Endocrine Abstracts

Adipogenesis is induced when intracellular cAMP increases and follows cell cycle arrest. During preadipocyte differentiation expression of the TSHR, which signals via cAMP and the inositol phosphate cascade, is upregulated. To investigate the role of TSHR activation in adipogenesis, we have developed an in vitro model in which constitutively active TSHR mutants (L629F, M453T) are introduced by retroviral vectors (RV).

We aimed to compare the effects of TSHR activation on preadipocyte cell lines derived from brown (BAT) and white (WAT) adipose tissue.

NIH3T3LI (murine WAT) and PAZ6 (human BAT) cell lines, non-modified or stably expressing the WT, L629F or M453T human TSHR were studied. Proliferation was assessed by direct counting (Coulter). Basal cAMP was measured by RIA. Spontaneous lipid accumulation was determined as the absorbance (490 nm) following oil red O stain.

Expression of activating TSHR mutants induced morphological changes. 3T3-L1 had a more rounded appearance, accompanied by significant increase in population doubling time (PDT). Proliferation of PAZ6 cells was severely inhibited, with WT and L629F expressing cells ceasing to grow as monolayers and forming aggregates, M453T barely proliferated.

In both cell types, the mutant TSHR expressing cells demonstrated increased spontaneous lipid accumulation. cAMP increased 127-220% in activating mutant expressing 3T3-L1 compared with non-mod & WT.

The results indicate that TSHR activation stimulates WAT and BAT adipogenesis – possibly via cell cycle arrest consequent to elevated cAMP. The model could be improved by using inducible RV, to vary TSHR expression, and resemble more closely the in vivo situation. Our preliminary results with such a system are promising.