Role of SMAD2 and SMAD3 on Adipose Tissue Development and Function
Authors:   ,   University of Tennessee Health Science Center, Kumari Roshan 
Published: November 2021

Introduction: Obesity and its associated metabolic syndrome are major medical problems worldwide including United States. Adipose tissue is the primary site of energy storage, playing important roles in health. Adipose tissue also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety, inflammation, and total energy homeostasis. Activin A and activin B play important roles in maintaining body composition and energy homeostasis. This dissertation highlights the role of activin/SMADs signaling in adipose tissue development, function, and maintenance. SMAD2/3 proteins are downstream mediators of transforming growth factor-β (TGFβ) family signaling, including activins, which regulate critical preadipocyte and mature adipocyte functions. Previous studies have demonstrated that Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity and the direct contributions of Smad2 and Smad3 in adipose tissues individually or in combination and the responses of these tissues to activin signaling are unknown. Additionally, our lab demonstrated that the combined loss of activin A and activin B have reduced adiposity in mice and appearance of brown-like cells in visceral white adipose tissue. However, the cell-autonomous role of activins on cell proliferation and differentiation remained unknown in vitro. My hypothesis was that activin signaling regulate adipocyte differentiation and functions via SMAD2/3-mediated mechanism(s) and that the individual or combined adipose-specific deletion of SMAD2/SMAD3 would result in reduced adiposity similar to activin deficient mice. Objective: Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 individually and in combination, on diet-induced adiposity and to establish whether preadipocytes isolated from subcutaneous and visceral white adipose tissues differ in their differentiation capacity. We also assessed the role of activins on cell proliferation and differentiation using an in vitro model. Research Design: To assess the adipose-selective requirements of Smad2, Smad3 and Smad2/3, we generated three lines of adipose-selective conditional knockout (cKO) mice including Smad2cKO, Smad3cKO, and Smad2/3 double cKO mice using Smad2 and/or Smad3 “floxed” mice intercrossed with Adiponectin-Cre mice. Additionally, we isolated preadipocytes and examined adipogenic activity of visceral and subcutaneous preadipocyte and the effects of activin on preadipocyte proliferation and differentiation in vitro. Furthermore, we used mouse embryonic fibroblasts (MEFs) from wild type mice and activin double knockout mice to study the cell autonomous role of activin on differentiation and cell fate. Results: Our results demonstrated that subcutaneous preadipocytes differentiate uniformly and almost all wildtype subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A promoted proliferation and suppressed differentiation of subcutaneous preadipocytes more robustly given that visceral adipocytes differentiate poorly at baseline. Additionally, global knockout of activin A and B promoted differentiation and browning in differentiated MEFs in vitro consistent with in vivo studies. Furthermore, we showed that Smad2cKO mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3cKO male mice displayed a trend of reduced body weight on high fat diet. On both (LFD and HFD) diets, Smad3cKO male mice displayed an adipose depot-selective phenotype, with significant reduction in subcutaneous fat mass but not visceral fat mass. Smad2/3cKO male mice did not show any difference in body weight or fat mass compared to control mice. Female mice with adipose-selective combined deletion of Smad2/3, displayed reduced body weight and reduction of fat mass in both visceral and subcutaneous depot with higher metabolic rate on HFD compared to control littermates. Conclusions: Our study demonstrated that Smad3 is an important contributor to the development and/or maintenance of subcutaneous white adipose tissue in a sex-selective fashion. Combined reduction of Smad2/3 protects female mice from diet induced obesity and is important for visceral and subcutaneous depots in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation. Activin treatment promoted proliferation of preadipocytes, while activin deficiency promoted differentiation and altered the phenotypic characteristics of White adipocytes to brown-like cells in vitro consistent with in vivo.