Abstract
Abstract: :
Purpose: Orbital fat expansion is a feature of Graves’ ophthalmopathy (GO). Differentiating preadipocytes express thyrotropin (TSH) receptor (TSHR) and become susceptible to modulation by TSHR agonists. We have simulated chronic TSHR signalling by retroviral transduction of preadipocytes with activating mutations. These mutations cause familial non–autoimmune thyrotoxicosis. Thyrocyte proliferation is increased by TSHR stimulation but over–stimulation inhibits proliferation. Methods: Retroviral vectors (pLNSX) were used to transduce human orbital preadipocytes, human adipo/osteoprogenitor MG 63 and HC C1 and murine preadipocyte 3T3–L1 cell lines, with Wild–type (WT) and activating mutant TSHRs L629F and M453T. Direct sequencing confirmed the appropriate transcripts, proliferation was determined by direct cell counting, basal and stimulated cAMP by RIA and adipogenesis by oil red–O staining following differentiation. Results: GO preadipocytes were inhibited to 77% (WT), 66% (L629F) and 34% (M453T) relative to non–transduced (p<0.0001). Non–GO preadipocyte proliferation was inhibited to 63% (WT) and 38% (L629F) relative to non–transduced (p=0.0036), insufficient numbers of the M453T cells accumulated to perform experiments. No effects on proliferation were seen in 3T3–L1, MG 63 or HC C1 cells. Elevations in basal cAMP were present in activating mutant non–GO, 3T3–L1, MG 63 but not in GO or HC C1 cells. Retroviral TSHR activation produced no spontaneous differentiation of GO and non–GO preadipocytes. PPARγ induced differentiation in cells whose proliferation was reduced to ∼30%, developed morphological characteristics of adipogenesis but no lipid accumulation (GO M453T/ non–GO L629F). In contrast a moderate reduction in proliferation (∼65%) increased lipid accumulation (GO L629F/ non–GO WT). Conclusions: TSHR activation alone is insufficient to induce spontaneous differentiation of human orbital preadipocytes but does inhibit their proliferation. A moderate reduction in proliferation accompanied increased lipid accumulation whereas a severe reduction in proliferation suppressed lipid accumulation. The reduced accumulation could be due to repression of terminal differentiation or stimulation of lipolysis. The model may have clinical parallels whereby TSHR stimulation stimulates adipogenesis in GO or reduces body fat in patients with activating TSHR mutations.
Keywords: orbit • proliferation