Role Of TSHR Expression In Adipocyte Differentiation And Its Association With Obesity

Obesity, a pathological body condition characterized by over accumulation of fat and long-term intaking of energy over its consumption, leading to a body weight over-gain. Researches showed that obesity increased the incidence of a variety of disease and was associated with increased risks of cardiovascular disease, metabolic syndrome and diabetes mellitus. However, the mechanism of obesity development is not fully understood, and has become a focus of extensive investigation. Therefore study on the pathogenesis of obesity is of significance for clinical prevention and control of a variety of metabolic diseases.Studies found that the incidence of obesity is the result of the interaction of a number of physiological, biochemical and behavioral factors. Adipose tissue plays a very important role in the incidence of obesity and the development process. Adipocyte differentiation is not only the physiological basis of normal body fat storage, but also the pathological basis of obesity. The core aspects of obesity is excessive differentiation and over-growth of fat cells, generating too multifunction disorder fat cells. Therefore, the molecular mechanism of fat cells’ growth, differentiation and apoptosis is becoming hot in obesity and its related diseases research area.Adipocytes derived from the pluripotent stem cells of mesoderm, which are the common precursor cells of bone cells, cartilage cells, muscle cells and fat cells. The specific mechanism determing these pluripotent stem cells into fat precursor cells is not yet clear, however, these pluripotent stem cells are capable to differentiate into fat precursor cells and thereafter mature adipocytes. A lot of reports mentioned about the specific regulatory mechanism of fat cell differentiation and maturation, which is actually a complex process of a series of fat cell-specific gene expression in differential times.Extrathyroidal Tshr expression, especially in adipocyte, has been confirmed, and found that it has biological functions. However, the mechanism of adipogenesis from fat precursor cells is not fully understood, especially with regard to its association with obesity. Further exploration on the role of Tshr in the process of fat formation, the regulatory mechanism and its association with obesity has become a focus of extensive investigations and is a major significance for clinical prevention and control of obesity. Experimental work is divided in three parts:Part I Possible Role of TSHR expression in adipocytes differentiationAim:To investigate the probable role of TSHR expression in adipogenesis of3T3-L1and3T3-F442A preadipocytes in vitro.Methods: 1.3T3-L1and3T3-F442A preadipocytes were induced to differentiate as described in literature. The morphology change was observed under inverted microscope and Oil-red-O staining was applied to further verify the preadipocyte differentiation.2. To further verify the preadipocyte differentiation, the expression levels of PPARy and ALBP, the two essential regulators and markers for adipocyte differentiation, were analyzed by RT-PCR and Western Blot.3. To explore the possible role of Tshr in adipogenesis, total mRNA and proteins were extracted from3T3-L1or3T3-F442A preadipocytes during the process of adipogenesis induction. Expression level of Tshr was determined by RT-PCR and Western Blot.4. Inverted phase contrast fluorescence microscope was exploited to analyze the expression level of Tshr between3T3-L1or3T3-F442A preadipocytes and adipocytes.5. To further investigate the role of Tshr in adipose differentiation, Tshr gene expression level of3T3-F442A preadipocytes was knocked down by introducing small hairpin RNA (shRNA). The shRNA targeting to Tshr was generated by using the lenti virus vector LV3according to the manufacturer’s instruction. After infection, stable transfectants of preadipocytes were selected. Its influence on PPARy and ALBP expressions and on adipose differentiation was evaluated subsequently.6. Then, biological role of bTSH (0,0.125,0.25,0.5,1.0,2.0μM) in3T3-F442A preadipocytes and adipocytes were assayed with cAMP direct immunoassay kit.7. Cells were cultured in differentiation medium plus bTSH (0.2μM,2μM) at the earlier four days and samples were harvested at day0, day2, day4, day8, day12, day14and day16. The effect of bTSH (0.2μM,2μM) on preadipocyte differentiation was assayed by OiI-Red-O staining. In order to test possible molecular mechanism, ERK and JNK activation of differentiated day2, day4, day8, day12and day16were checked by Western Blot.8. Differentiated adipocytes were further stimulated by bTSH (0.2μM,2μM) to detect the phosphorylation level of ERK and JNK by Western Blot.Results:1. Lipid droplets could be detected by Oil-red-O staining as early as day-4post differentiation induction, and reached its peak on day-12.2. The mRNA and protein levels of PPARy and ALBP were significantly increased from day-4post differentiation induction, compared with untreated cells (day-0induction)(P﹤0.05).3. To explore the possible role of Tshr in adipogenesis, Tshr protein level during the differentiation of3T3-L1and3T3-F442A preadipocytes were analyzed. Tshr expression level was significantly increased from day-4, compared with untreated cells (day-0induction)(P﹤0.05).4. Tshr protein expression was analyzed by inverted phase contrast fluorescence microscope following immuno-fluorescence staining. Tshr protein expression was significantly higher in differentiated adipocytes (day-12post induction) than that of the undifferentiated preadipocytes (day-0induction).5. To investigate the role of Tshr in adipose differentiation, Tshr gene expression was knocked down by introducing small hairpin RNA (shRNA) in3T3-F442A preadipocytes, and its effect on adipose differentiation was evaluated subsequently. When induced to differentiate, shRNA treated cells exhibited less intracellular lipid accumulation and lower protein levels of PPAR y and ALBP compared to vector treated control cells at each time point. These results indicated that Tshr-mediated signaling has the effect on adipose differentiation of3T3-F442A preadipocytes.6. Both3T3-F442A preadipocytes and adipocytes were stimulated with bTSH at different dose (0,0.125,0.25,0.5,1.0,2.0μM). Cell lysates were collected to detect cAMP contents with the cAMP Direct Immunoassay Kit according to the manufacturer’s instructions. Results showed that cAMP increased both in preadipocytes and in adipocytes, suggesting that Tshr expressed in preadipocytes and adipocytes was both functional.7. Preadipocytes stimulated with bTSH showed delayed differentiation, Oil-red-O staining extract measurement at OD510nm reduced in bTSH involved cells in comparison with control cells. bTSH stimulation inhibited the phosphorylation of ERK and JNK induced by insulin, suggesting that the mechanism of differentiation inhibition by bTSH maybe related with the interference of insulin signaling pathway (ERK and JNK phosphorylation).8. JNK and ERK were both activated under insulin, which were inhibited by bTSH in a dose-dependent manner at early differentiation stage (Day2and Day4). Statistic analysis showed significant difference was detected on day2and4(P<0.05). Data analysis by Image J soft was confirmed the results mentioned above.Conclusions:1. Tshr expression was increased during adipogenesis of both3T3-L1and3T3-F442A.2. Tshr expression silenced by shRNA inhibited adipogenesis of3T3-F442A in vitro.3. bTSH stimulation could inhibit early adipogenesis of3T3-F442A, the mechanism of which may lie in inhibition of insulin signaling (ERK and JNK phosphorylation). Part II The correlation between TSHR expression and occurrence of obesityAim:To investigate the association of TSHR expression with obesity occurrence in vivo.Methods:1. Animal study with obesity modelAdult male C57/BL6mice at8weeks old were purchased from the Experimental Animal Center of Shandong University. Animal experiments were in according to the ’Principles of laboratory animal care’established by the National Institutes of Health, and approved by the "Animal Care and Use Committee" of the Shandong University (number ECAESDUSM2011037). One group mice were fed with control diet contained5%fat, while the other group was fed with high-fat diet contained20%fat. The body weight of each animal was monitored once a week until the difference of body weight reached more than30%.The animals were sacrificed after deep anesthesia. Blood was collected in EDTA-coated tubes after fasting the mice for12hr. Serum cholesterol (CHOL), triglycerides (TG), aspartate transaminase (AST) and serum glucose (GLU) levels were measured. Visceral adipose tissues near the kidneys were collected for protein extraction.2. Human clinic sample studyAdipose tissue samples were collected from patients with benign cervical lesions who underwent surgeries in the Department of Otolaryngology Division of Head and Neck Surgery in Shandong Provincial Hospital Affiliated to Shandong University. These patients were divided in4groups based on their BMI (Body Mass Index, calculated as body weight (BW, kg) over squared height in meter):slim group, BMI≤20; normal group,20<BMI<25; overweight group,25≤BMI≤30; obesity group, BMI≥30. Written informed consent was obtained from all patients before surgery. All patients were operated under general anesthesia. Adipose tissues were got from the subcutaneous areas in the necks. The study was approved by the Ethics Committees of Shandong University (number MECSDUMS2011055).Results: 1. Animal study with obesity model(1) At the end of experiments, the average body weight was increased in obese mice (37.66±3.64) g compared to the control mice (25.82±1.05) g.31.44%difference was detected between control and obese mice. Significant difference could be found (P<0.05).(2) Serum CHOL, TG, AST and GLU levels were measured in obese mice compared with the control. The successful establishment of obese model was confirmed by the significantly higher serum CHOL levels in obesity mice (3.41±0.24) mmol/L after14weeks of diet induction, compared with control mice (1.61±0.31) mmol/L. Significant difference also could be found for GLU level between obesity mice (8.55±0.6) mmol/L and control mice (4.36±0.83) mmol/L (P<0.05). There is no significant difference between control mice and obesity mice in serum TG or AST quantification.(3) In each group, Tshr protein expression in visceral adipose tissues was shown to increase with body weight. The average Tshr protein level in obese mice was significantly increased in comparison with control mice (P<0.05).2. Human clinic sample studyThe TSHR protein expression in human adipose tissue was assayed by Western Blot and the correlation between BMI and TSHR protein expression in human visceral adipose tissues was analyzed. We found that the TSHR expression level was higher in people with BMI>30than that of those with20<BMI<25. Our results showed that TSHR protein expression in human visceral adipose tissues tend to increase with the increase in BMI.Conclusions:Tshr expression level was significantly higher in obesity fat samples both from aimal and human than that in their controls, suggesting that overexpression of Tshr in adipocytes may be a promising marker of obesity. Part Ⅲ Mechanism analysis of correlation between TSHR expression and obesityAim:To investigate the possible mechanism between TSHR expression and obesity occurrence.Methods:1.3T3-L1and3T3-F442A preadipocytes were induced to differentiate as described in literature. Confocal laser scanning microscope was exploited to analyze the expression level of Tshr located in cell membrane between3T3-L1or3T3-F442A preadipocytes and adipocytes based on in vitro culture systerm.2. Serum TT3, TT4, FT3and FT4contents of control mice and obesity mice were measured:Adult male C57/BL6mice at8weeks old were purchased from the Experimental Animal Center of Shandong University. Animal experiments were completed according to the’Principles of laboratory animal care’established by the National Institutes of Health, and approved by the "Animal Care and Use Committee" of the Shandong University (number ECAESDUSM2011037). One group mice were fed with control diet contained5%fat, while the other group was fed with high-fat diet contained20%fat. The body weight of each animal was monitored once a week until the difference of body weight reached more than30%. The animals were sacrificed after deep anesthesia. Blood was collected in EDTA-coated tubes after fasting the mice for12hr. Serum TT3, TT4, FT3and FT4levels were measured. Thyroid and visceral adipose tissues near the kidneys were collected and immersed into liquid nitrogen for quick frozen sections.3. Immuno fluorescence detection of Tshr between thyroid and visceral adipose tissues of control and obesity mice:quick frozen sections of thyroid and visceral adipose tissues from control and obesity mice were exploited for Immunofluorescence detections. Tshr expression level was measured under confocal laser scanning microscope.4. Serum TT3, TT4, FT3and FT4contents of clinic specimen with different BMI were measured:Serum samples were collected from patients with benign cervical lesions who underwent surgeries in the Department of Otolaryngology Division of Head and Neck Surgery in Shandong Provincial Hospital Affiliated to Shandong University. These patients were divided in4groups based on their BMI (Body Mass Index, calculated as body weight (BW, kg) over squared height in meter): slim group, BMI<20; normal group,20<BMI<25; overweight group,25<BMI<30; obesity group, BMI>30. Written informed consent was obtained from all patients before surgery. The study was approved by the Ethics Committees of Shandong University (number MECSDUMS2011055). Serum TT3, TT4, FT3and FT4levels were measured.5. Determination of cAMP levels in preadipocyte and adipocyte after TSH treatment with different doses.Results:1. Level of Tshr in cell surface expression was analyzed based on confocal laser scanning microscope imaging:Both Preadipocytes and mature adipocytes at the same horizon in3T3-L1and3T3-F442A were selected to analyze the Tshr cell surface expression level, we found that under the same excitation light intensity, Tshr fluorescencethe strength of mature adipocytes is significantly higher than that of preadipocytes.2. Determination of serum TT3, TT4, FT3and FT4contents of control mice and obesity mice:The serum TT3, FT3levels in obesity mice was (1.62±0.16) nmol/L,(6.38±0.58) pmol/L after14weeks of diet induction,,while in control mice was (1.65±0.12) nmol/L and (6.28±0.52) pmol/L. No significant difference was found (P>0.05). However, significant difference was found in TT4, FT4levels between obesity mice(11.32±1.04) nmol/L,(4.8±0.44) pmol/L and control mice (66.32±6.28) nmol/L,(28.56±2.94) pmol/L (P﹤0.05).3. Immunofluorescence detection of Tshr between thyroid and visceral adipose tissues in control and obesity mice:both control mice and obesity mice had higher Tshr protein expression level in their thyroid; but obese mice had higher Tshr expression level in their adipose tissure than that in control mice.4. Determination of serum TT3, TT4, FT3and FT4contents in clinic specimen with different BMI were measured:Serum TT3, TT4, FT3and FT4contents in clinic specimen with different BMI were all within normal biological reference interval, confirmed the normal thyroid function of all our samples. However, we found that serum TT3, FT3, TT4, FT4content gradually decreased with BMI increasing, suggesting that changes of thyroid hormone within the normal range correlated negatively with body weight.5. Both3T3-F442A preadipocytes and adipocytes were stimulated with bTSH at different dose (0,0.125,0.25,0.5,1.0,2.0μM). Cell lysates were collected to detect cAMP contents with the cAMP Direct Immunoassay Kit according to the manufacturer’s instructions. Results showed that cAMP increased both in preadipocytes and in adipocytes.Conclusions:So far, reports on the role of Tshr during obesity formation have been scarcely found. Our data showed that differentiated adipocyte has higher Tshr cell surface expression than preadipocyte, adipocyte in visceral fat in obese mice also has higher Tshr cell surface expression than that in normal control mice. Therefore, we speculate that because of large volume and surface area of fat tissue, large ratio of differentiated adipocytes will result in the limited TSH distributing to adipocyte instead of thyroid, so that the production of T4and T3will be reduced, in the end, result in adipocyte accumulation or obesity. On the other hand, Tshr in cell surface of preadipocyte is able to associtate with TSH and trigger TSH signaling, which will increase cellular cAMP level, consequently resulting in PKA activation, and activation of CREB (cyclic AMP response element binding protein), while, it has been reported that CREB was important in preadipocyte differentiation. Our previous data showed that knocking down TSHR expression will result in less lipid accumulation in adipocyte. We can speculate that during later differentiation stage of preadipocyte, or pre-mature adipocyte, TSH binding to TSHR in the cell surface will activate PKA and CREB, thereafter, trigger the expression of some genes related with lipid accumulation.