title: Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.
abstract: Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gbetagamma signalling may be inhibitory but failed to induce adipogenesis using activated Gsalpha (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARgamma (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARgamma1 was reduced and PPARgamma2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsalpha signalling impedes FOXO1 phosphorylation and thus inhibits PPARgamma transcription and the alternative promoter usage required to generate PPARgamma2, the fat-specific transcription factor necessary for adipogenesis.
authors.affiliation: School of Medicine, Centre for Endocrine and Diabetes Sciences, Cardiff University, Heath Park, Cardiff, UK.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Carol
authors.initials: C
authors.lastname: Paddon
authors.forename: Mark D
authors.initials: MD
authors.lastname: Lewis
authors.forename: Fiona
authors.initials: F
authors.lastname: Grennan-Jones
authors.forename: Marian
authors.initials: M
authors.lastname: Ludgate
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D050156:Adipogenesis / Q000502:physiology*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D047495:PPAR gamma / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D010766:Phosphorylation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013234:Stem Cells / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D047495:PPAR gamma; D020033:Protein Isoforms; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs