The congenital obesity, beta-adrenergic receptor (betaAR) stimulation of lipolysis and thermogenesis are impaired. Both white and brown adipocytes express beta1-, beta2-, and beta3AR (the latter is expressed exclusively in fat). Our preliminary data demonstrate that beta1AR and beta3AR are impaired in white and brown adipocytes from genetically obese (ob/ob) mice compared to lean controls. This defect may contribute to the obese state. In addition, the stimulatory G-protein (Gs) alpha subunit, to which betaARs are coupled, is depressed in obese animals. Since elevated corticosteroid (CORT) levels are an essential feature promoting obesity, and betaAR subtype expression can be modulated by CORTs, regulation of betaAR subtype gene transcription by this steroid may be an important component of obesity. There are 4 major aims of this research plan. (1) Changes in betaAR and Gsalpha will be monitored during the development of obesity. The mRNA levels of the beta1AR, beta2AR beta3AR and Gsalpha genes, radioligand binding and beta-agonist- stimulated adenylyl cyclase activity will be measured in white and brown adipose tissue from lean and obese mice from 1 week to 14 weeks of age. Adipocyte-specific markers will also be monitored. (2) The effect of CORT on expression of betaAR subtypes and Gsalpha genes in brown and white fat, and their effects on obesity will be assessed in 2 ways: (i) Adrenalectomized obese mice will be studied over the same developmental period outlined in Aim 1. (ii) Lean and obese mice will be treated chronically with the CORT antagonist RU38486 over the same period as in Aim 1. Results from Aims 1 and 2 will provide a basis for examining more specific molecular and cellular mechanisms regulating betaAR and Gsalpha gene expression. (3) Established murine preadipocyte cell lines of white and brown fat origin will be used to examine the molecular mechanisms regulating transcription of betaAR subtypes and Gsalpha genes during adipocyte differentiation and CORT exposure. Nuclear run-on transcription, mRNA half-life, and cycloheximide sensitivity studies will determine specific molecular mechanisms responsible for observed changes in individual betaAR and Gsalpha transcripts. These data will be related to results obtained from lean and obese mice in vivo in Aims 1 and 2. (4) Transfection studies with betaAR promoter-reporter constructs in pre- and differentiated adipocytes are designed to identify regulatory elements involved in adipocyte differentiation and CORT regulation of betaAR transcription. Together, these integrated studies should more directly define the physiological, cellular and molecular mechanisms regulating betaAR signal transduction in adipocytes during the development of obesity.
