The Role Of Preadipocytes CD36 In Adipose Tissue Metabolic Inflammation And The Underlying Mechanism

Objective:Metabolic syndrome（MS）is a group of chronic disease syndromes caused by multiple systemic metabolic disorders including obesity,insulin resistance,type 2 diabetes（T2DM）,cardiovascular disease,and nonalcoholic fatty liver disease（NAFLD）.MS has become a serious problem of human health throughout the world.MS is characterized by insulin resistance and the persistence of low-grade metabolic inflammation.Adipose tissue is considered to be a major source of metabolic inflammation and preadipocytes account for 15-50%of adipose tissue cells.Recent studies have also shown that it is undifferentiated preadipocyte rather than mature adipocyte can become the main contributor to recruit macrophages and secrete pro-inflammatory factors in adipose tissue.CD36is a scavenger receptor that regulates a variety of immune and metabolic functions and is closely related to the development of MS.Therefore,this study will be to investigate the role of preadipocytes CD36 in metabolic inflammation and MS,and next to explore its molecular mechanism.Methods:The first part:General health survey data were collected from 28035 patients with health checkups in the First Affiliated Hospital and Jinsan Affiliated Hospital of Chongqing Medical University.Firstly,hs-CRP was divided into quartiles to analyze the changes of blood pressure,blood glucose,blood lipids and liver function under different degrees of inflammation.Then,to determine the inflammation levels of patients with different degrees of obesity,the subjects were categorized into quartiles for body mass index（BMI）or waistline.Human adipose tissue samples were obtained from patients undergoing abdominal surgery or abdominal puncture in the Second Affiliated Hospital of the Medical University.According to the presence or absence of obesity and MS,the patients were divided into normal control group（normal group）and obesity with metabolic syndrome group（MS group）.The pathological changes of human adipose tissue were observed by HE staining.The protein levels of CD36andF4/80inhumanadiposetissueweremeasuredby immunohistochemical staining.The localization of CD36 on preadipocytes in human adipose tissue was detected by using of a dual-staining of CD36and Pref-1.The second part:Six-week-old C57BL/6J mice were randomly divided into C57BL/6J normal-chow diet group（CTL,n=8）and C57BL/6J high-fat diet group（HFD,n=8）.Six-week-old wild-type（WT-HFD,n=8）mice and CD36-knockout mice（CD36KO-HFD,n=8）were fed a high-fat diet.All mice were fed for 14 weeks and glucose tolerance test and insulin resistance test were performed at one week before the end of feeding.After the end of feeding,the body weight was weighed and epididymal adipose tissue was collected.The localization of CD36 on preadipocytes in human adipose tissue was detected by using of a dual-staining of CD36 and Pref-1.The pathological changes of mice adipose tissue were observed by HE staining.The protein levels of F4/80 in mice adipose tissue were measured by immunohistochemical staining.The third part:After treatment of 3T3-L1 preadipocytes with FFA,CD36 protein level was detected by Western blot and single standard flow.The lentiviral vector was transfected to establish CD36 overexpression3T3-L1 preadipocytes line.Oil red O and Bodipy staining were used to analyze intracellular lipid accumulation.Cellular proteins were extracted and Western blot was used to measure the protein expressions of CD36,ACC,PACC,NF-κB p65,NLRP3,caspase-1 and IL-1.Cellular RNA was extracted and the expression of inflammatory/chemokine mRNA was determined by QPCR.Transwell cell migration assay was used to observe the effect of preadipocytes CD36 overexpression on the migration of THP-1 cells.The fourth part:Mitochondrial reactive oxygen species（mtROS）levels were detected by MitoSOX staining to evaluate mitochondrial oxidative stress in 3T3-L1 preadipocytes.Mitochondrial membrane potential（MMP）was measured by JC-1 staining to determine mitochondrial function in 3T3-L1 preadipocytes.Intracellular Ca²⁺content was observed by Fluo-4/AM staining and mitochondrial calcium levels were analyzed by a Fluo-4/AM and mitotracker co-staining.After 2APB was used to inhibit IP3R-mediated calcium release from endoplasmic reticulum into mitochondria,the expression of preadipocytes inflammation/chemokine mRNA was detected by QPCR and intracellular lipid accumulation was analyzed by Bodipy staining.Results:The first part:The health survey data showed that aseptic inflammation was positively correlated with various metabolic diseases（P<0.05）.BMI and waistline were positively correlated with aseptic inflammation（P<0.05）.The results of adipose tissue staining showed that the expression of CD36 and F4/80 in adipose tissue from MS patients was increased compared with normal patients.The colocalization efficiency of CD36 and Pref-1 was higher,indicating the expression of CD36 was induced in preadipocytes of patients with MS（P<0.05）.The second part:Compared with the CTL group,HFD feeding resulted in obesity,fatty liver,impaired glucose tolerance and insulin resistance in C57BL/6J mice（P>0.05）.F4/80 staining showed that HFD feeding induced an increase of macrophage infiltration to adipose tissue and led to adipose tissue inflammation（P<0.05）.Colocalization analysis of CD36 and pref-1showed that CD36 expression was up-regulated in preadipocytes of HFD group mice（P<0.05）.DNA agarose gel electrophoresis results showed that CD36KO mice models were successfully constructed.Compared with WT-HFD,CD36KO did not alleviate body weight and fat weight in HFD-fed mice（P>0.05）.However,CD36KO reduced HFD-induced impaired glucose tolerance and insulin resistance（P<0.05）.CD36KO also protected mice from HFD-induced adipose tissue macrophage infiltration as well as（P<0.05）.The third part:Western blot results showed that CD36 expression was not detectable in preadipocytes,while FFA increased CD36 expression in3T3-L1 preadipocytes in a time-dependent manner（P<0.05）.CD36-PE single-stream flow also showed that FFA increased the localization of CD36 on the membrane in 3T3-L1 preadipocytes（P<0.05）.After 3T3-L1preadipocytes transfected with a lentiviral vector containing CD36overexpressing gene,CD36 overexpression increased the uptake of FA,promoted lipid synthesis,inhibited lipid degradation（P<0.05）.Finally,CD36 overexpression promoted lipid accumulation in preadipocytes（P<0.05）.CD36 overexpression also promoted inflammatory response through activated NF-κB p65 and NLRP3 inflammasome（P<0.05）.The fourth part:MitoSOX and JC-1 staining showed that CD36overexpression decreased MMP and promoted mtROS production compared with NC,indicating that CD36 overexpression induced mitochondrial dysfunction（P<0.05）.Intracellular Ca²⁺content and distribution experiments showed that CD36 overexpression increased the levels of intracellular total Ca²⁺and mitochondrial Ca²⁺.Treatment of3T3-L1 preadipocytes with IP3R inhibitor 2APB inhibited the release of calcium from the endoplasmic reticulum to mitochondria,which protects cells from mitochondrial dysfunction induced by CD36 overexpression.2APB also reduced inflammation and lipid accumulation.These suggested that preadipocyte CD36 promoted IP3R-mediated calcium transport from the endoplasmic reticulum to mitochondria,causing mitochondrial calcium overload and induction of inflammatory response（P<0.05）Conclusions:In vivo,preadipocytes CD36 was increased in MS patients or MS mice models,accompanied by increased macrophages infiltration in adipose tissue.CD36 gene deletion protected mice from HFD-induced insulin resistance and macrophages infiltration in adipose tissue.In vitro,free fatty acids induced the expression of CD36 in preadipocytes.Preadipocytes CD36 overexpression can induce mitochondrial calcium overload by increasing IP3R-mediated calcium transport from the endoplasmic reticulum to mitochondria.Mitochondrial calcium overload induced mitochondrial dysfunction and mtROS production,and then promoted the activation of NLRP3 inflammasomes.Thereby,these aggravated the inflammatory response of preadipocytes and macrophages infiltration,participating in the development of metabolic inflammation of adipose tissue.