| Background:subclinical hypothyroidism (SCH), the most common endocrine and metabolic disease, which was defined as high serum thyrotropin (thyroid stimulating hormone, TSH) levels whereas free thyroxin (FT4) within normal levels, and it was regarded as the early stage of clinical hypothyroidism (CH). In adults, the prevalence of SCH ranges from 4% to 10% of the population in different regions, especially, the prevalence was as high as 20% in over 60 years old women. Researches show that: SCH has been a popular epidemiological trend, whose prevalence continues to grow. It has become the important reason for the secondary hypertension and dyslipidemia, and it is the independent risk factor for diseases such as coronary heart disease, fatty liver disease and so on. In addition, the large-scale population studies and our previous epidemiological and clinical studies have shown that:serum TSH level was positively correlated with blood cholesterol and triglyceride levels. SCH is becoming a global health problem for its increasing prevalence and potential deleterious effects.SCH was often concomitant with lipid metabolic abnormalities. However, the molecular mechanism of lipid metabolic disorders in SCH has not been fully clarified. Liver has a crucial role in metabolic homeostasis, especially, in lipid metabolism. Therefore, we chose the hepatic lipid metabolic disorders in SCH as the main target in our study.Endoplasmic reticulum (ER), an important organelle in hepatic cells, provides a specialized environment for the production and post-translational modification of lipid biosynthesis. Endoplasmic reticulum stress (ERS), which is defined as disruption of ER homeostasis by various factors, leading to the accumulation of misfolded protein in the endoplasmic reticulum (ER) and activating the unfoled protein response (UPR). When the ER is stressed, Bip is dissociated from these stress sensors IRE1? (inositol requiring transmembrane kinase and endonuclease1?), PERK (PKR-like ER kinase) and ATF6a (activating transcription factor 6a), triggering the downstream signaling cascade, leading to activate the IREla/XBP-1 (mRNA-encoding-X-box-binding protein) pathway, PERK/eif2a pathway and ATF6a pathway, and finally, leading to trigger diseases. Now there are a large number of studies have shown that ERS is involved in impairment of energy metabolism, apoptosis, lipotoxicity, insulin resistance and inflammation, which has a closely correlation with lipid metabolic disorder disease, such as, diabetes mellitus, cardiovascular diseases, and NAFLD.The SREBPs are key regulators of lipid homeostasis and play crucial role in de nova lipogenesis. SREBPs are basic-helix-loop-helix-leucine zipper(bHLHLZ) transcription factors bound to ER membrane as inactive precursor. SREBP-1 regulates fatty acid and triglyceride metabolism whileas SREBP-2 controls cholesterol metabolism and LDL receptor expression. SREBP activity is controlled within ER by interaction of SREBP cleavage activating protein (SCAP) with insulin regulated proteins; Insigs. It has been shown the ER stress regardless of its source (disturbance of calcium homeostasis, glycosylation or the redox state) induces the rapid cleavage of precursor form of SREBP-1c and expression of SREBP-1c target genes independent of insulin. Furthermore ER stress induces proteolytic activation of SREBPs by increasing the turnover of Insigl and over expression of Insigs have been shown to reduce hepatic lipogenesis. Recent evidences have shown that ER stress can increase de nova lipogenesis and lipid droplet formation in hepatocytes by upregulating the transcription of genes encoding key lipogenic trans-activators and enzymes. The expression of other lipogenic related transcription factor ChREBP is also enhanced by ER stres.So, is ERS also involved in the lipid metabolic disorders in SCH? Up to now, there have not been relevant reports.Both hemi-thyroid electrocauterisation and thyroidectomy has been adopted to set up SCH animal model. Nevertheless, some inadequacies still existed in these two methods:traumatic processes could trigger inevitable damage to animals, meanwhile they both requested researchers had skilled operation experience.Therefore, establishing an appropriate, noninvasive and ideal SCH animal model will be conductive to investigate the pathological characteristics of SCH. Particularly, exploring the molecular mechanism of lipid metabolic disorders in SCH may improve the theory on lipid metabolism associated with thyroid disease. Objective:1. To establish an novel SCH mouse model.2. To evaluate the whole body metabolism and lipid metabolism in SCH mouse model.3. To evaluate whether exists hepatic ERS in SCH mouse model.4. To explore the relationship between ERS and lipid metabolic disorders in SCH mouse model. Methods:1. A common antithyroid drug — methimazole (MMI,0.08 mg/kg·d) was applied to C57BL/6 mice to construct a noninvasive SCH mice model. After MMI were respectively administered for 12 weeks,16 weeks and 20 weeks, serum free triiodothyronine (FT3) and free thyroxin (FT4) were detected by determined by competition radioimmunoassay (RIA) binding assays kit and serum TSH was determined by a mouse Elisa kit. Alanine Transaminase (ALT) and Aspartate Transaminase (AST) of all mice were determined to evaluate liver function.2. Food intake, physical activity, VCO2 (production of carbon dioxide), VO2 (oxygen consumption), RQ (respiratory quotient), heat production and BMR (basic metabolism rate) data of mice were recorded by the metabolic chambers.3. Assessment of lipid metabolism in SCH mice. Serum lipid profiles were measured in clinical clinical laboratory. The content of total cholesterols and triglycerides in liver were assayed using a total cholesterol assay kit and a triglyceride assay kit. Filipin staining was used to determine hepatic cholesterol accumulation and Oil red O staining was used to determine hepatic lipid accumulation. The mRNA expressions of key molecules involved in hepatic lipid metabolism were detected by Real-time PCR.4. Assessment of hepatic ERS in mice. The expression of Bip, phospho-IRE1?, total IRE1?, XBP1u, XBP1s, phospho-eif2a, total eif2a and ATF6a were detected by Western blot.5. HepG2 cells were treated with or without bTSH or tunicamycin for 6 hours to observe ERS. The expression of Bip, phospho-IRE1?, total IRE1?, XBP1u, XBP1s were detected by Western blot.6. SCH mice were injected intraperitoneally with 4-Phenylbutyrate (4-PBA, a chemical chaperone which can inhibit ERS in cells) for 4 weeks. Then we evaluated the situation of hepatic ERS, and meanwhile, we also detected the lipid metabolism status in mice.Results:1. SCH mouse model was successfully constructedWhen using MMI (0.08 mg/kg·d) for 12 weeks, the mice showed the diagnostic variation of SCH compared to the controls, serum TSH levels in SCH group significantly increased (P< 0.05), and serum thyroid hormone (FT3 and FT4) levels were undistinguishable (P> 0.05). Administering MMI in their drinking water for 16 weeks, SCH state persisted. But, for 20 weeks, serum FT4 in SCH mice was lower than that in controls (P< 0.05), which indicated that model mice had entered into the phase of clinical hypothyroidism (CH). These results suggested that after the application of MMI, mice indeed appeared a process from SCH to CH, and the SCH state persisted nearly eight weeks.2. The whole body metabolic situation of SCH mouse modelFor the sake of comprehensively assessing the SCH mouse model, metabolic chambers were used to evaluate the whole body metabolic situation. When using MMI for 16 weeks, model mice were kept in SCH state, physical activity as well as heat production of SCH mice was no discrepancy with that of controls. Consistent with the clinical traits of SCH disease, production of carbon dioxide (VCO2), oxygen consumption (VO2), respiratory quotient (RQ),heat production and basic metabolism rate (BMR) were all no difference between SCH mice and control mice These results suggested that the entire body metabolism were no discrepancy between SCH mice and control mice, which conformed to the clinical features of SCH.3. Dyslipidemia and Hepatic lipid metabolic disorders in SCH miceAt the 12th week, the mice were just beginning to be SCH. Serum lipid profiles and hepatic cholesterol content of SCH mice were no remarkable difference with that of control mice. But hepatic triglyceride (TG) contents of SCH mice increased significantly than that of control mice (P<0.05). Simultaneously, more lipid droplet accumulation was observed in the liver of SCH mice by oil red O staining. At the 16th week, the status of SCH was persisting for 4 weeks. Serum TC, LDL-C and TG levels in SCH mice increased significantly relative to controls (P<0.05). Hepatic cholesterol and triglyceride contents in SCH mice were significantly higher than controls (P<0.05). The Filipin staining and oil red O staining were both further confirmed this above results. HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase) is the rate-limiting enzyme for cholesterol synthesis. CYP7A1 (Cytochrome P450, Family 7, Subfamily A, Polypeptide 1) is the rate-limiting enzyme for cholesterol converting into bile acids. In comparison with controls, the mRNSA expression of HMGCR in SCH mice increased significantly while the expression of CYP7A1 decreased. Also, expressions of the key genes involved in TG synthesis, such as SREBP1C and PPARa, were significantly up-regulated.4. Hepatic ERS was induced in SCH mice.The expressions of p-IRE1? (the active form of IRE1?) and XBP-ls (an active spliced form of XBP-1) in SCH mice were higher than that of controls. But, the expression of ATF6a and p-eif2? did not show obvious differences in two groups, which indicated that IRE1?/XBP1s pathway may play an important role in hepatic ERS in SCH. To eliminate the influence of MMI, and confirm the direct effect of TSH on ERS, HepG2 cells were treated with different concentrations of bTSH. Besides, TM (tunicamycin, an ERS inducer) was applied as a positive control. Interestingly, after bTSH treatment, the protein levels of Bip, p-IRE1? and XBP-1s in hepG2 cells increased in a dose-dependent manner, which was consistent with the results observed in vivo.5. ERS might play an indispensable role in lipid metabolism disorders in SCH4-PBA (a chemical chaperone which can inhibit ERS in cells) was used to alleviate TSH-induced ERS for 4 weeks to observe serum lipid profile and liver lipid metabolism. The serum lipid profile in 4-PBA treated SCH mice markedly fell back to normal ranges by inhibiting ERS. Satisfactorily, we could also observe the similar effect in the determination of liver lipid contents. The treatment of 4-PBA obviously ameliorated the increase of hepatic cholesterol and triglyceride contents in SCH mice (P<0.05). Filipin staining and oil red O staining supported the above findings. Therefore, blocking ERS using 4-PBA in mice could significantly alleviate SCH-induced lipid metabolic disorders. Together, these findings manifested that hepatic ERS played an important role in lipid metabolic abnormalities in SCH. Conclusions:1. A novel appropriate, noninvasive and ideal SCH mouse model could be established by using MMI.2. ERS might play an indispensable role in lipid metabolism disorders in SCH3. Blocking ERS in SCH could significantly alleviate SCH-induced lipid metabolic disorders. |
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