Expression of the brown adipocyte-specific gene, uncoupling protein 1 (UCP1), is increased by both PPAR stimulation and cAMP activation through their ability to stimulate the expression of the PPAR coactivator PGC1 . In HIB1B brown preadipocytes, combination of the PPAR agonist, rosiglitazone, and the cAMP stimulator forskolin synergistically increased UCP1 mRNA expression, but PGC1 expression was only increased additively by the two drugs. The PPAR antagonist, GW9662, and the PKA inhibitor, H89, both inhibited UCP1 expression stimulated by rosiglitazone and forskolin but PGC1 expression was not altered to the same extent. Reporter studies demonstrated that combined rosiglitazone and forskolin synergistically activated transcription from a full length 3.1？kbp UCP1 luciferase promoter construct, but the response was only additive and much reduced when a minimal 260？bp proximal UCP1 promoter was examined. Rosiglitazone and forskolin in combination were able to synergistically stimulate promoters comprising of tandem repeats of either PPREs or CREs. We conclude that rosiglitazone and forskolin act together to synergistically activate the UCP1 promoter directly rather than by increasing PGC1 expression and by a mechanism involving cross-talk between the signalling systems regulating the CRE and PPRE on the promoters. 1. Introduction Nonshivering thermogenesis in brown adipose tissue (BAT) in response to a cold environment is initiated by sympathetic neural stimulation of -adrenergic receptors on brown adipocytes which elevate intracellular cyclic AMP (cAMP) and, via the protein kinase A (PKA) pathway, increase the expression and thermogenic activity of uncoupling protein 1 (UCP1) [1]. UCP1 is BAT specific and responsible for uncoupling oxidative phosphorylation by enabling protons to return to the mitochondrial matrix without ATP synthesis, thereby producing heat. UCP1 expressing BAT has recently been identified in humans and has been proposed as a target for activation to increase energy expenditure and prevent or treat obesity [2]. UCP1 expression has been suggested to be regulated by the cAMP-inducible peroxisome proliferator activated coactivator 1 (PGC1 ) which interacts with a BAT determination factor, PRDM16, to increase the expression of a number of BAT-selective genes including Cidea [3]. We have also shown that the cAMP-inducible transcription factor C/EBP stimulates PGC1 expression in white and brown adipocytes by binding to the cAMP response element (CRE) on the PGC1 proximal promoter [4, 5] while others have demonstrated that PKA activation of
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