell extracts were incubated with IgG or polyclonal anti-PGC-1α (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 22.Jun H.J. Joshi Y. Patil Y. Noland R.C. Chang J.S. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism.Diabetes. 2014; 63: 3615-3625Crossref PubMed Scopus (28) Google Scholar) overnight at 4°C, followed by incubation with protein A agarose beads for 3 h at 4°C. After washings, immunoprecipitates were subjected to Western blot analysis using anti-PGC-1α antibody. Protein concentration was determined using Bio-Rad DC protein assay reagents according to the manufacturer's instructions. Data are presented as mean ± SEM. Student t-test or two-way ANOVA was used to compare the difference between groups using GraphPad Prism 6 software. Values of P < 0.05 were considered statistically significant. We previously reported that alternative 3′ splicing of the PPARGC1A gene produces an additional transcript encoding a shorter isoform of PGC-1α named NT-PGC-1α (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and that NT-PGC-1α is sufficient to maintain normal BAT function and activate cold-induced thermogenesis in mice selectively deficient in full-length PGC-1α (21.Kim J. Fernand V.E. Henagan T.M. Shin J. Huypens P. Newman S. Gettys T.W. Chang J.S. Regulation of brown and white adipocyte transcriptome by the transcriptional coactivator NT-PGC-1alpha.PLoS One. 2016; 11: e0159990PubMed Google Scholar, 22.Jun H.J. Joshi Y. Patil Y. Noland R.C. Chang J.S. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism.Diabetes. 2014; 63: 3615-3625Crossref PubMed Scopus (28) Google Scholar, 23.Chang J.S. Fernand V. Zhang Y. Shin J. Jun H.J. Joshi Y. Gettys T.W. NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis.J. Biol. Chem. 2012; 287: 9100-9111Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To evaluate whether NT-PGC-1α is necessary for BAT function and adaptive thermogenesis, we generated mice with selective loss of NT-PGC-1α. A neomycin-based gene-targeting vector was generated to delete a 108 bp intron sequence between exons 6 and 7 of the murine PPARGC1A gene that contains an alternative 3′ splice site for NT-PGC-1α (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) (Fig. 1A). The insertion/recombination event resulted in a fused exon 6–7 without the intron. It is predicted that this fused exon 6–7 prevents generation of NT-PGC-1α as well as additional NT-PGC-1α-b (also known as PGC-1α4) and NT-PGC-1α-c isoforms produced from the alternative exon 1b (23.Chang J.S. Fernand V. Zhang Y. Shin J. Jun H.J. Joshi Y. Gettys T.W. NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis.J. Biol. Chem. 2012; 287: 9100-9111Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 27.Ruas J.L. White J.P. Rao R.R. Kleiner S. Brannan K.T. Harrison B.C. Greene N.P. Wu J. Estall J.L. Irving B.A. et al.A PGC-1alpha isoform induced by resistance training regulates skeletal muscle hypertrophy.Cell. 2012; 151: 1319-1331Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). The neo cassette was subsequently removed by crossing to the Zp3-Cre transgenic mouse that expresses Cre recombinase in growing oocytes (24.Lewandoski M. Wassarman K.M. Martin G.R. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line.Curr. Biol. 1997; 7: 148-151Abstract Full Text Full Text PDF PubMed Google Scholar). The resulting heterozygous NT-PGC-1α+/− mice were mated to produce homozygous NT-PGC-1α−/− mice. The mutant allele was confirmed using PCR analysis of genomic DNA isolated from NT-PGC-1α+/+, NT-PGC-1α+/−, and NT-PGC-1α−/− mice (Fig. 1B). The efficacy of the gene targeting was further evaluated by qPCR analysis with the previously verified isoform-specific primers (19.Zhang Y. Huypens P. Adamson A.W. Chang J.S. Henagan T.M. Lenard N.R. Burk D. Klein J. Perwitz N. Shin J. et al.Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}.J. Biol. Chem. 2009; 284: 32813-32826Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23.Chang J.S. Fernand V. Zhang Y. Shin J. Jun H.J. Joshi Y. Gettys T.W. NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis.J. Biol. Chem. 2012; 287: 9100-9111Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Both PGC-1α and NT-PGC-1α transcripts were detected in BAT, muscle, heart, WAT, brain, and liver of WT littermates, but NT-PGC-1α transcripts were absent in NT-PGC-1α−/− mice (Fig. 1D), demonstrating that the mutant allele blocks an alternative splicing event producing the NT-PGC-1α transcript. Pups lacking NT-PGC-1α were born at the expected Mendelian ratio at room temperature (22°C). After weaning, mice were housed at 22°C or near thermoneutral temperature (28°C). H&E-stained BAT sections at 22°C rev
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