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


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
doi: 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:

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


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2019 Číslo 11