Inducible UCP1 silencing: A lentiviral RNA-interference approach to quantify the contribution of beige fat to energy homeostasis

English version

Autoři: Nicole Wen Mun Khor ^aff001; Michael M. Swarbrick ^aff001; Jenny E. Gunton ^aff001
Působiště autorů: The Westmead Institute for Medical Research, Westmead, Sydney, Australia ^aff001; Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia ^aff002; Faculty of Medicine, University of New South Wales, Sydney, Australia ^aff003; Faculty of Medicine and Health, The University of Sydney, Sydney, Australia ^aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0223987

Souhrn

Energy consuming, heat-producing beige adipocytes, located in classic white adipose tissue (WAT), hold promise for the treatment of obesity. Few reports have quantitatively assessed the contribution of browned 'WAT' to energy expenditure. There is a need for methods to examine beige-fat thermogenesis, independently of classical brown fat. The aim of this study is to optimize an inducible lentiviral shRNA to conditionally knock-down Ucp1 and assess the effects on 'browned' WAT. Primary adipocytes from mouse inguinal WAT converted into thermogenic adipocytes when stimulated with β-adrenergic agonist and thiazolidinedione. There was increased UCP1 protein and importantly increases in various indicators of mitochondrial bioenergetics. Next, we determined optimal transfection conditions for the UCP1-shRNA lentiviral system and subsequently applied this to 'browned' WAT. UCP1 knockdown decreased the brown/beige-fat gene profile and decreased mitochondrial respiration. In summary, this study optimizes lentiviral UCP1-shRNA technology in vitro. This technique could be applied to inguinal fat depots in vivo. This would allow investigation of contribution of depots to whole-body metabolism to help elucidate the physiological relevance of beige fat.

Klíčová slova:

Fats – Mitochondria – Doxycycline – Adipocytes – Thermogenesis – Brown adipose tissue – Adipocyte differentiation

Zdroje

1. World Health Organisation. Obesity—Situation and trends 2015. Available from: http://www.who.int/gho/ncd/risk_factors/obesity_text/en/.

2. Lee P, Swarbrick MM, Ho KK. Brown adipose tissue in adult humans: a metabolic renaissance. Endocrine reviews. 2013;34(3):413–38. doi: 10.1210/er.2012-1081 23550082.

3. Rosen ED, Spiegelman BM. What we talk about when we talk about fat. Cell. 2014;156(1–2):20–44. doi: 10.1016/j.cell.2013.12.012 24439368; PubMed Central PMCID: PMC3934003.

4. Pfeifer A, Hoffmann LS. Brown, beige, and white: the new color code of fat and its pharmacological implications. Annual review of pharmacology and toxicology. 2015;55:207–27. doi: 10.1146/annurev-pharmtox-010814-124346 25149919.

5. Bartness T, Vaughan C, Song C. Sympathetic and sensory innervation of brown adipose tissue. International journal of obesity. 2010;34:S36–S42. doi: 10.1038/ijo.2010.182 20935665

6. Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, et al. Functional brown adipose tissue in healthy adults. New England Journal of Medicine. 2009;360(15):1518–25. doi: 10.1056/NEJMoa0808949 19357407

7. van Marken Lichtenbelt WD, Schrauwen P. Implications of nonshivering thermogenesis for energy balance regulation in humans. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2011;301(2):R285–R96. doi: 10.1152/ajpregu.00652.2010 21490370

8. Rothwell N, Stock M. Luxuskonsumption, diet-induced thermogenesis and brown fat: the case in favour. Clin Sci (Lond). 1983;64(1):19–23.

9. Lidell ME, Betz MJ, Enerback S. Brown adipose tissue and its therapeutic potential. Journal of internal medicine. 2014;276(4):364–77. doi: 10.1111/joim.12255 24717051.

10. Cohen P, Levy JD, Zhang Y, Frontini A, Kolodin DP, Svensson KJ, et al. Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch. Cell. 2014;156(1):304–16.

11. Yang T, Burrows C, Park JH. Development of a doxycycline-inducible lentiviral plasmid with an instant regulatory feature. Plasmid. 2014;72:29–35. doi: 10.1016/j.plasmid.2014.04.001 24727543

12. GEHealthcare. SMARTvector Lentiviral shRNA [Technical Manual ]. In press 2014.

13. Granneman JG, Li P, Zhu Z, Lu Y. Metabolic and cellular plasticity in white adipose tissue I: effects of β3-adrenergic receptor activation. American Journal of Physiology-Endocrinology and Metabolism. 2005;289(4):E608–E16. doi: 10.1152/ajpendo.00009.2005 15941787

14. Petrovic N, Shabalina IG, Timmons JA, Cannon B, Nedergaard J. Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPARγ agonist. American Journal of Physiology-Endocrinology And Metabolism. 2008;295(2):E287–E96. doi: 10.1152/ajpendo.00035.2008 18492776

15. Brand M, Nicholls D. Assessing mitochondrial dysfunction in cells. Biochem J. 2011;435:297–312. doi: 10.1042/BJ20110162 21726199

16. Bugge A, Dib L, Collins S. Measuring respiratory activity of adipocytes and adipose tissues in real time. Methods of Adipose Tissue Biology Part B: Methods of Adipose Tissue Biology. 2014;538:233.

17. Ma SW, Foster DO. Uptake of glucose and release of fatty acids and glycerol by rat brown adipose tissue in vivo. Canadian journal of physiology and pharmacology. 1986;64(5):609–14. doi: 10.1139/y86-101 3730946

18. Betz MJ, Bielohuby M, Mauracher B, Abplanalp W, Müller H-H, Pieper K, et al. Isoenergetic Feeding of Low Carbohydrate-High Fat Diets Does Not Increase Brown Adipose Tissue Thermogenic Capacity in Rats. PLoS ONE. 2012;7(6):e38997. doi: 10.1371/journal.pone.0038997 PMC3374780. 22720011

19. Harms M, Seale P. Brown and beige fat: development, function and therapeutic potential. Nature medicine. 2013;19(10):1252–63. doi: 10.1038/nm.3361 24100998.

20. Zhou Z, Toh SY, Chen Z, Guo K, Ng CP, Ponniah S, et al. Cidea-deficient mice have lean phenotype and are resistant to obesity. Nature genetics. 2003;35(1):49–56. doi: 10.1038/ng1225 12910269

21. Cypess AM, Weiner LS, Roberts-Toler C, Elía EF, Kessler SH, Kahn PA, et al. Activation of human brown adipose tissue by a β3-adrenergic receptor agonist. Cell metabolism. 2015;21(1):33–8. doi: 10.1016/j.cmet.2014.12.009 25565203

22. Wu J, Cohen P, Spiegelman BM. Adaptive thermogenesis in adipocytes: Is beige the new brown? Genes & development. 2013;27(3):234–50.

23. Bartesaghi S, Hallen S, Huang L, Svensson P-A, Momo RA, Wallin S, et al. Thermogenic activity of UCP1 in human white fat-derived beige adipocytes. Molecular Endocrinology. 2014;29(1):130–9.

24. Nedergaard J, Cannon B. UCP1 mRNA does not produce heat. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids. 2013;1831(5):943–9.

25. Weyer C, Tataranni PA, Snitker S, Danforth E, Ravussin E. Increase in insulin action and fat oxidation after treatment with CL 316,243, a highly selective beta3-adrenoceptor agonist in humans. Diabetes. 1998;47(10):1555–61. doi: 10.2337/diabetes.47.10.1555 9753292

26. Redman LM, de Jonge L, Fang X, Gamlin B, Recker D, Greenway FL, et al. Lack of an effect of a novel β3-adrenoceptor agonist, TAK-677, on energy metabolism in obese individuals: a double-blind, placebo-controlled randomized study. The Journal of Clinical Endocrinology & Metabolism. 2007;92(2):527–31.

27. Larsen TM, Toubro S, van Baak MA, Gottesdiener KM, Larson P, Saris WH, et al. Effect of a 28-d treatment with L-796568, a novel β3-adrenergic receptor agonist, on energy expenditure and body composition in obese men. The American journal of clinical nutrition. 2002;76(4):780–8. doi: 10.1093/ajcn/76.4.780 12324291

28. Cawthorne MA, Sennitt MV, Arch J, Smith SA. BRL 35135, a potent and selective atypical beta-adrenoceptor agonist. The American journal of clinical nutrition. 1992;55(1):252S–7S.

29. Lee KY, Russell SJ, Ussar S, Boucher J, Vernochet C, Mori MA, et al. Lessons on conditional gene targeting in mouse adipose tissue. Diabetes. 2013;62(3):864–74. doi: 10.2337/db12-1089 23321074

30. Guerra C, Navarro P, Valverde AM, Arribas M, Brüning J, Kozak LP, et al. Brown adipose tissue–specific insulin receptor knockout shows diabetic phenotype without insulin resistance. Journal of Clinical Investigation. 2001;108(8):1205. doi: 10.1172/JCI13103 11602628

31. Wang QA, Scherer PE, Gupta RK. Improved methodologies for the study of adipose biology: insights gained and opportunities ahead. Journal of lipid research. 2014;55(4):605–24. doi: 10.1194/jlr.R046441 24532650

32. Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, Shadrach JL, et al. Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proceedings of the National Academy of Sciences. 2011;108(1):143–8.

33. Lee P, Swarbrick MM, Greenfield JR. The Sum of All Browning in FGF21 Therapeutics. Cell metabolism. 2015;21(6):795–6. doi: 10.1016/j.cmet.2015.05.018 26039444

34. Schulz TJ, Huang P, Huang TL, Xue R, McDougall LE, Townsend KL, et al. Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat. Nature. 2013;495(7441):379–83. doi: 10.1038/nature11943 23485971

35. Emanuelli B, Vienberg SG, Smyth G, Cheng C, Stanford KI, Arumugam M, et al. Interplay between FGF21 and insulin action in the liver regulates metabolism. The Journal of clinical investigation. 2015;125(1):458. doi: 10.1172/JCI80223 25654556

36. Wu J, Boström P, Sparks LM, Ye L, Choi JH, Giang A-H, et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell. 2012;150(2):366–76. doi: 10.1016/j.cell.2012.05.016 22796012

37. Clarke KJ, Adams AE, Manzke LH, Pearson TW, Borchers CH, Porter RK. A role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial uncoupling protein 1 (UCP1). Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2012;1817(10):1759–67.

Inducible UCP1 silencing: A lentiviral RNA-interference approach to quantify the contribution of beige fat to energy homeostasis

Souhrn

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