The beta3-adrenergic receptor (beta3AR) is a G protein- coupled receptor (GPCR) expressed predominantly in adipose tissue, where it mediates the effects of noradrenaline-stimulated lipolysis in white adipose tissue (WAT) and thermogenesis in brown adipose tissue (BAT). Selective agonists for the beta3AR can prevent or reverse obesity and insulin resistance in canine and rodent models, and are under pharmaceutical development for humans. In addition to potent stimulation of lipolysis, beta3AR agonists promote the appearance of thermogenically active brown adipocytes in typical white adipose depots but the mechanism responsible for this effect is not understood. Completion of the Aims in our previous funding period established that the beta3AR in adipocytes is constitutively coupled to both Galphas and Galphai, leading to the activation of the protein kinase A (PKA) and MAP kinase (ERK1/2) pathways, respectively, and that the adipocyte-specific expression of beta3AR is controlled in large part by C/EBPalpha and we defined the C/EBP site. In probing the regulation of the PKA and MAPK pathways in adipocytes by beta3AR, we have recently discovered that betaARs activate p38 MAPK kinase in adipocytes in a PKA-dependent manner, and that one of the important physiological endpoints of this regulation is the induction of the main thermogenic protein in brown fat, UCP1. Two main hypotheses are proposed. That the signaling cascade from PKA to MKK3/6 and p38 involves Rac1/cdc42 and the mixed- lineage kinases (MLKs), and that the p38 MAPK pathway is required in tandem with PKA to appropriately activate and recruit transcription factors to the UCP1-promoter, with the ultimate end point being increased thermogenesis. There are three Aims proposed: (1) to characterize the signal transduction pathway linking the betaARs in adipocytes to the p38 MAP kinase; to test the hypothesis that Rac1/Cdc42 and the MLKs are activated by PKA and, in turn, directly activate MKK3/6, the immediate upstream kinase for p38 MAPK; (2) to test the hypothesis that beta3AR- stimulation of p38 MAP kinase activity regulates PPAR-dependent transcriptional processes in adipocytes. We will test whether phosphorylation of PPARgamma and/or PGC-1 are required steps in the transactivation of the UCP1 gene, and test the hypothesis that a dual signaling cascade requiring PKA both directly and indirectly to modulate CREB and PPARgamma triggers UCP1 transcription; (3) to test the hypothesis that the orphan receptor RORgamma is a second critical transcriptional activator for the tissue-specific expression of the beta3AR gene in adipocytes. Experiments in this aim will also test the hypothesis that alterations in the profile of C/EBP proteins contribute to the obesity/diabetes dependent down-regulation of expression of the beta3AR and beta1AR genes in adipocytes.
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