| Background:With the development of economy, the change of living environment and habits, the prevalence of obesity in China and even the global is increasing. The number of obesity is nearly doubled since 1980. There are more than 1.4 billion people are overweight, including more than 200 million men and nearly 300 million women were obese in 20 years old and older adults till 2008. Obesity is affecting more and more people’s health:overweight and obesity is the sixth largest risk of death in globle, There are many diseases closely related to obesity, such as diabetes, hyperlipidemia, hypertension, coronary heart disease and some cancers(breast cancer and colorectal cancer). Obesity and the health problems caused by it bring heavy economic burden to various countries of the world’s.In adults, the pathological physiology of obesity is the increasing size of adipocytes, that is, the increasing of the content of intracellular triglyceride which is caused by the increasing of triglyceride synthesis and decreasing of lipolysis. In this study, we focus on the the lipolysis of triglyceride catabolism. Lipolysis means that triglycerides are lysised into free fatty acids and glycerol step by step, which are gulated by the many enzymes and factor. Once upon a time, hormone sensitive Lipase (HSL) was thought the only rate-limiting enzyme in the process, until the Adipose Triglyceride Lipase (ATGL) was found.ATGL was discovered in 2004 by three teams and the subsequent studies have found that it is a kind of important enzyme in the process of lipolysis:ATGL are responsible for the hydrolysis of triglycerides to diglyceride and then launched the decomposition of triglycerides. ATGL gene knock out mice appeared multiple organ triglyceride accumulation and the function of the heart was affected so seriously that it cause early death of mice. In human body, the mutation of ATGL gene can cause neutral adipose accumulation in organs. The study found that ATGL play an important role in the process of both basl lipolysis and the lipolysis stimulated by adrenaline. ATGL is an important rate-limiting enzymei in the process of lipolysis.Subclinical hypothyroidism (SCH) is defined as the increasing of thyroid stimulating hormone (TSH) levels in serum with free thyroxine levels within the normal range. Clinical data showed that subclinical hypothyroidism is associated with hyperlipidemia, obesity, etc, TSH and weight are positive related.TSH is one of the important protein molecular regulating the thyroid function in the body. It is combined with thyroid hormone receptor (TSHR) and then works. Researches has found that TSHR also expresses in adipose tissue, and TSH also has a certain function in regulating lipid metabolism:TSH can promote preadipose cells to differentiate into mature adipocytes. Other studies also found that TSH could promote the decomposition of triglycerides. Our research attempts to observe the effect of TSH on ATGL in vivo and in vitro and tried to explore the possible molecular mechanism.Objectives:1. The adipocytes have been verified to express functional TSHR. This topic proposed by observing the TSHR ATGL-/-mice adipocytes and expressed in TSH ATGL in mature adipocytes to explore the effect of TSH may play a role in the process of lipolysis and reveal the possible molecular mechanism of subclinical hypothyroidism cause obesity.2. By using cAMP agonist forskolin and PKA inhibitors H89 to observe whether cAMP/ PKA pathways involved in the effect of TSH on ATGL, and try to reveal the possible mechanism of this action.Methods:1. Cell culture:In accordance with the training programmes,3T3-Ll preadipocytes were induced to differentiated into mature adipocytes. In the process of cell differentiation, ATGL expression in cells during this process was observed. Treated mature adipocytes with different concentrations of TSH for different time course. After treatment, the ATGL expression in cells was observed.2. Animal Model:Tshr-/-mice were raised in barrier (SPF) environments, day and night, 12 hours a day and night cycle, free feed and water intake.4-6 weeks after weaning for genotyping, and litter male mice divided into three groups:Tshr+/+, Tshr+/- and Tshr-/-group, Tshr-/-mice were supplemented with T4 exogenously.3. Oil red staining was used to describe the lipid droplets in adipocytes.4. RT-PCR was used to detect the ATGL mRNA expression.5. Western Blotting was used to detect the ATGL protein expression.6. Immunofluorescence methods were used to describe the ATGL expression and distribution in adipocytes.7. Immunohistochemical was used to detect the ATGL expression and distribution in adipose tissue.8. SAS and SPSS statistical software were used to analysis the experimental data. Main measurement data using x-±s description, a few of groups were compared using analysis of variance. Variance of irregular data using Kruskal Wallis rank and inspection.Results:1 After knockout of Tshr gene in mice, the expression of ATGL in the epididymal adipocytes was increased significantly.Immunohistochemical was used to observe the content of ATGL in adipocytes in Tshr-/-mice and Tshr+/+mice, by using epididymal adipose tissue paraffin section. Immunohistochemical results show that, around the epididymis of mice in the adipose tissue paraffin section, lipid droplet is in the middle of adipocyte, and nucleus is located against the cell membrane. ATGL is distributed in the cytoplasm, it’s also close to the cell membrane, brown-yellow stained. Compared with Tshr+/+mice, expression of ATGL of Tshr-/-mice was increased. At the same time, we use Western blotting and RT-PCR to detect the expression of ATGL in epididymal adipose tissue. Compared with the Tshr+/+mice, both protein and mRNA expression of ATGL of Tshr-/-mice were significantly increased, that is, the expression of ATGL can be inhibited by TSH in adipocytes.2. The expression of ATGL was inhibited by in mature 3T3-L1 cells2.1 In the process of differentiation, the protein expression of ATGL in 3T3-L1 cells increased gradually. In 3T3-L1 preadipocytes, ATGL expression is rarely. With the increasing of differentiation, that is, while preadipocytes were differentiated into mature adipocytes, the expression of ATGL gradually increased. To the 16th day, while 80%--90% preadipocytes differentiated into mature adipocytes, the expression of ATGL is the highest.2.2 TSH suppression the expression of ATGL in mature 3T3-L1 adipocytes. We use 0.1μM, 1μM,2μM bTSH to treat mature adipocytes for 24 hours and 48 hours respectively and then discovered that compared with the control group, bTSH inhibited the expression of ATGL in mature adipocytes significantly. Processing after 24 hours,0.1μM,1 and 2μMbTSH treatment group compared with control group, the intracellular ATGL protein content decreased by 31.1% respectively (p< 0.01),57.3%(p<0.01) and 58.1%(p < 0.01); Processing after 48 hours, compared with control group, the 0.1μM, 1μM and 2μM bTSH treatment group decreased the intracellular ATGL protein content by 45.1%(p<0.01), 60.5%(p< 0.01) and 77.1%(p< 0.01) respectively. The differences were statistically significant. The effect of bTSH on ATGL is significantly obvious, especially while the cells were treated after 48 hours (p< 0.01). But the inhibition effect of bTSH on ATGL with 0.1μM and 1μM were not time-dependent:using 0.1μM bTSH and 1μM bTSH to treat cells for 24 hours and 48 hours respectively, the differences of two group were not obvious, only if the application of 2μM bTSH for 48 hours, the decrease of ATGL protein expression was more prominent than that of 24 hours.Mature adipocytes treated with 2μM bTSH. After 48 hours, RT-PCR was used to observe the RNA content of ATGL, we found that ATGL RNA content in cells about 70% less than that of control group, the difference was statistically significant (p< 0.01). ALL results showed that bTSH can inhibit expression of ATGL in mature adipocytes, this effect was dose-dependent.3 TSH activated PKA pathway to inhibit the expression of ATGL in adipocytes.3.1 Forskolin (cAMP agonists) inhibits the expression of ATGL in mature adipocytes. After 3T3-L1 cells were differentiated into mature adipocytes, they were treated with 2.5uM and 5uM forskolin respectively. After 24 hours, Western blotting methods was used to detect the ATGL expression in cells. The results show that compared with control group, ATGL expression in cells after treated with 2.5 uM forskolin was decreased by 47%(p< 0.01), and that of 5uM group was 52%(p< 0.01).3.2 H89 (PKA inhibitors) had no effect on ATGL expression in mature adipocytes.After 3T3-L1 cells were differentiated into mature adipocytes, they were treated with 5 uM and 10 uM H89 after 24 hours respectively. Western blotting method was used to detect the expression of ATGL in cells. The results showed that, compared with control group, the content of ATGL proteins in the two treatment groups had no changes (p> 0.05).3.3 TSH activating PKA, and then to inhibit the expression of ATGL in mature adipocytes. Cells were pretreated with 10uM H89 for 1 hour, and then treated with 2uM bTSH. The other cells, add 2 separately 2uM bTSH and 5uM forskolin and 10uM H89. The remaining dish works as blank control group. Cell were treated for 24 hours. We use Western blotting method and immunofluorescence method to detect the expression of ATGL within a cell. Western blotting shows that, compared with the control group, the expression of ATGL in bTSH group and forskolin group were obviously suppressed (p< 0.01, p< 0.01). In H89 group, the protein content of ATGL has no obvious change. Similarly, After treated with H89 before bTSH, the expression of ATGL did not change significantly, compared with the control group. Immunofluorescence results are consistent with that of Western blotting. ATGL is located within the cytoplasm of mature adipocytes, and compared with the control group, the fluorescence intensity of ATGL in bTSH and forskolin group decreased significantly, ATGL of H89 group showed no change compared with the control group Similarly, after treated with H89 and before treated with bTSH, fluorescence of intracellular ATGL did not change significantly compared with the control group. These results suggest that, after treated with H89, the PKA activity was inhibited. And PKA cannot be activated after TSH combined with TSHR PKA, making it impossible to play the inhibition of TSH on ATGL.Conclusions:1. TSH regulated the catabolism of triglyceride in adipocytes.2. TSH inhibited the expression of ATGL in mature adipocytes.3. TSH inhibited the expression of ATGL by stimulating cAMP/PKA pathway.Significance:The number of subclinical hypothyroidism is gradually increased. Subclinical hypothyroidism is associated with many diseases, but the evidence to diagnose and treat it is poor. This study enriched the out-thyroid function of TSH. It is supported that TSH play a part role in the metabolism of triglyceride, which offers a new understanding to us. |
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