Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2008) 15 P148

SFEBES2008 Poster Presentations Diabetes, metabolism and cardiovascular (51 abstracts)

Thyrotropin receptor activation and preadipocyte biology; dissecting the effects of Gsα and Gβγ signalling on 3T3-L1 preadipocytes

Lei Zhang , Carol Paddon , Fiona Grennan-Jones & Marian Ludgate

Cardiff University, Cardiff, UK.

TSH receptor (TSHR) expression increases >100-fold during adipogenesis and signals via CREB. Since CREB activation has been reported to be ‘necessary and sufficient to induce adipogenesis’, we investigated whether TSHR activation, a feature of most thyroid dysfunction, has a role in adipocyte biology and evaluated the contribution of Gsα and Gβγ signalling.

Retroviral vectors for WT or constitutively active mutant TSHR* or gsp were introduced into 3T3L1 preadipocytes. Experiments were performed in complete (CM) or differentiation medium (DM), containing a PPARγ agonist. Proliferation was assessed by counting; adipogenesis by oil red O (ORO) staining and QPCR measurement of differentiation markers. Other measurements included cAMP (RIA) and pCREB (western blot).

In CM, non-modified cell population doubling time was 20.45(0.46) h, WT were not significantly different, gsp, and TSHR* proliferated more slowly (P<0.01). Unstimulated cAMP and pCREB levels were modestly (180 to 300%) but significantly (P<0.02) increased in TSHR* and gsp, compared with WT or non-modified. Despite a significant increase in basal levels of PPARγ transcripts, spontaneous adipogenesis, assessed by morphology and differentiation markers, did not occur in the TSHR* or gsp expressing cells, even with addition of a phosphodiesterase inhibitor.

After ~10 days in DM, non-modified and WT cells displayed ORO positive lipid droplets and increased PPARγ (5-fold) and GPDH transcripts (50-fold). In contrast, TSHR* expressing cells had significantly fewer lipid droplets, reduced ORO staining and less induction of GPDH. The gsp cells were devoid of droplets, GPDH was not upregulated. In all populations levels of pCREB were 150 to 300% higher after 10 days in DM, compared with CM.

Our results do not confirm previous reports on the role of CREB - but do support the negative impact of Gsα - in adipogenesis. They suggest that Gβγ signalling partially abrogates Gsα inhibition of differentiation.

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