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An Optimized Immunoblotting Protocol for Accurate Detection of Endogenous PGC-1α Isoforms in Various Rodent Tissues

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Nuclear Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1966))

Abstract

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays a central role in the response and adaptation to environmental and nutritional stimuli by initiating tissue-specific transcriptional reprogramming. Since its discovery in 1998, the field of PGC-1α biology has grown exponentially and a large body of research has elucidated the diverse roles of PGC-1α in brown adipose tissue thermogenesis, fatty acid oxidation, muscle fiber type switching, hepatic gluconeogenesis, and circadian clock regulation, etc. In addition, recent research has identified a splice variant(s) of PGC-1α in humans and rodents. The common misconception relating to PGC-1α is that it migrates at a predicted molecular weight of ~90 kDa by SDS-PAGE gel electrophoresis. However, several recent studies have provided solid evidence that the biologically relevant molecular weight of PGC-1α is ~110 kDa. In this chapter, we describe an optimized immunoblotting protocol that is developed to detect the low abundance protein PGC-1α and its alternatively spliced isoform named NT-PGC-1α in various rodent tissues. We also describe an optimized immunoprecipitation protocol that can isolate and concentrate endogenous PGC-1α and NT-PGC-1α. The protocols presented here will hopefully allow investigators to report accurate and reliable data regarding PGC-1α isoforms.

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References

  1. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, Spiegelman BM (1999) Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98(1):115–124

    Article  CAS  Google Scholar 

  2. Lehman JJ, Barger PM, Kovacs A, Saffitz JE, Medeiros DM, Kelly DP (2000) Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest 106(7):847–856

    Article  CAS  Google Scholar 

  3. Schreiber SN, Emter R, Hock MB, Knutti D, Cardenas J, Podvinec M, Oakeley EJ, Kralli A (2004) The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial biogenesis. Proc Natl Acad Sci U S A 101(17):6472–6477

    Article  CAS  Google Scholar 

  4. Vega RB, Huss JM, Kelly DP (2000) The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 20(5):1868–1876

    Article  CAS  Google Scholar 

  5. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM (1998) A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92(6):829–839

    Article  CAS  Google Scholar 

  6. Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puigserver P, Isotani E, Olson EN, Lowell BB, Bassel-Duby R, Spiegelman BM (2002) Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature 418(6899):797–801

    Article  CAS  Google Scholar 

  7. Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Schutz G, Yoon C, Puigserver P, Spiegelman B, Montminy M (2001) CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature 413(6852):179–183

    Article  CAS  Google Scholar 

  8. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM (2001) Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413(6852):131–138

    Article  CAS  Google Scholar 

  9. Liu C, Li S, Liu T, Borjigin J, Lin JD (2007) Transcriptional coactivator PGC-1alpha integrates the mammalian clock and energy metabolism. Nature 447(7143):477–481

    Article  CAS  Google Scholar 

  10. Zhang Y, Huypens P, Adamson AW, Chang JS, Henagan TM, Lenard NR, Burk D, Klein J, Perwitz N, Shin J, Fasshauer M, Kralli A, Gettys TW (2009) Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1{alpha}. J Biol Chem 284(47):32813–32826

    Article  CAS  Google Scholar 

  11. Chang JS, Fernand V, Zhang Y, Shin J, Jun HJ, Joshi Y, Gettys TW (2012) NT-PGC-1alpha protein is sufficient to link beta3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis. J Biol Chem 287(12):9100–9111

    Article  CAS  Google Scholar 

  12. Jun HJ, Joshi Y, Patil Y, Noland RC, Chang JS (2014) NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism. Diabetes 63(11):3615–3625

    Article  CAS  Google Scholar 

  13. Chang JS, Jun HJ, Park M (2016) Transcriptional coactivator NT-PGC-1alpha promotes gluconeogenic gene expression and enhances hepatic gluconeogenesis. Physiol Rep 4(20):e13013

    Article  Google Scholar 

  14. Chang JS, Ha K (2017) An unexpected role for the transcriptional coactivator isoform NT-PGC-1alpha in the regulation of mitochondrial respiration in brown adipocytes. J Biol Chem 292(24):9958–9966

    Article  CAS  Google Scholar 

  15. Puigserver P, Rhee J, Lin J, Wu Z, Yoon JC, Zhang CY, Krauss S, Mootha VK, Lowell BB, Spiegelman BM (2001) Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPARgamma coactivator-1. Mol Cell 8(5):971–982

    Article  CAS  Google Scholar 

  16. Sano M, Tokudome S, Shimizu N, Yoshikawa N, Ogawa C, Shirakawa K, Endo J, Katayama T, Yuasa S, Ieda M, Makino S, Hattori F, Tanaka H, Fukuda K (2007) Intramolecular control of protein stability, subnuclear compartmentalization, and coactivator function of peroxisome proliferator-activated receptor gamma coactivator 1alpha. J Biol Chem 282(35):25970–25980

    Article  CAS  Google Scholar 

  17. Olson BL, Hock MB, Ekholm-Reed S, Wohlschlegel JA, Dev KK, Kralli A, Reed SI (2008) SCFCdc4 acts antagonistically to the PGC-1alpha transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis. Genes Dev 22(2):252–264

    Article  CAS  Google Scholar 

  18. Baldelli S, Aquilano K, Ciriolo MR (2014) PGC-1alpha buffers ROS-mediated removal of mitochondria during myogenesis. Cell Death Dis 5:e1515

    Article  CAS  Google Scholar 

  19. Lancel S, Hassoun SM, Favory R, Decoster B, Motterlini R, Neviere R (2009) Carbon monoxide rescues mice from lethal sepsis by supporting mitochondrial energetic metabolism and activating mitochondrial biogenesis. J Pharmacol Exp Ther 329(2):641–648

    Article  CAS  Google Scholar 

  20. Zhu JH, Gusdon AM, Cimen H, Van Houten B, Koc E, Chu CT (2012) Impaired mitochondrial biogenesis contributes to depletion of functional mitochondria in chronic MPP+ toxicity: dual roles for ERK1/2. Cell Death Dis 3:e312

    Article  CAS  Google Scholar 

  21. Greene NP, Lee DE, Brown JL, Rosa ME, Brown LA, Perry RA, Henry JN, Washington TA (2015) Mitochondrial quality control, promoted by PGC-1alpha, is dysregulated by Western diet-induced obesity and partially restored by moderate physical activity in mice. Physiol Rep 3(7):e12470

    Article  Google Scholar 

  22. Zeng T, Zhang CL, Song FY, Zhao XL, Xie KQ (2014) CMZ reversed chronic ethanol-induced disturbance of PPAR-alpha possibly by suppressing oxidative stress and PGC-1alpha acetylation, and activating the MAPK and GSK3beta pathway. PLoS One 9(6):e98658

    Article  Google Scholar 

  23. Kawalec M, Boratynska-Jasinska A, Beresewicz M, Dymkowska D, Zablocki K, Zablocka B (2015) Mitofusin 2 deficiency affects energy metabolism and mitochondrial biogenesis in MEF cells. PLoS One 10(7):e0134162

    Article  Google Scholar 

  24. Rockl KS, Hirshman MF, Brandauer J, Fujii N, Witters LA, Goodyear LJ (2007) Skeletal muscle adaptation to exercise training: AMP-activated protein kinase mediates muscle fiber type shift. Diabetes 56(8):2062–2069

    Article  CAS  Google Scholar 

  25. Iacovelli J, Rowe GC, Khadka A, Diaz-Aguilar D, Spencer C, Arany Z, Saint-Geniez M (2016) PGC-1alpha induces human RPE oxidative metabolism and antioxidant capacity. Invest Ophthalmol Vis Sci 57(3):1038–1051

    Article  CAS  Google Scholar 

  26. Zhao Q, Zhang J, Wang H (2015) PGC-1alpha limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species. Biosci Rep 35(5):e00252

    Article  CAS  Google Scholar 

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Acknowledgments

The authors thank Jeho Shin for technical assistance and Ms. Cindi Tramonte for administrative support. This work was supported by the National Institutes of Health grants R01DK104748 (J.S.C.) and R01DK096311 (T.W.G.). The work used Cell Biology & Bioimaging and Genomics Core facilities that are supported in part by COBRE (NIH8 1P30GM118430-01) and NORC (NIH P30-DK072476) center grants from the National Institutes of Health.

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Authors and Affiliations

  1. Laboratory of Nutrient Sensing and Adipocyte Signaling, Pennington Biomedical Research Center, Baton Rouge, LA, USA

    Thomas W. Gettys

  2. Laboratory of Gene Regulation and Metabolism, Pennington Biomedical Research Center, Baton Rouge, LA, USA

    Ji Suk Chang

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  1. Thomas W. Gettys
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  2. Ji Suk Chang
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Correspondence to Ji Suk Chang .

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Editors and Affiliations

  1. School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA

    Mostafa Z. Badr

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Gettys, T.W., Chang, J.S. (2019). An Optimized Immunoblotting Protocol for Accurate Detection of Endogenous PGC-1α Isoforms in Various Rodent Tissues. In: Badr, M. (eds) Nuclear Receptors. Methods in Molecular Biology, vol 1966. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9195-2_2

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  • DOI: https://doi.org/10.1007/978-1-4939-9195-2_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9194-5

  • Online ISBN: 978-1-4939-9195-2

  • eBook Packages: Springer Protocols

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