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Regulation of brown adipocyte gene expression by protein kinase A and PPAR gamma signalling pathways

Published online by Cambridge University Press:  14 October 2011

H. Y. Chen  and
M. A. Lomax
H. Y. Chen
Affiliation:
Division of Nutritional Sciences, University of Nottingham, Nottingham, UK
M. A. Lomax
Affiliation:
Division of Nutritional Sciences, University of Nottingham, Nottingham, UK
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Abstract

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The brown thermogenic genes, uncoupling protein 1 (UCP1) and peroxisome proliferator activated receptor gamma coactivator 1 α (PGC1α) are regulated by protein kinase A (PKA)-dependent transactivation of the cAMP response elements (CRE) in the enhancer and proximal promoters(Reference Cao, Daniel and Robidoux1, Reference Karamitri, Shore and Docherty2). Agonists PPARγ also increases UCP1 and PGC1α gene expression(Reference Petrovic, Shabalina and Timmons3). The aim of the study is to establish the interaction between PKA and PPARγ signalling pathways on stimulation of UCP1 and PGC1α in response to forskolin, an activator of cAMP, and rosiglitazone, a PPARγ agonist.

Brown preadipocytes (HIB-1B) were transfected with either the UCP1 (3.1 kb) or PGC1α (2.6 kb) promoter luciferase reporter construct in the presence and absence of forskolin and rosiglitazone. UCP1 and PGC1α transcriptional activity were measured by luciferase assay and gene expression by real-time PCR (RT-PCR). All data were analysed by ANOVA.

Forskolin and rosiglitazone significantly increased UCP1 (P<0.001) and PGC1α (P<0.001) transcriptional activity. (Fig. 1). When forskolin and rosiglitazone were combined together, there was a synergistic increase in UCP1 and PGC1α (P<0.001) transcription. These results were confirmed in experiments measuring gene expression by RT-PCR. Inclusion of a PKA inhibitor (H89) down regulated both forskolin and rosiglitazone stimulated PGC1α and blocked forskolin stimulated UCP1 expression whereas the PPARγ antagonist (rosiglitazone) only inhibited UCP1 expression when forskolin and rosiglitazone were combined.

It is concluded that the PKA- and PPARγ-dependent pathways interact to induce synergistic regulation of UCP1 and PGC1α expressions.

Fig. 1.

Luciferase activities of 3.1 kb UCP1 and 2.6 kb PGC1α promoters. Values are from three independent experiments. ***P<0.001.

References

1.Cao, WH, Daniel, KW, Robidoux, J et al. (2004) p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene. Mol Cell Biol 24. 3057–3067.Google Scholar
2.Karamitri, A, Shore, AM, Docherty, K et al. (2009) Combinatorial transcription factor regulation of the cyclic AMP-response element on the Pgc-1alpha promoter in white 3T3-L1 and brown HIB-1B preadipocytes. J Biol Chem. 284, 20738–20752.CrossRefGoogle Scholar
3.Petrovic, N, Shabalina, IG, Timmons, JA et al. (2008) Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPAR gamma agonist. Am J Physiol Endocrinol Metab. 295, E287–E296.CrossRefGoogle Scholar
Figure 0

Fig. 1. Luciferase activities of 3.1 kb UCP1 and 2.6 kb PGC1α promoters. Values are from three independent experiments. ***P<0.001.