Characterization of early steps in adipocyte differentiation in C3H10T1/2 cells and the inhibitory activity of the aryl hydrocarbon receptor

2,3,7,8-tetrachlorodibenzo-p-dioxin (TODD) suppresses adipocyte differentiation in C3H10T1/2 cells by activation of the aryl hydrocarbon receptor (AhR). The AhR is endogenously activated in response to changes in cell adhesion. The mechanism of AhR activation is a key objective in characterizing a role of the AhR in adipogenesis. A central hypothesis of this thesis is that the AhR is a natural repressor of adipogenesis, either endogenously or via TODD-dependent processes. This work has resulted in the identification of key regulatory steps in AhR activation in the absence of an external activator like TCDD. This work characterizes loss of cell-cell contact as an initial activation signal for the AhR. This hypothesis is examined in 10T1/2 cells under suspension and low density cultures. Endogenous AhR activation, by disruption of cell-cell contact and its nuclear activity, was not affected by the competitive inhibitor α-naphthoflavone. This provides evidence for a process that does not involve a TCDD-like natural ligand. Hormonal mixture of insulin, methylisobutylxanthine and dexamethasone stimulates adipogenesis including morphological differentiation while insulin and serum change are not essential. Cell cycle progression from G1 to G2/M and DNA replication occurred concurrently during this initial 24 h after hormonal stimulation of IDM. DNA replication was shown to be a required step for adipocyte commitment, independent of subsequent cell divisions. Morphological commitment to adipogenesis is induced by MIX stimulation during this period. This change comprised disruption of actin stress fibers and focal complexes, and was fully blocked by TCDD treatment. I have developed a low density model of adipogenesis that demonstrates that cell-cell contact is not a requisite for adipogenesis. Low density adipogenesis required insulin along with DM, but was not suppressed by TCDD activation. This model also demonstrates that single, isolated cells either arrest and differentiate, or proliferate. I have also provided evidence that AhR overexpression independent of TCDD suppresses both PPARγ expression and adipogenesis in parallel. In the same cells hormonal stimulation dramatically lowered the natural AhR transcription. This provides strong evidence that the lowering of the AhR is a key part of the onset of increased PPARγ1 induction and subsequent adipogenesis.