Regulation of in Vitro Adipogenesis by All-trans Retinoic Acid (P21-075-19)

Abstract

Abstract Objectives Vitamin A, referred to as retinol, is an essential nutrient that affects the cell growth and differentiation including adipogenesis. Although previous studies using supraphysiological doses (over 1 mM) of all-trans retinoic acid (atRA) demonstrated anti-adipogenic activity, effects of atRA at various levels on differentiation of 3T3-L1 preadipocytes have not been extensively investigated. Methods Various doses of atRA were supplemented during the differentiation of 3T3-L1 preadipocytes. Lipid accumulation was assessed by Oil-Red-O staining and cellular triglyceride levels. Expression levels of adipogenic markers and cell cycle regulators were measured by quantitative real-time PCR or Western blot analyses. In addition, cell proliferation, cell cycle, and cytotoxicity were analyzed. Results Our study showed that the amount of cellular triglyceride and intensities of Oil-Red-O staining were decreased by supplementing atRA (1 and 10 mM) but increased by low concentrations of atRA (0.01 to 100 nM) compared with the control. Also PPARg and FABP4 were gradually over-expressed by atRA up to 1 nM but decreased at over the 1 nM concentrations. Moreover, mitotic clonal expansion (MCE) and consequential growth-arrest were analyzed as important steps in adipogenesis of 3T3-L1 cells. The 1 nM group showed more cell proliferation and thereafter a higher ratio of G0/G1 phase on day 2. Protein levels of S/G2-phase factors were dose-dependently increased by atRA up to 1 nM on day 1, but the factors were highly expressed in higher doses on day 2. G0/G1 markers were higher at the higher doses of atRA on day 1; whereas, they were highly expressed in mild or medium doses on day 2. Conclusions Our data indicate that atRA controls adipogenesis with accompanied changes in expression of PPARgamma and FABP4, cell proliferation and follow-up growth-arrest. These results indicate that atRA can function both as a negative and positive regulator of adipogenesis depending on dosages, providing a strategy for achieving proper nutritional balance for treatment of obesity. Funding Sources USDA-NIFA and KRIBB Research Initiative Program.