The Role of Thyrotropin Receptor Activation in Adipogenesis and Modulation of Fat Phenotype

Abstract

Evidence from clinical and experimental data suggests that thyrotropin receptor (TSHR) signaling is involved in energy expenditure through its impact on white adipose tissue (WAT) and brown adipose tissue (BAT). TSHR expression increases during mesenchymal stem cell (MSC) differentiation into fat. We hypothesize that TSHR activation [TSHR*, elevated thyroid-stimulating hormone, thyroid-stimulating antibodies (TSAB), or activating mutation] influences MSC differentiation, which contributes to body composition changes seen in hypothyroidism or Graves' disease (GD). The role of TSHR activation on adipogenesis was first investigated using ex vivo samples. Neck fat (all euthyroid at surgery) was obtained from GD (n = 11, TSAB positive), toxic multinodular goiter (TMNG, TSAB negative) (n = 6), and control patients with benign euthyroid disease (n = 11, TSAB negative). The effect of TSHR activation was then analyzed using human primary abdominal subcutaneous preadipocytes (n = 16). Cells were cultured in complete medium (CM) or adipogenic medium [ADM, containing thiazolidinedione (TZD), PPARγ agonist, which is able to induce BAT formation] with or without TSHR activation (gain-of-function mutant) for 3 weeks. Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, qPCR measurement of terminal differentiation marker (LPL). BAT [PGC-1α, uncoupling protein 1 (UCP1), and ZIC1], pre-BAT (PRDM16), BRITE- (CITED1), or WAT (LEPTIN) markers were analyzed by semiquantitative PCR or qPCR. In ex vivo analysis, there were no differences in the expression of UCP1, PGC-1α, and ZIC1. BRITE marker CITED1 levels were highest in GD followed by TMNG and control (p for trend = 0.009). This was associated with higher WAT marker LEPTIN level in GD than the other two groups (p < 0.001). In primary cell culture, TSHR activation substantially enhanced adipogenesis with 1.4 ± 0.07 (ORO), 8.6 ± 1.8 (foci), and 5.5 ± 1.6 (LPL) fold increases compared with controls. Surprisingly, TSHR activation in CM also significantly increased pre-BAT marker PRDM16; furthermore, TZD-ADM induced adipogenesis showed substantially increased BAT markers, PGC-1α and UCP1. Our study revealed that TSHR activation plays an important role in the adipogenesis process and BRITE/pre-BAT formation, which leads to WAT or BAT phenotype. It may contribute to weight loss as heat during hyperthyroidism and later transforms into WAT posttreatment of GD when patients gain excess weight.

Keywords: BRITE adipocytes; adipogenesis; body composition; brown adipose tissue; thyrotropin receptor; white adipose tissue.

Figures

Figure 1

Ex vivo analysis of deep neck fat. Samples were snap frozen, and total RNA was isolated. Gene transcripts were measured by qPCR, (A) thyrotropin receptor (TSHR); (B)ZIC1 (brown adipose tissue marker), shown as transcript copy number (TCN) per 1,000 copies of housekeeper gene APRT (adenosine phosphoribosyl transferase); standard PCR was used to analyze (C)LEPTIN (white adipose tissue marker), (D)CITED1 (BRITE marker), densitometry were measured and corrected to housekeeping gene (GAPDH). Representative photos were shown (gels with all samples had been included in Figure S1 in Supplementary Material). Post-ANOVA test for linear trend of CITED1 was performed (p = 0.009). Results expressed as mean ± SD of all samples studied (each performed in duplicate) (**p ≤ 0.01; ***p < 0.001).

Figure 2

Adipogenesis in subcutaneous preadipocytes was assessed by foci counting (n = 11, representative photos were shown with arrows indicating differentiating adipocytes), LPL transcripts measured by qPCR (n = 9) and oil red O (ORO, n = 4). Result presented as fold increase in TSHR* populations relative to empty vector controls. The table reports raw data for qPCR results expressed as transcript copy number per 1,000 copies of housekeeper gene APRT (adenosine phosphoribosyl transferase), together with foci numbers and ORO optical density values (mean ± SEM). Histograms = mean ± SEM of all samples studied (each performed in duplicate) (*p < 0.05; **p ≤ 0.01; ***p < 0.005).

Figure 3

Preadipocytes/fibroblasts from subcutaneous fat (n = 6) expressing activating thyrotropin receptor (TSHR) mutant L629F (TSHR*) and equivalent empty vector controls were cultured until confluent (day 0) before changing to differentiation medium for 22 days. Total RNA was isolated before or after adipogenesis. PRDM16 (relative expression ratio) (A), PGC-1α (B), and uncoupling protein 1 (UCP1) (C) transcripts were measured by qPCR. Results are expressed as comparative qPCR (relative ratio to APRT) of PRDM16 or absolute qPCR (PGC-1α and UCP1) of transcript copy number (TCN) per 1,000 copies of housekeeper gene APRT (adenosine phosphoribosyl transferase). Histograms = mean ± SEM of all samples studied (each performed in duplicate). Two-tailed t-test or Mann–Whitney test used for statistic analysis (*p < 0.03; **p = 0.01).