1.TSH Promotes Adiposity By Inhibiting The Browning Of White Fat 2.Monocyte Fraction In Peripheral Blood Is Associated With Nonalcoholic Fatty Liver Disease

BackgroundThe high prevalence of obesity and its comorbidities have inspired an increase in research on adipose tissues.There are three types of adipose tissue.White adipose tissue(WAT)is characterized by large unilocular lipid-droplet-containing white adipocytes,which are generally believed to store excess energy and act as an active endocrine organ that secretes adipocytokines to regulate diverse physiological functions.Brown adipose tissue(BAT)is morphologically and functionally different from WAT.BAT is composed of multiple lipid droplets and is rich in mitochondria that express uncoupling protein 1(UCP1).BAT specializes in energy consumption via thermogenesis to maintain body temperature.This process depends on UCP1,which uncouples mitochondrial oxidative phosphorylation to produce heat.Beige or brite adipocytes distribute in classical WAT depots,sharing some characteristics with brown adipocytes.Beige adipocytes have low basal levels of UCP1 in their mitochondria,when activated,they can express UCP1 at high levels,similar to classical brown fat.Browning,which is defined as stimulating the development of beige adipocytes in WAT,might ameliorate the adverse effects of excessive WAT and could help improve metabolic health.The development of obesity is associated with the balance between BAT and WAT;BAT dissipates energy as heat,while WAT stores excess energy.In some rodent models,a propensity towards obesity is associated with decreased BAT activity,while resistance to obesity correlates with increased BAT function or the development of beige cells into WAT.Thyroid-stimulating hormone(TSH)is a type of hypophyseal hormone.TSH receptors(Tshrs)have been identified in many tissues in addition to the thyroid,including adipose tissues,brain,kidneys,testes,heart,thymus and bone,indicating that TSH might exhibit other biological effects in addition to its functions on the thyroid gland.A large number of studies have demonstrated the correlation between serum TSH levels and adiposity.In some epidemiological studies,serum TSH levels are positively associated with body mass index(BMI)in euthyroid subjects.Serum TSH levels and degree of obesity are positively correlated in patients with subclinical hypothyroidism or metabolic syndrome.In addition,TSH levels are higher in obese patients than in non-obese patients.However,the putative connection between TSH and white adipose browning has not been elucidated.In this study,we report that elevated TSH increases adiposity by reducing energy expenditure;and that fat-specific or global Tshr knockout limits weight gain,resists high-fat diet-induced obesity and promotes the browning of both epididymal white adipose tissue(eWAT)and inguinal subcutaneous white adipose tissue(iWAT),likely via AMPK/PRDM16/PGCla.We identify an important role for TSH in adiposity and energy metabolism through inhibition of the browning of white fat.Objectives1.To determine the effects of TSH on white fat content,glucose and lipid metabolism in mice.Subclinical hypothyroidism mice,thyroid-specific Tshr-knockout mice injected with TSH,adipocyte-specific Tshr-knockout mice and global Tshr-knockout mice were used to study the effects of TSH on obesity and related metabolic disorders.2.To determine the influence of TSH on the metabolic rate of mice by monitoring the metabolic cage of four animal models.3.To study the effects of TSH on the browning of white fat and its molecular mechanism.4.To verify the effects of TSH on the browning of white fat by stimulating 3T3-L1 cell and SVF primary cell by TSH.5.To study the effects of TSH and AMPK on white fat browning using 3T3-Ll cells and SVF cells transfected with TSHR SiRNA or treated with AMPK agonists and inhibitorsMethods1.Animal models:a subclinical hypothyroidism model was established by feeding small dose of Methimazole.Global TSHR knockout model were purchased from The Jackson laboratory,the fat specific knockout and thyroid specific TSHR knockout model were established by the CRE-LOXP system.2.Cell culture:3T3-L1 cells and SVF adipocytes were cultured according to the standard culture procedure and induced into brown-like adipocytes.3.The expression of UCPI,PRDM16,PAT2,P2rx5 and PGC1 alpha mRNA was detected by RT-PCR.4.The expression of UCP1,PRDM16,PGC1 alpha,p-AMPK and t-AMPK were detected by Western blotting.5.The expression of UCP1 and CD 137 was detected by immunofluorescence and immunohistochemistry.6.TSHR gene silencing:TSHR specific siRNA transfected 3T3-L1 cells and primary SVF cells to silence TSHR expression.7.AMPK continued to activate or inhibit:AICAR and compound C were transfected into 3T3-L1 cells and primary SVF cells to continuously activate or inhibit AMPK expression.8.Metabolic cages were used to detect the metabolic rate of mice:metabolic cages were used to detect the metabolic rates of four mouse models.9.Statistical analysis:The data are presented as the means with standard errors of the mean(s.e.m.),and significant differences were analyzed using two-tailed Student’s t-tests or 2-way ANOVA with Repeated Measures and Bonferonni post-hoc tests.Results1.TSH promotes adiposity and related metabolic disorders.Subclinical hypothyroidism and thyroid specific knockout TSHR mice injected with TSH have higher bodyweight than their wild type controls,and the content of epididymal and inguinal white fat increased significantly,but the content of brown fat was not changed.Accordingly,their glucose tolerance and insulin tolerance deteriorated and serum triglyceride increased.2.Elevated TSH decreased metabolism rate of mice.The subclinical hypothyroidism mice and the thyroid specific TSHR knockout mice injected with TSH have lower metabolic rate,which showed a decrease in oxygen consumption,carbon dioxide production,total energy expenditure(EE)and resting energy expenditure(REE).3.TSHR knockout resists adiposity and related metabolic disorders.Global TSHR knockout and fat specific TSHR knockout mice showed lower weight,better glucose tolerance,insulin tolerance and lower triglyceride level,Global TSHR knockout mice resisted high fat diet induced obesity.4.TSHR knockout increased the metabolic rate of mice.Global TSHR knockout and fat specific TSHR knockout mice showed higher metabolic rate,which showed increased oxygen consumption,carbon dioxide production,total energy expenditure(EE)and resting energy expenditure(REE).5.TSHR knockout induced the browning of white fat in maice.The epididymal and inguinal subcutaneous white fat content of the TSHR knockout mouse decreased.Further HE staining found that the white fat contained brown-like adipocytes in the eWAT and iWAT of TSHR knockout mice,and the content of UCP1 increased significantly,the expression of beige fat markers increased obviously.This phenomenon is particularly evident under cold stimulation.6.Tshr knockout induces the browning of WAT via AMPK/PRDM16/PGCla.In vivo experiments confirmed that knockout TSHR caused the increase of P-AMPK expression,which promoted the increase of PGC1a expression in PRDM16 and downstream.In vitro experiments further verified that UCP1/PRDM16/PGC1αexpression increased after TSHR gene silence or stimulated by AMPK agonists in brown-differentiated 3T3-L1 cells and SVF primary cell,accordingly PGCla and UCP1 decreased when given the AMPK inhibitor.ConclusionTSH inhibits the phosphorylation of AMPK and inhibits the activity of PRDM16 and downstream PGC1α by binding to TSH receptor,and then inhibits the expression of UCP1 and the browning of white fat,which promotes the development of adiposity.BackgroundGlobally,nonalcoholic fatty liver disease(NAFLD)represents the most commonliver disease.The hallmark of NAFLD is characterized by excessive hepatocyte fat accumulation despite no history of alcohol abuse or other causes for secondary hepatic steatosis.NAFLD confers an increased risk of atherosclerosis,cardiovasculardisease,insulin resistance,obesity and type 2 diabetes mellitus.It is thought that low-grade inflammation brought about by the activation of inflammatory pathways may be a key contributor to the development of NAFLD.Monocytes and monocyte-derived macrophages are important sources of cytokines in the liver and play important roles in NASH progression and resolution.Based on the surface expression of CD 16(FcyRIII receptor)and CD 14(lipopolysaccharide(LPS)receptor),human monocyte subpopulations can be defined as “classical” CD14++ CD16-/‘intermediate” CD14++ CD16+ and “non-classical”CD 14+ CD16++ monocytes.These three subsets differ significantly in terms of theirfunctions and phenotypes ? Classical monocytes are the largest population of monocytes and have the highest phagocytic potential as well as display a marked response to LPS.Non-classical monocytes tend to have high migratory capabilities but limited phagocytic qualities.Nevertheless,non-classical monocytes are able to promote inflammation and the secretion of important inflammatory cytokines.Current research suggests that they possess dendritic cell-like features and appear to carry out antigen-presenting functions by mediating the inflammatory process and infection pathogenesis.Increasing evidence showed that intermediate monocytes are associated with mmy autoimmune and inflammatory conditions such as sepsis,active rheumatoid arthritis,coronary artery disease,liver disease,acute Kaw^aki disease and sarcoidosis.Recently Kim HL et al reported that peripheral blood samples of patients with NAFLD were found to have an elevated total monocyte fraction.Another study showed that non-classical monocyte fraction increased in NAFLD and contributed on the presence of NAFLD.However,the relationship between intermediate monocytesubsets and the NAFLD development has yet to be elucidated.The current study aims to address the information gap that exists regarding the association between intermediate monocytes and NAFLD.Additionally,we sought to determine the possibility of an association between different monocyte populations and established NAFLD biomarkers.Patients and methodsPatients.Subjects enrolled in this study were recruited from a large,long-term follow up epidemiological study cohort residing in Ningyang,Shandong province,China.998 participants were enrolled in the study and written informed consent was obtained from each subject.After exclusion,53 patients with NAFLD were included in the final investigation.208 healtiiy subjects from the study cohort were used as controls.In total,261 samples of monocyte subsets results were obtainedData collection.Clinically relevant information pertaining to the underlying disease,medication history,alcohol intake and smoking history were collected using questionnaires administered by trained personnel.Biochemical tests of subjects were performed after 12 hours of fasting.Blood lipid profile,fasting plasma glucose,insulin and liver enzymes were analyzed using standardized procedures.A diagnosis of NAFLD was performed by ultrasonography.The subpopulations of monocytes were distinguished using flowcytometry.DStatistics analysis.Normally distributed quantitative variables were expressed as mean ± SD values,while percentages were used to present categorical variables.The independent-sample student^ t-test was used to compare the continuous variables between subjects with NAFLD and heathy controls.The %2 test was used to compare categorical variables between subjects with and without NAFLD.Correlations between each biochemical parameter and monocyte subsets were examined by multiple linear/ logistic regression analysis via the enter/forward stepwise method.Statistical significance was defined as p<0.05 and all statistical tests were two-tailed.Statistical IBM SPSS statistical software(version 19.0)was utilized for all data analysis procedures.Results1.Baseline characteristics of the subjects.No significant differences were found in the age and gender ratio between the two groups and most of metabolic parameters including BMI,waist circumference,waist-to-hip ratio,diastolic pressure,fasting glucose,insulin,HOMR-IR and triglyceride were worse in patients with NAFLD in comparison to the healthy controls.Moreover,TNF-a levels in patients with NAFLD was significantly higherthan those in the control group.2.Association between NAFLD and monocyte subpopulationsSubjects with NAFLD displayed an increased intermediate monocyte fraction and decreased classical monocyte fraction.However,the differences in non-classicalmonocyte fraction between the two groups was not statistically significant.And subsequent statistical analysis revealed that the intermediate monocyte fraction,BMI,GGT and TNFa significantly contributed on the presence of NAFLD.Multiple linear regression analysis show that the classical,nonclassical and intermediate monocytes fraction were strongly associated witii age,TG and waist circumstance.ConclusionOur study revealed a novel correlation between intermediate monocytes fraction and NAFLD,which is likely related to the aggravation of NAFLD.