AUTHOR=Zhang Zhiyin , Wu Qihan , He Yang , Lu Peng , Li Danjie , Yang Minglan , Gu Weiqiong , Liu Ruixin , Hong Jie , Wang Jiqiu 

TITLE=IRX3 Overexpression Enhances Ucp1 Expression In Vivo

JOURNAL=Frontiers in Endocrinology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2021.634191

DOI=10.3389/fendo.2021.634191

ISSN=1664-2392

ABSTRACT=<sec><title>Objective</title><p>The Iroquois homeobox 3 (<italic>IRX3</italic>) gene was recently reported to be a functional downstream target of a common polymorphism in the <italic>FTO</italic> gene, which encodes an obesity-associated protein; however, the role of <italic>IRX3</italic> in energy expenditure remains unclear. Studies have revealed that the overexpression of a dominant–negative form of IRX3 in the mouse hypothalamus and adipose tissue promoted energy expenditure by enhancing brown/browning activities. Meanwhile, we and others recently demonstrated that <italic>IRX3</italic> knockdown impaired the browning program of primary preadipocytes <italic>in vitro</italic>. In this study, we aimed to further clarify the effects of overexpressing human <italic>IRX3</italic> (h<italic>IRX3</italic>) on brown/beige adipose tissues <italic>in vivo</italic>.</p></sec><sec><title>Methods</title><p>Brown/beige adipocyte-specific h<italic>IRX3</italic>-overexpressing mice were generated and the browning program of white adipose tissues was induced by both chronic cold stimulation and CL316,243 injection. Body weight, fat mass, lean mass, and energy expenditure were measured, while morphological changes and the expression of thermogenesis-related genes in adipose tissue were analyzed. Moreover, the browning capacity of primary preadipocytes derived from h<italic>IRX3</italic>-overexpressing mice was assessed. RNA sequencing was also employed to investigate the effect of h<italic>IRX3</italic> on the expression of thermogenesis-related genes.</p></sec><sec><title>Results</title><p>h<italic>IRX3</italic> overexpression in embryonic brown/beige adipose tissues (<italic>Rosa26</italic><sup>h</sup><italic><sup>IRX3</sup></italic>;<italic>Ucp1-</italic>Cre) led to increased energy expenditure, decreased fat mass, and a lean body phenotype. After acute cold exposure or CL316,243 stimulation, brown/beige tissue h<italic>IRX3</italic>-overexpressing mice showed an increase in <italic>Ucp1</italic> expression. Consistent with this, induced h<italic>IRX3</italic> overexpression in adult mice (<italic>Rosa26</italic><sup>h</sup><italic><sup>IRX3</sup></italic>;<italic>Ucp1-</italic>Cre<sup>ERT2</sup>) also promoted a moderate increase in <italic>Ucp1</italic> expression. <italic>Ex vitro</italic> experiments further revealed that h<italic>IRX3</italic> overexpression induced by <italic>Ucp1</italic>-driven Cre recombinase activity upregulated brown/beige adipocytes <italic>Ucp1</italic> expression and oxygen consumption rate (OCR). RNA sequencing analyses indicated that h<italic>IRX3</italic> overexpression in brown adipocytes enhanced brown fat cell differentiation, glycolysis, and gluconeogenesis.</p></sec><sec><title>Conclusion</title><p>Consistent with the <italic>in vitro</italic> findings, brown/beige adipocyte-specific overexpression of h<italic>IRX3</italic> promoted <italic>Ucp1</italic> expression and thermogenesis, while reducing fat mass.</p></sec>