The ability of catecholamines to stimulate cAMP production and lipolysis from adipose tissue is depressed in congenital and diet- induced obese rodents. In both white (WAT) and brown (BAT) adipocytes, 3 beta-adrenergic receptor (AR) sub-types are expressed: beta1-, beta2-, and adipose-specific beta3AR. We have shown that depressed cAMP induction in obesity is linked to impaired expression of beta3AR and beta1AR in WAT and BAT. However, selective beta3- agonists can prevent or reverse obesity in vivo, and the expression of adipocyte betaARs can be restored. Since agonist-stimulation of betaARs is generally associated with down-regulation, the mechanism(s) responsible for the apparent beta3-induced up-regulation of adipocyte betaARs is unknown. The unique anti-obesity effects of beta3-agonists may be related, in part, to preliminary evidence that the beta3AR may couple to a pertussis toxin (PTX)-sensitive G protein as well as to Gas, and to adipose-specific regulation of betaAR genes. This proposal will test the hypothesis that the beta3AR utilizes dual signal transduction mechanisms in WAT and BAT, and determine promoter elements of the beta3AR gene responsible for differentiation-and beta-agonist-regulated expression.
Aim 1 : G protein(s) which couple to beta3AR in adipocytes will be identified biochemically by the agonist-dependent incorporation of the photo- activated GTP analog azidoanilido-[32P]-GTP (AAGTP). Galpha proteins labeled with AAGTP will be identified by Galpha-subunit-specific antisera. The effects of PTX on beta3AR-stimulated lipolysis, and on AAGTP labeling will also be assessed.
Aim 2 : The ability of beta3AR agonist to activate signal transduction pathways other than cAMP production, such as intracellular Ca+2 mobilization and phospholipid metabolism, will be investigated.
Aim 3 : The transcription and functional activity of the beta3AR in response to norepinephrine or the beta3- selective agonist CL316,243 will be examined in immortalized WAT and BAT cell lines. In addition, the effect of adipocyte differentiation by the PPARgamma2 and RXR nuclear receptors (potent activators of fat cell differentiation) on beta3AR expression will be determined. Levels of beta1-and beta2AR will also be monitored, and the results interpreted in relation to data obtained from beta3AR-treated mice.
Aim 4 : Regulatory regions responsible for differentiation-dependent and beta-agonist-regulated transcription will be determined. Beta3AR promoter-reporter constructs will be transiently tranfected into the cell lines under conditions described in Aim 3. Candidate regulatory sequences will be further analyzed by co-transfection studies, and by gel shift assays using oligonucleotides comprising these transcription factor binding sites.
Showing the most recent 10 out of 11 publications
