| BackgroundObesity is a chronic and low-grade inflammation disease caused by excessive fat accumulation or abnormal distribution in the body.In recent years,the overweight rate and obesity rate of the Chinese population are both rising with the rapid development of economy and the accelerating pace of life,and people are increasingly concerned about overweight and obesity.There are about 2.1 billion overweight or obese people in the world until 2016.While the obesity rate in China is about 17%at present,with 89.6 million obese people,and China becomes the country with the largest number of obese people in the world.Obesity is not only an independent disease,but also an important risk factor for a series of chronic diseases such as hypertension,diabetes,cardiovascular disease,and even some malignant diseases such as cancer.Thus,the global epidemic of obesity and the resulting metabolic syndrome have become a global public health crisis in the 21st century.Therefore,it is of great theoretical and practical significance for the prevention and treatment of obesity and related metabolic diseases to explore the important molecular mechanism of the occurrence and development of obesity and identify the key candidate target genes.In the development of obesity,adipose tissue is essential for the inflammatory response.Adipose tissue contains a variety of immune cells,and macrophages are the major immune cells contributing to the development of obesity.Macrophages also play a key role in promoting inflammation and causing insulin resistance.In obese adipose tissues,quantity of macropahges can be increased by 50%compared with the normal control,which have become the hot topic in the field of the immunological etiology of obesity.The macrophages infiltrate into fat called adipose tissue macrophages(ATMs).ATMs have traditionally been categorized into two distinct and mutually exclusive activation phenotypes:classical activation phenotype(M1)and alternative activation phenotype(M2).M1 macrophages mainly secrete pro-inflammatory factors,which cause chronic inflammation of adipocyte,leading to glucose metabolism disorder and insulin resistance.However,M2 macrophages act to suppress WAT inflammation and promote insulin sensitivity.M2 macrophages could directly secret catecholamines to activate brown fat and induce beige fat formation within WAT,thereby promoting adaptive thermogenesis and energy expenditure.There is a dynamic balance between the proportion of M1 and M2 macrophages in adipose tissue,and the factors regulating the dynamic transformation of M1/M2 are closely related to the development of obesity.The heterogeneity and plasticity of ATMs are the key factors contributing to obesity.Therefore,the identification of the novel regulators involved in maintaining the functional homeostasis of ATMs would provide effective solutions to control the obesity progression.T cell immunoglobulin domain and mucin domain 4(Tim-4)is a member of the Tim gene family that was discovered in 2001.Tim-4 is selectively highly expressed on macrophages and dendritic cells,but not on T cells.Several studies have confirmed that Tim-4 can inhibit macrophage activity and participate in liver immune homeostasis and autoimmune diseases.These data indicate the importance of Tim-4 in immunological disease,however the role of Tim-4 in obesity is completely unknown.Although it has been reported that M2-like tissue-resident macrophages express high levels of Tim-4,but its role in macrophage polarization remains unclear.Based on the key role of macrophages in the development of obesity and the negative regulation of Tim-4 on macrophages,we put forward the hypothesis that Tim-4 might be involved in the development of obesity by regulating the function of ATMs.ObjectivesThis study aims to explore the role and molecular mechanism of Tim-4 in the development of obesity and seek new targets for clinical treatment of obesity.The specific purpose is as follows:1.To determine the effect of obesity microenvironments on Tim-4 expression.2.To explore the roles of Tim-4 in obesity.3.To determine the effect of Tim-4 on macrophage polarization.4.To elucidate the mechanism of Tim-4 regulating macrophage polarization.5.To clarify the role of Tim-4 in obesity by regulating macrophages.Methods and results1.The effect of obesity microenvironments on Tim-4 expression1.1 Enhanced Tim-4 expression in adipose tissue from obese miceFirstly,we set up two kinds of mice obesity models:high-fat diet(HFD)induced obesity in the wild-type(WT)mice and spontaneously obesity model in db/db mice.Then we detected the expression of Tim-4 in the adipose tissue of HFD mice,db/db mice and normal diet WT mice by qPCR and WB assay.The results showed that the expression levels of Tim-4 mRNA and protein in adipose tissues from obese mice were significantly increased compared with the control group.These results suggest that obesity microenvironments can enhance the expression of Tim-4 in adipose tissue.1.2 Increased expression of Tim-4 on ATMs from obese miceTo determine the expression of Tim-4 in SVFs,we isolated stromal vascular fraction cells(SVFs)from subcutaneous adipose tissues of db/db mice and their control mice.qPCR and flow cytometry were performed to detect the expression of Tim-4 mRNA and protein in SVFs.The results showed that both SVFs and ATMs expressed higher levels of Tim-4 in adipose tissues of obese mice.Immunofluorescence(IF)also further confirmed that the expression of Tim-4 was up-regulated in adipose tissue macrophages of db/db obese mice.All these data above indicated that the expression of Tim-4 in ATMs of obese mice was up-regulated.1.3 Serum of obese mice promotes Tim-4 expression on macrophagesTo further clarify the correlation between the expression of Tim-4 and obesity,we used the serum of obese mice(proportion of medium:20%)to mimic obesity microenvironments to simulate the bone morrow-derived macrophage(BMDM)of mice in vitro.The expression of Tim-4 in BMDM was detected by qPCR,WB and flow cytometry separatively.The results showed that expression of Tim-4 on BMDM was up-regulated after stimulation with serum from obese mice.The above results demonstrated that obesity microenvironments could enhance the expression of Tim-4 in macrophages,suggesting that Tim-4 might be involved in the development and progress of obesity by regulating macrophages.2.The role of Tim-4 in obesity2.1 Identification of Tim-4 knockout miceIn order to verify the role of Tim-4 in obesity,Tim-4 knockout mice were included in this study.Tim-4-/-mice were genotyped and Tim-4 expression was detected in SVFs,BMDM,and peritoneal resident macrophages by qPCR,WB,and flow cytometry.The results showed the efficient knockout of Tim-4 in Tim-4-/-mice,which prepared the basis for the following experiments.2.2 Tim-4 knockout promotes HFD-induced obesity2.2.1 Tim-4 knockout aggravates the obesity induced by HFDIn order to clarify the roles of Tim-4 in obesity,we established the obesity model by using Tim-4-/-mice and WT mice fed with HFD for 20 weeks.During the process of obesity preparation,we took the weight measurements every week.The results showed that Tim-4 knockout mice began to have a significant trend of weight increase after 14 weeks of high-fat diet feeding.After 20 weeks,Tim-4-/-mice were found to have higher adipose tissue weights including subcutaneous,epididymal and visceral adipose tissues,and the size of each adipocyte from Tim-4-/-mice were significantly larger than control mice.In addition,in order to determine the ability of blood glucose regulation,the glucose tolerance and insulin resistance test were performed after 20 weeks of high-fat diet feeding.The results showed that the Tim-4-/-mice appeared a trend of decreased glucose tolerance and increased insulin resistance.These results suggested that Tim-4 knockout aggravate the obesity induced by HFD.2.2.2 Tim-4 knockout aggravates obesity-associated metabolic deteriorationWe also examined the expression of lipid metabolism and glucose metabolism associated molecules in the serum of two obesity models.Firstly,serum lipid levels(TG,T-CHO,LDL-C)in Tim-4-/-mice were higher than those in control group.In addition,ELISA kit was used to detect the levels of insulin,leptin and adiponectin in serum,and the results showed that Tim-4-/-mice displayed increased resistance to insulin and leptin,while the levels of adiponectin in serum were significantly decreased,which could improve insulin sensitivity,indicating that Tim-4 knockout could aggravate obesity-associated metabolic deterioration.The above results suggest that Tim-4 knockout promote the progress of HFD-induced obesity.2.3 The role of Tim-4 knockout in energy balance of HFD-induced obesitySince adipose tissue plays an important role in energy metabolism homeostasis,we tested the energy metabolism level of two groups of obese mice after 20 weeks of high-fat diet.We found that the oxygen consumption level of Tim-4-/-mice was significantly reduced,suggesting that Tim-4 knockout decrease the energy expenditure.In order to clarify whether Tim-4 regulates the energy metabolism of adipose tissue,we separated brown and white adipose tissue of two groups respectively.We found that Tim-4 knockout could reduce the expression of UCP1,suggesting that Tim-4 knockout decrease the thermogenesis function of adipose tissues.In addition,beige adipocyte is also an important way to enhance energy metabolism.Therefore,the expression of beige adipocyte biomarkers in subcutaneous adipose tissues was detected by qPCR.We found that the levels of UCP-1,Ear2,Tmem26 and CD137 mRNA were significantly reduced in adipose tissues from Tim-4 knockout mice.All the above results indicated that Tim-4 knockout could decrease energy expenditure.3.The effect of Tim-4 on macrophage polarizationPolarization and recruitment of ATMs play a key role in the progress of obesity.In the obese fat tissue,the proportion of M1/M2 macrophages is imbalance,which leads to chronic inflammation in fat tissues.The local inflammation of adipose tissues also further recruits macrophages from surrounding tissues,in turn promoting the development of obesity.3.1 The effect of Tim-4 on macrophage phenotype in SVFs of HFD-induced obesityIncreased levels of TNF-a and IL-6 in the serum from Tim-4-/-mice imply that Tim-4 knockout promotes systemic inflammation.SVFs were extracted from the subcutaneous adipose tissues of the two groups,and the ratio of M1/M2 macrophages in SVFs was detected by qPCR and flow cytometry.qPCR results showed that the SVFs from Tim-4-/-mice expressed higher levels of M1 macrophage markers,such as iNOS,TNF-? and Ccl2,'but expressed low levels of M2 macrophage markers,such as CD206,Argl,and Retnla.Flow cytometry results also showed that the proportion of M1 macrophages(CD11b+CD11c+)was up-regulated,while percentage of M2 macrophages(CD11b+CD206+)was down-regulated in ATMs of Tim-4-/-mice.In addition,IF results showed that iNOS was up-regulated,but Ym-1 was down-regulated in the ATMs of Tim-4-/-mice.Finally,WB results were consistent with those of IF assay.These results suggest that Tim-4 knockout reduce the proportion of M2 macrophages in SVFs of HFD mice,thereby promoting the inflammatory response.3.2 Tim-4 regulates the M1/M2 balance3.2.1 Tim-4 knockout promotes the expression of pro-inflammatory cytokines in macrophagesIn order to further clarify the regulatory effect of Tim-4 on macrophages,BMDM of Tim-4-/-and WT mice were treated with LPS(100ng/ml)or IL-4(20ng/ml)for 24h,and the expression of inflammatory cytokines in macrophages was detected.qPCR results showed that BMDM of Tim-4-/-mice highly expressed M1 macrophage markers,such as iNOS,TNF-a and Ccl2,but lowly expressed the markers of M2 macrophages,such as CD206,Argl and Retnla.WB results showed that the expression of iNOS was up-regulated after LPS stimulation,while the expression of Ym-1 was down-regulated after IL-4 stimulation in BMDM of Tim-4-/-mice.These results suggest that Tim-4 knockout can promote the expression of pro-inflammatory cytokines in macrophages.3.2.2 Tim-4 knockout promotes macrophages polarized to Ml phenotypeNext,flow cytometry was used to detect the phenotype changes of macrophages in the two groups after LPS or IL-4 stimulation.After LPS stimulation,Tim-4 knockout increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages.The above results are consistent with those in vivo,suggesting that Tim-4 knockout promote macrophages polarized to M1 phenotype.Finally,flow cytometry was used to detect the phenotype of BMDM after stimulation with the serum of obese mice.The results showed that the proportion of M1 phenotype was up-regulated and the proportion of M2 phenotype was down-regulated in BMDM from Tim-4-/-mice.The above results proved that Tim-4 could regulate the proportion balance of M1/M2 macrophages.4.The mechanism of Tim-4 in regulating macrophage polarization4.1 The effect of Tim-4 on NF-?B signaling pathwayTo further clarify the effect of Tim-4 on the NF-?B pathway in the obesity microenvironments,BMDMs of Tim-4-/-and WT mice were treated with serum from db/db obese mice or controls.It was found that the serum of the obese mice could promote the phosphorylation of NF-?B p65,and stronger activation of NF-?B p65 was found in BMDMs from Tim-4-/-mice.Dual luciferase reporter assay showed that Tim-4 overexpression can inhibit the activation of NF-?B.WB results showed that LPS stimulation significantly enhanced the activation of IKK?/?,IKB? and p65 of BMDMs from Tim-4-/-mice.The above results demonstrated that Tim-4 knockout promoted the activation of NF-?B signaling pathway.4.2 Tim-4 regulates macrophage polarization through the NF-?B pathwayIn order to clarify the role of the NF-?B pathway in Tim-4 mediated macrophage polarization,NF-?B pathway inhibitor was introduced to treat BMDMs under stimulation with LPS or IL-4.qPCR results showed that Tim-4 knockout promoted the expression of pro-inflammatory molecules such as iNOS,TNF-?,and IL-1?,but no significant difference was found in NF-?B inhibitor group.The flow cytometry results showed that the proportion of M1 macrophages was significantly up-regulated and that of M2 macrophages was down-regulated in BMDM of Tim-4-/-mice.However,no obvious differences were found in NF-?B inhibitor treated group.The above results confirmed that Tim-4 regulated the polarization of macrophages through the NF-?B pathway.5.The role of Tim-4 in obesity by regulating macrophages5.1 The effects of Tim-4 on WAT browningWe then sought to assess the metabolic cross-talk between M2-polarized macrophages and adipocytes,and determine whether such cross-talk might be influenced by Tim-4 ablation in macrophages.Mature adipocytes differentiated from 3T3-L1 cell lines were used to co-culture with IL-4-stimulated BMDMs from WT or Tim-4-/-mice.IL-4-treated BMDMs co-culture significantly increased the expression of UCP1 and beige adipocyte markers,including Cd137,Ear2 and Tmem26,in differentiated beige adipocytes.However,Tim-4 deficiency robustly weakened the ability of IL-4-stimulated BMDMs to upregulate these signatures of beige adipocytes.Thus,Tim-4 abrogation inhibits M2 polarization of ATMs and consequently enables less efficient in WAT browning.5.2 The influence of Tim4-/-ATMs transfer on obesity progressionWT male mice were fed with HFD for 10 weeks to induce obesity.Then these obese WT mice were used to given WT-ATMs or Tim-4ko-ATMs isolated from the epididymal WAT(epWAT)by peritoneal injection,and PBS was used as a negative control.Every 4 d injections were conducted for a total of 6 times.Significant deterioration in glucose tolerance but not insulin resistant was observed in Tim-4ko-ATMs transfered mice.The weight of epWAT in Tim-4ko-ATMs transfer group was higher than that in WT-ATMs group,but no significant difference was found.Other metabolic parameters,such as insulin,TG,showed worsen in the Tim-4ko-ATMs transfered mice as compared to WT-ATM-injected mice.mRNA levels of browning markers(UCP-1,Tmem26 and Ear2)were prominently reduced in the epWAT of Tim-4ko-ATM transfered mice.qPCR results showed that gene expression associated with inflammatory M1 macrophages(TNF-a and IL-6)indeed was increased,whereas that associated with anti-inflammatory M2 macrophages(Arg1,Retnla)was decreased in the epWAT in Tim-4ko-ATM injected mice.Accordingly,we conclude that local injection of pro-inflammatory Tim-4ko-ATMs into adult mice under a HFD feeding could inhibit epWAT browning and decrease energy expenditure.In summary,this study reveals the enhanced expression of Tim-4 in obesity which inhibits the HFD-induced obesity.In addition,we prove that Tim-4 regulates macrophage polarization through the NF-?B pathway.This study would provide a new potential target for the intervention of obesity. |
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