The Role And Mechanism Of USP20 In Improvement Of Lipid Dysmetabolism After Sleeve Gastrectomy

BackgroundObesity is a complex chronic disease pathologically characterized by an excessive accumulation of adipose tissue in the body.Statistics indicate that China has more than 90 million obese individuals,accounting for approximately 15%of the global obese population and making China the country with the largest number of obese people worldwide.While the World Health Organization has stated that obesity has extremely obvious clinical manifestations,its damage is far from being understood.The disease causes slow damage to human health over a long time span,reduces labor efficiency,and imposes a heavy social burden.Although the positive correlation between the incidence of obesity and individual mortality was first recognized two decades ago,the global prevalence of obesity remains on the rise.It is predicted that one in five adults worldwide will be obese by 2025.Lipids are a class of hydrophobic organic compounds that include triglycerides(TG),cholesterol,phospholipids,and steroids.They serve important biological functions in the human body,such as energy storage,hormone regulation,nerve impulse transmission,and transport of fat-soluble substances.Normal lipid metabolism plays a key role in maintaining lipid homeostasis in the body.Severe lipid metabolism disorders exist in patients with obesity,with the main manifestation in the liver being a disruption of the balance of lipids.This leads to lipid accumulation,which in turn induces hepatotoxicity and non-alcoholic fatty liver disease.The primary manifestation in the blood is dyslipidemia,which includes the elevation of free fatty acid,total cholesterol(TC),and TG levels.Consequently,inflammation and oxidative stress occur at corresponding sites and cause further development of more severe pathophysiological outcomes.Bariatric surgery can effectively reduce body weight and improve lipid metabolism disorders in patients with obesity.It also reduces the risk of obesity-related complications such as diabetes mellitus,hypertension,coronary heart disease,stroke,and malignancies.Sleeve gastrectomy(SG)is a surgical technique in bariatric surgery.Owing to its positive treatment effects and advantages of low operative difficulty,minimal invasiveness,few complications,and non-alteration of the normal sequence of food passage through the digestive tract,SG has gradually surpassed Roux-en-Y gastric bypass,which was traditionally the gold standard of bariatric surgery,as the most widely performed bariatric procedure in China and other countries in recent years.The ubiquitin-proteasome system is the most important post-translational modification in eukaryotic cells.It involves both ubiquitination and deubiquitination,and is in dynamic equilibrium in normal organism.Deubiquitinases participate in the deubiquitination process and stabilize the labeled substrate protein by enzymolysis of the ubiquitin chain.As an important member of the deubiquitinase family,ubiquitin-specific peptidase 20(USP20)regulates the stability of a variety of proteins through the ubiquitin-proteasome pathway.Recent studies have revealed the important role of USP20 in improving lipid metabolism and treating metabolic diseases,including hyperlipidemia,hepatic steatosis,obesity and diabetes.Heat shock protein A2(HSPA2)is a member of the evolutionarily conserved heat shock protein superfamily.HSPA2 was originally identified as a testis-specific chaperone protein that plays an important role in spermatogenesis.Subsequent studies have shown that the polymorphism of HSPA2 gene is highly associated with obesity,and its homozygous genotype is susceptible to obesity,suggesting that HSPA2 may be involved in the occurrence and development of obesity and its related metabolic abnormalities.It was previously believed that the effect of SG on improving lipid metabolism is dependent on reduced energy intake and weight loss during the long-term postoperative period.However,an increasing number of studies have demonstrated that SG can improve lipid metabolism during the early postoperative period in a manner independent of reduced energy intake and weight loss,and that this effect can be maintained for a long time postoperatively.Researchers now believe that SG is not merely a restrictive surgical procedure that involves the removal of the fundus and most of the stomach body without changing the route of food passage through the gastrointestinal tract,but that it also improves lipid metabolism through complex neural,humoral,and immune regulatory networks.In-depth investigation of the specific mechanisms of SG in improving lipid metabolism and the quest for non-invasive methods for the treatment of diseases and disorders related to lipid metabolism are important in the fields of weight loss and metabolism and in the translation of this knowledge to clinical settings.ObjectiveThe present study aimed to achieve the following:(1)Investigate lipid metabolism disorders that coincide with diet-induced obesity;(2)Determine whether USP20-HSPA2 axis is involved in regulating lipid metabolism and mediating the effect of SG on the improvement of lipid metabolism;(3)Perform an in-depth exploration of the molecular mechanisms by which USP20-HSPA2 axis mediates the effect of SG on the improvement of lipid metabolism;(4)Evaluate whether USP20-HSPA2 axis could serve as targets for pharmacotherapy.MethodsA diet-induced obesity mouse model was first established,and adult mice were selected and divided into an SG surgery group and a Sham surgery group.High-fat feeding was continued for 10 weeks after surgery.Lipid metabolism disorders and postoperative improvements in mice were assessed by hematoxylin and eosin(H&E)staining,Oil Red O staining,and TG and TC measurements.To investigate whether USP20 mediates the effect of SG on the improvement of lipid metabolism,we measured the expression and spatial distribution of USP20 in liver tissues by western blotting(WB),real-time fluorescent quantitative polymerase chain reaction(RT-qPCR),and immunohistochemistry(IHC),and subsequently constructed USP20 overexpression and knockdown models to elucidate the regulatory role of USP20 in lipid metabolism.In the in vivo experiment,diet-induced obesity mice were divided into SG group and SG+Flag-USP20 group.SG group was injected with control virus and performed SG surgery.SG+Flag-USP20 group was injected with adeno-associated virus to up-regulate the expression level of USP20 and performed SG surgery.The effect of USP20 up-regulation on the improvement of lipid metabolism in SG was observed.Different bile acid components were subsequently used to stimulate HepG2 cells in vitro to ascertain the upstream mechanisms underlying the changes in USP20 expression levels.We used liquid chromatography tandem-mass spectrometry(LC-MS/MS)to determine substrates of USP20 by identifying proteins that co-immunoprecipitated with USP20.The interaction mechanisms of USP20 and HSPA2 were explored using WB,RT-qPCR,immunoprecipitation,and fluorescence co-localization.To investigate whether HSPA2 mediates the effect of SG on the improvement of lipid metabolism,we measured the expression and spatial distribution of HSPA2 in liver tissues by WB,RT-qPCR,and IHC,and subsequently constructed HSPA2 overexpression and knockdown models to elucidate the regulatory role of HSPA2 in lipid metabolism.Lastly,the gene expression levels of lipid-metabolism-related pathways were evaluated using RT-qPCR.Results1.Preoperatively,there were no significant differences in body weight,serum cholesterol,or serum triglyceride levels between the Sham and SG surgery groups.At 10 weeks post-operation,the Sham surgery group had a significantly higher body weight than the SG surgery group and exhibited obesity.Liver tissues of the Sham surgery group manifested a typical fatty liver appearance,while the liver appearance of the SG surgery group was close to that of a normal liver.H&E and Oil Red O staining of liver tissues revealed the presence of significantly more fat vacuoles and lipid droplets in the Sham surgery group than in the SG surgery group,and H&E staining of adipose tissue showed a significantly higher number of adipocytes in the Sham surgery group than in the SG surgery group.The Sham surgery group had higher liver tissue and serum TG and TC levels than the SG surgery group.2.USP20 expression was downregulated at both the transcriptional and translational levels in the SG surgery group when compared with the Sham surgery group.Oil Red O staining of cells revealed a significantly larger number of intracellular lipid droplets in cells overexpressing USP20 than that of the control group,while the number of intracellular lipid droplets in USP20-knockdown cells was significantly lower than that of the control group.Intracellular TC and TG levels were significantly higher in USP20-overexpressing cells than in the control group,and significantly lower in USP20-knockdown cells than in the control group.3.Ten weeks after surgery,the body weight of SG+Flag-USP20 group was significantly higher than that of SG group with an obese body type.The liver in SG+Flag-USP20 group had a typical appearance of fatty liver,while the liver in SG group had a similar appearance to normal liver.Liver H&E and Oil Red O staining showed that the number of fat vacuoles and lipid droplets in the SG+Flag-USP20 group was significantly higher than that in the SG surgery group.Compared with SG group,SG+Flag-USP20 group had higher TG and TC levels in liver tissue.4.Results of the in vitro experiment revealed that the expression level of USP20 was downregulated in lithocholic acid stimulated cells,while cholic acid,chenodeoxycholic acid,and deoxycholic acid had no significant effects on USP20 expression.5.Through LC-MS/MS,62 proteins and 52 proteins were identified in the experimental and control groups,respectively.After the elimination of 40 non-specific binding proteins,22 proteins that bound specifically to USP20 remained.Subsequently,through screening and validation of these proteins,HSPA2 was identified as a possible substrate of USP20.6.Results of WB showed that Flag-USP20 and Myc-HSPA2 co-immunoprecipitated with each other when they were co-expressed.Immunofluorescence staining revealed that Flag-USP20 and Myc-HSPA2 mainly co-localized in the cytoplasm of co-transfected HEK293T cells.In addition,USP20 and HSPA2 could be co-immunoprecipitated at the endogenous level of HEK293T cells.The ubiquitination level of Myc-HSPA2 was increased by ubiquitin overexpression and decreased by Flag-USP20 co-expression.7.Results of the in vitro experiment showed that USP20 overexpression led to the upregulation of HSPA2 protein expression,whereas USP20 knockdown resulted in the downregulation of HSPA2 protein expression.Knockdown or overexpression of HSPA2 had no significant effect on USP20 protein expression.USP20 overexpression significantly prolonged HSPA2 half-life in the protein degradation experiment.At the mRNA level,overexpression or knockdown of USP20 had no significant effect on the expression level of HSPA2.Similarly,overexpression or knockdown of HSPA2 had no significant effect on the expression level of USP20.8.When compared with the Sham surgery group,the SG surgery group showed a downregulation of HSPA2 protein expression,whereas the mRNA expression level was not significantly different between the two groups.Oil Red O staining of cells revealed the presence of significantly more intracellular lipid droplets in HSPA2-overexpressing cells than in the control group,while the number of intracellular lipid droplets in HSPA2-knockdown cells was significantly lower than that of the control group.Intracellular TC and TG levels were significantly higher in HSPA2-overexpressing cells than in the control group,and were significantly lower in HSPA2-knockdown cells than in the control group.9.In the in vitro experiment,HSPA2 overexpression significantly upregulated the expression levels of FAS,ACC1,and SCD,and downregulated the expression levels of ABCA1 and ABCG1,HSPA2 knockdown led to the downregulation of expression levels of FAS,ACC1,and SCD,and upregulation of expression levels of ABCA1.Conclusions1.SG reduced body weight,attenuated steatosis and lipid accumulation in the liver,and improved lipid metabolism abnormalities in diet-induced obese mice.2.USP20 is involved in the regulation of lipid metabolism.Up-regulation of USP20-HSPA2 axis partially reversed the ameliorating effect of SG on lipid metabolism in vivo and improvement in lipid metabolism after SG is mediated in part by USP20.3.Serum lithocholic acid level was significantly increased after SG surgery,which inhibited USP20 expression.4.USP20 interacts with HSPA2 and stabilizes HSPA2 through deubiquitination.5.The expression level of HSPA2 was reduced after SG surgery.HSPA2 is involved in the regulation of lipid metabolism.Improvement in lipid metabolism after SG is mediated by HSPA2.6.The USP20-HSPA2 axis regulates lipid metabolism through the fatty acid synthesis pathway and cholesterol efflux.7.SG improves lipid dysmetabolism by downregulating the USP20-HSPA2 axis in diet-induced obese mice.