In rodents, TCDD treatment causes a general depletion of body fat with a parallel increase in serum triglycerides and diabetic-like insulin resistance. Mouse embryo fibroblast (MEF) lines can be induced to differentiate to fat cells (adipocytes) by a hormonal mixture (IDM) containing insulin (I), a glucocorticoid (dexamethasone [D]) and a means to elevate cAMP (methylisobutylxanthine [M]) in combination with a stimulant for the peroxisome proliferation activation receptor-type gamma2 (PPARgamma2), usually a thiazolidinedione (TZD). Previous work has shown that a pre-adipocyte line 3T3-LI undergoes this differentiation without TZD and this is blocked by TCDD at an early stage. We have shown that the TZD stimulated adipogenesis in primary MEF's and equivalent cell lines is also blocked by TCDD. In contrast MEF's cultured from mice deficient in the Ah-receptor (AhR(-/-)EF) no longer respond to TCDD, consistent with the AhR being required in this suppression. MEF's express a form of cytochrome P450 (CYPlBl) that is expressed constitutively under AhR control and which is highly induced TCDD. MEF's from CYPlBl-deficient mice are completely converted to fat cells with IDM stimulation without the need for external PPARgamma2 stimulation. The proposed research addresses the role of the AhR in regulating PPARgamma2 and CYPlBl during normal adipogenesis and in inhibiting this process when activated by TCDD. We will test the hypothesis that TCDD inhibits the increased activity of PPARgamma2 following IDM/TZD stimulation, which is critical to the initiation of differentiation. The research will focus on effects of endogenous AhR and TCDD specifically during the period of proliferative arrest (2 days after IDM stimulation) where nuclear regulatory factors such as c/EBPbeta and SREBP-1 stimulate expression of PPARgamma2. This will be linked to measurement of other potential early initiators of the differentiation pathway that respond to PPARgamma2 (RB-family proteins, PP2Ac immunophilins). We will establish that these effects correlate with the subsequent progression of adipogenesis after a further 5 days. We will further test the possibility that CYPlBl checks adipogenesis by metabolizing an endogenous activator of PPARgamma2 and that elevation of this process by TCDD contributes to the anti-adipogenic effect. This research will provide insight into general mechanisms through which TCDD alters differentiation, as well as provide a noel approach to the mechanism of adipogenesis and the action of TZD drugs, now a treatment for Type II disease.
