| With the increasing consumption and standard of living,the number of obese people is increasing every year,and the diseases caused by obesity pose a great threat to human health.Obesity usually leads to insulin resistance(IR),which subsequently leads to type 2 diabetes(T2D),cardiovascular disease(CVD),nonalcoholic fatty liver disease(NAFLD),retinopathy,and nephropathy,among other complications.Currently,NAFLD is the leading cause of chronic liver disease worldwide and a major risk factor for cirrhosis,hepatocellular carcinoma and liver-related mortality,yet its pathogenesis has not been fully understood.As the largest energy storage site in the body,adipose tissue is highly plastic and plays an important role in regulating systemic metabolism and energy balance,not only by secreting adipokines and various lipids and metabolites,but also by secreting a large number of exosomes(Exos).Adipose-derived exosomal not only promote insulin resistance and T2 D,but also induce myocardial damage.The mechanism of the role of white adipose tissue-derived exosomal in the development of NAFLD,an important causative factor of obesity,has not been elucidated.Pre-laboratory in vivo imaging experiments in small animals confirmed that white adipose tissue-derived exosomal injected tail vein into mice accumulate heavily in the liver and that injection of visceral adipose tissue-derived exosomal in obese mice promoted insulin resistance while leading to hepatic lipid accumulation.Therefore,we hypothesized that exosomes secreted by white adipose tissue might be involved in the development of NAFLD by promoting hepatic lipid accumulation.Based on the complexity of the components in exosomes,we propose to screen the exosomal protein components involved in the development of NAFLD by exosome proteomic analysis and to investigate the mechanisms.The experimental results are as follows:1.Proteomic analysis of white adipose tissue-derived exosomal in miceSubcutaneous adipose tissue(SAT)and visceral adipose tissue(VAT)of wild-type mice on normal diet(Chow),wild-type mice on high fat diet(HFD)and mice with leptin mutation(ob/ob)were cultured and isolated in vitro.Exosomes were obtained by differential centrifugation,and exosome morphology was observed by transmission electron microscopy,exosome particle size was analyzed by nanoparticle tracking analysis(NTA),and exosome marker proteins were identified by immunoelectron microscopy and immunoblotting,and DIA(data-independent acquisition,DIA)was used to identify exosome marker proteins.DIA(data-independent acquisition)proteomics analysis was used to identify differential proteins in each group of exosomes.The results showed that the exosomes isolated in this study were all in the form of cupshaped vesicles with diameters of 20-200 nm,and the marker proteins CD9,CD63 and CD81 were expressed on the exosomes,which proved that the white adipose tissue-derived exosomal were successfully obtained.Proteomic analysis screened that CD36 was highly expressed in the VAT groups of ob/ob mice and HFD mice,and there were statistical differences between HFD-VAT and ob/ob-VAT,respectively,and Chow-VAT.Based on the important role of CD36 in fatty acid transport,it was hypothesized that white adipose-derived exosomal CD36 might play an important role in hepatic lipid accumulation;therefore,this study was followed up to investigate the role of exosomal CD36 in promoting hepatic lipid accumulation in white adipose tissue.2.CD36 is highly expressed in white adipose tissue and its exosomal in obese miceSAT and VAT and their secreted exosomes were isolated from three different mice,and CD36 expression was observed by immunoblotting.The results showed that SAT and VAT and their secreted exosomes from the three different mouse were able to express CD36,and the expression of VAT was higher than SAT in homozygous mice,and showed high expression in SAT and VAT in HFD mice and ob/ob mice compared with Chow mice.Correspondingly,in Chow and HFD mice,VAT-derived exosomal expressed higher CD36 than SAT-derived exosomal,while ob/ob mice showed high expression of CD36 in both VAT-and SAT-derived exosomal.It is suggested that white adipose tissue CD36 can be secreted extracellularly in the form of exosomes.3.Presence of CD36 in mouse and human serum exosomalSerum specimens were collected from Chow,HFD and ob/ob mice,as well as from healthy(BMI≤24)and obese(BMI≥27)populations,and serum exosomal were obtained by differential centrifugation.The morphology and particle size of exosomes were typical of exosomes,and the number of serum exosomal was significantly higher in HFD mice than in Chow mice,correspondingly,the number of serum exosomal was significantly higher in the obese population than in the healthy population.Immunoblotting also showed that all serum exosomal expressed exosomal marker proteins CD9,CD63 and CD81,while immunoblotting,immunoelectron microscopy and Elisa assay further analyzed the expression and content of serum exosomal CD36.The enrichment of CD36 on the surface of serum exosomal was confirmed by immunoelectron microscopy.Immunoblotting showed that serum exosomal CD36 expression was significantly higher in the obese population than in the healthy population,and significantly higher in ob/ob mice than in HFD and Chow mice.Elisa results showed that serum exosomal CD36 levels were significantly higher in the obese population than in the healthy population,and significantly higher in ob/ob mice than in HFD and Chow mice,and that serum soluble CD36(s CD36)was significantly higher in the same serum volume.CD36(s CD36)and serum exosomal CD36 levels were almost equal for the same serum volume,indicating that serum s CD36 was mainly concentrated in serum exosomal.In addition,we analyzed the adipose marker proteins Adipo Q and FABP4 in serum exosomal and showed that FABP4 was significantly higher in the obese population than in the healthy population,suggesting that white adipose tissue-derived exosomal can enter the circulation.4.White adipose tissue-derived exosomal CD36 promotes hepatic lipid uptakeTo investigate whether the white adipose tissue-derived exosomal CD36 is involved in hepatic lipid accumulation,we used Cd36 knock out(Cd36 KO)mice to inject white adipose tissue-derived exosomal by tail vein,and detected the distribution and expression of CD36 by frozen section,immunofluorescence and immunoblotting.The results showed that CD36 was clearly detected in the liver of Cd36 KO mice,indicating that the white adipose tissue-derived exosomal CD36 could enter the liver tissue of Cd36 KO mice.To continue to investigate whether the white adipose tissue-derived exosomal CD36 entering the liver affected hepatic lipid accumulation,we injected 250 μl of exosome into Cd36 KO mice through the tail vein every 24 h for two injections,and mice were executed after 60 h.The liver and other tissues were removed and stored at-80°C.It was found by oil red O staining that the injection of white adipose tissuederived exosomal significantly promoted liver lipid accumulation compared with the PBS-injected group.It is suggested that the white adipose tissue-derived exosomal CD36 plays an important role in hepatic lipid accumulation.Meanwhile,we isolated primary hepatocytes from WT and Cd36 KO mice and co-incubated the primary hepatocytes with ob/ob mice VAT-EXO,and observed the expression of CD36 and hepatocyte marker protein albumin(Alb)by immunofluorescence after 24 h.The results showed that most of the cells expressed Alb and CD36 was able to be distributed in primary hepatocytes of Cd36 KO mice,suggesting that the white adipose tissue-derived exosomal CD36 was able to be taken up by hepatocytes.To further investigate the role of exosomal CD36 in promoting hepatocyte lipid accumulation,we incubated primary hepatocytes from Cd36 KO mice with SATEXO and VAT-EXO from Chow,HFD and ob/ob mice for 24 h,respectively,and established a hepatocyte lipid accumulation model using oleic acid(OA)induction,followed by Bodipy staining showed that large lipid droplets and an increase in the number of lipid droplets occurred when primary hepatocytes from Cd36 KO mice were co-incubated with white adipose tissue-derived exosomal from ob/ob mice.In summary,this study demonstrated that white adipose tissue can secrete exosomes into the circulation and accumulate in liver tissue,and that the white adipose tissue-derived exosomal CD36 promotes lipid accumulation in hepatocytes.The present study investigated the important role of exosomes in intertissue communication,and the results enriched the understanding of white adipose tissue-derived exosomal in regulating hepatic lipid accumulation,providing important data to further investigate the mechanism of obesity-induced NAFLD and having theoretical significance for elucidating the pathogenesis of NAFLD. |
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