| Background:Obesity, the excess accumulation of white adipose tissue (WAT), is a major health hazard worldwide, and the epidemic incidence of obesity is increasing. Obesity is an established risk factor for metabolic diseases, including type 2 diabetes, hypertension, and nonalcoholic fatty liver disease. Obesity-related metabolic diseases are accompanied by abnormal serum lipid parameters, which are usually caused by higher TG levels and ectopic TG accumulation. WAT contains white adipocytes, which specialize in the storage of energy as triglycerides (TGs) and in the secretion of numerous adipokines that affect numerous aspects of metabolism. The etiology of obesity is complicated, with both genetic and environmental factors influencing its development and susceptibility. In this study, we identified a hormone involved in obesity.Thyrotropin (thyroid stimulating hormone, TSH) is a hypophyseal hormone, the major role of which is to stimulate thyrocyte proliferation, iodide uptake, hormonogenesis, and the release of thyroid hormones. Numerous studies have confirmed the association between serum TSH levels and obesity. Epidemiological evidence has indicated a positive correlation between elevated serum TSH concentrations and body mass index (BMI) in euthyroid subjects. In patients with metabolic syndrome and/or subclinical hypothyroidism (SCH, a type of thyroid disease accompanied only by elevated serum TSH levels), serum TSH levels are correlated with the severity of obesity. Furthermore, obese people exhibit higher serum TSH levels than non-obese people.However, the molecular mechanism by which TSH affects obesity has not been fully elucidated.Expression of the TSH receptor (TSHR), once thought to be limited to thyrocytes, has been detected in numerous extrathyroidal tissues, including liver and adipose tissues. Our previous study indicated that TSH promotes 3T3-L1 preadipocyte differentiation. Additionally, knocking downTshr blocked the effects of TSH on preadipocyte differentiation. Similar results were observed in rat preadipocytes, human orbital preadipocyte fibroblasts, and mouse embryonic stem cells.Obesity is a hypertrophic disease resulting from an increase in the number or size of individual adipocytes. We previously demonstrated that TSH could increase the number of adipocytes by promoting preadipocytes to differentiate into mature adipocytes. The number of adipocytes is set during childhood and adolescence. Adipocyte hypertrophy due to increased TG synthesis was recently shown to be the determinant of the development of adult obesity.Brook showed an increase in adipose cell size among all obese sub-jects, but the total number of adipose cells was only increased in obese children and in adults who developed obesity during child-hood. Thus, adipocyte size is a major determinant of obesity in adults. Still, the role of TSH in regulating TG synthesis among differ-entiated adipocytes has not been completely established.Glycerol-3-phosphate acyltransferase (GPAT) is the rate-limiting enzyme involved in TG synthesis. GPAT3 is the major GPAT iso-form expressed in adipocytes and plays a crucial role in adipogenesi. The over-expression of GPAT3 in mammalian cells resulted in increased TG formation, whereas the targeted knockdown of GPAT3 in 3T3-L1 cells significantly impaired adipogenesis.Recent studies have indicated that the expression and activity of GPAT3 are regulated by insulin, alcohol, the glucocorticoid receptor, and thiazolidinediones (peroxisome proliferator-acti-vated receptor y agonists). However, whether TSH affects the adi-pogenesis of differentiated adipocytes through GPAT3 has not been previously investigated. In the present study, we tested the hypothesis that TSH might upregulate GPAT3 expression, resulting in adipo-genesis and obesity. This study provides a more comprehensive understanding of the pathophysiological effects of TSH on adipogen-esis and suggests a novel interpretation of the mechanism that causes metabolic syndrome (MS) or SCH patients to develop obesity and obesity-related metabolic diseases.Objectives:1. To determine TSH regulates triglyceride content in adipose tissue, using SCH mouse models and differentiated 3T3-L1 adipocytes to explore the effects of TSH on TG synthesis.2. To determine the effect of TSH on adipogenesis, we conducted a systematic and quantitative analysis of a panel of genes involved in adipogenesis in WAT.3. To determine the possible mechanism of TG synthesis elevation induced by TSH in differentiated 3T3-L1 adipocytes.4. To determine the possible mechanism of adipogenesis induced by TSH in TSHR knockout mice.5. To determine the effect of conditioned media from 3T3-L1 adipocytes transfected by TSHR,PPAR y,GPAT3 siRNA or CA,DN-AMPK on adipogenesis.Methods:1. Cell culture1)3T3-L1 cells:cells were grown in media consisting of high glucose DMEM medium supplemented with 10% fetal bovine serum and induction differentiation medium according to guidance from ATCC and relative references 2) Primary human/mouse adipocytes:human/mouse thyroid tissues was cut into pieces and digested in a mixture of type Ⅰ collagenase and trypsin for 40-60 min at 37℃ then cultured in DMEM/F12 with newborn fetal serum.2. Animal modelSCH mice models:mice were given methimazole, a drug that inhibits thyroid hormone synthesis [MMI,0.04 mg/kg BW*d] in the drinking water, adjusted the MMI dose according to the body weight. TSHR knockout mice were bought from Jackson library.3. RT-PCR:was adopted to determine the expression of GPAT3 、PPAR y mRNA.4. Protein extraction and immunoblotting analysis:was adopted to determine the expression of GPAT3、PPARγ、p-AMPK、t-AMPK protein.5. Immunohistochemistry and Immunofluorescence:were adopted to determine the expression of GPAT3、PPAR γ.6. GPAT3,PPAR γ,TSHR silence:using siRNA to silent GPAT3,PPAR y or TSHR expression7. Activated or inhibited AMPK:using CA-AMPK or DN-AMPK to activated or inhibited AMPK expression.8. GPAT3, PPAR y activity:using luciferase report system to detected GPAT3, PPAR γ activity.8. ELISA:was adopted to determine the expression of adiponectin.Results:1. TSH regulates triglyceride content in adipose tissueThe SCH mice exhibited higher body, total fat pad weight and BMI levels. We found an apparent increase in total fat pad mass and adipocyte size in SCH mice compared with those in control mice. The Epi-WAT mass to body weight ratio revealed a 25% relative increase in SCH mice compared with their littermate controls. In addition, both the Epi-WAT TG content and serum TG levels were increased in SCH mice.In vitro, TSH stimulation significantly increased the number of lipid droplets, and the intracellular TG contents increased in a dose-and time-dependent manner in vitro. These results are consistent with the increase in TG caused by TSH treatment.2. Regulation of TG synthesis by TSH through GPAT3In vivo, we found TSHR-ko mice had lower GPAT3 mRNA and protein levels. In vitro, after treating differentiated 3T3-L1 adipocytes with TSH, the GPAT3 mRNA levels were enhanced, and the protein levels increased in a dose-and time-dependent manner. Immunofluorescence analysis indicated that after TSH treatment, the increased GPAT3 protein was mainly located in the cytoplasm in a dose-dependent manner. After we transfected GPAT3 siRNA into differentiated 3T3-L1 adipocytes, GPAT3 silencing markedly attenuated TSH-induced adipogenesis. The Oil Red O staining was weak and the TG content significantly dropped relative to the group treated with only TSH.3. PPAR y is indispensable in TSH-induced adipogenesis.In vivo, we found TSHR-ko mice had lower PPAR y mRNA and protein levels. In vitro, after treating differentiated 3T3-L1 adipocytes with TSH, the PPARγ mRNA levels were enhanced, and the protein levels increased in a dose-and time-dependent manner. Immunofluorescence analysis indicated that after TSH treatment, the increased PPARγ protein was mainly located in the nucleus in a dose-dependent manner. After we transfected PPAR y siRNA into differentiated 3T3-L1 adipocytes, PPAR y silencing markedly attenuated TSH-induced adipogenesis, the TG content in PPAR y silencing group significantly dropped relative to the group treated with only TSH.4. TSH-stimulated adipogenesis in differentiated adipocytes is dependent on AMPK.In vivo, we found AICAR administrated mice had lower PPAR γ/GPAT3 mRNA and protein levels. In vitro, After we transfected CA-AMPK into differentiated 3T3-L1 adipocytes, CA-AMPK markedly attenuated TSH-induced adipogenesis and PPAR γ/GPAT3 protein levels. After we transfected DN-AMPK into differentiated 3T3-L1 adipocytes, DN-AMPK markedly promoted TSH-induced adipogenesis and PPAR γ/GPAT3 protein levels.5. Tshr ablation ameliorates HFD-induced obesity.For the in vivo study,the body weights of Tshr-/- mice were less than those of the Tshr+/+ mice. In Tshr-/- mice, due to the absence of TSHR expression, the Epi-, retroperitoneal-adipose mass and adipocyte size were significantly decreased compared with those of the control mice. Decreased GPAT3 protein levels were observed in the adipose tissue of Tshr-/- mice. As a result, the intracellular and plasma TG contents were downregulated. In vitro, we transfected 3T3-L1 adipocytes with Tshr siRNA. Compared with the non-targeting siRNA, Tshr siRNA inhibited the effects of TSH on the regulation of TG levels and lipid droplet content. Furthermore, Tshr siRNA inhibited the effects of TSH on the upregulation of PPARy and GPAT3 expression, although AMPK phosphorylation increased.Conclusions:In this study, we demonstrated that TSH, by binding to TSHR, inhibits AMPK Thr172 phosphorylation, activates the PPARy transcriptional program, and subsequently increases GPAT3 expression in mature adipocytes, contributing to the pathogenesis of obesity. |
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