Adipose tissue is crucial for normal metabolic function, and abnormal proliferation of adipocytes plays an substantial role in syndromes of obesity. The 3T3-L1 cell line has served as an important model of adipocyte differentiation. Although retinoic acid (RA) promotes differentiation of a variety of cell types, RA inhibits adipose conversion of 3T3-L1 preadipocytes. The goal of this research is to understand the molecular basis of RA action in this system. It is hypothesized that RA regulates gene transcription via a specific subset of retinoid receptors (RARs and RXRs). The first specific aim of this project is to determine the role of specific retinoid receptor subtypes and isoforms in adipocyte differentiation. 3T3-L1 preadipocytes express multiple RARs and RXRs, but their expression pattern and correlation with RA effects strongly suggests that RARgamma1 and RXRalpha have particularly important roles in adipocyte differentiation. This will be tested by creating stable 3T3-L1 cell lines which constitutively express the RARs and RXRs and studying the differentiation of these cells in the presence or absence of RA. Inducible promoters and antisense approaches will also be employed, especially for receptors whose expression leads to terminal differentiation, since the differentiated cells do not divide and therefore cannot be selected. Regardless of which receptors mediate the effects of RA, it is important to understand which genes are the targets of RA action. The second specific aim is to identify and functionally characterize genes which are regulated by RA early in the process of adipose conversion. Differential display will be used to identify and clone genes whose expression is dramatically changed by RA within 24h of initiation of adipocyte differentiation. Some, including the ras recision gene (rrg) lysyl oxidase, which counteracts ras transformation of 3T3 fibroblasts, have already been isolated. Of particularly interest will be RA-responsive genes which are not normally regulated during adipose conversion, since one model of RA action predicts that such genes mediate the inhibitory effects of RA on adipoctye differentiation. To test the hypothesis that these genes are crucial for prevention of adipocyte differentiation by RA, the full-length cDNAs will be cloned and expressed in 3T3-L1 preadipocytes, whose ability to differentiate will be determined. The third specific aim is to delineate the developmental and tissue expression of adipose-specific retinoid-responsive genes. In situ hybridization will be used to follow the embryonic and neonatal expression of adipocyte- specific genes known from the above studies to be expressed early in adipose conversion. These studies will provide new insight into the ontogeny of adipose tissue. Together, the proposed studies will provide insight into early molecular events and the role of retinoid receptors and ligands in fat cell growth and differentiation. This knowledge is vital to the development of rational approaches to the prevention and treatment of human obesity, an endemic disorder which is a costly cause of morbidity and mortality in the U.S.
