Effects And Mechanism Of MBL On Inflammation And Insulin Resistance In Obesity

BackgroundObesity is a huge risk to human health.The fight against obesity has become one of the main means of defending human health.Obesity is often accompanied by the development of low-grade chronic inflammation in adipose tissue,which leads to obesity-related metabolic diseases.Adipose tissue dysfunction can lead to abnormally high levels of serum free fatty acids,and excess free fatty acids produce insulin resistance by interfering with insulin signalling.Mannan-binding lectin（MBL）,a plasma protein secreted by hepatocytes,plays an important role in natural immune defense.In recent years,new studies have identified an important role for MBL in the regulation of immune homeostasis in our body.Previous studies have confirmed that MBL plays a negative regulatory role at different stages of induction differentiation of 3T3-L1 adipocytes,and this regulatory effect is closely linked to autophagy.It is suggested that MBL has a regulatory role in the development of obesity,but the mechanism is unclear.In this study,we investigated the role and mechanism of MBL in regulating inflammation and insulin resistance in high-fat-induced obesity using in vivo and in vitro assays,which may provide new ideas for the prevention and treatment of obesity and related metabolic diseases.ObjectiveTo investigate the role and mechanisms of MBL on inflammation and insulin resistance in obesity.Methods1.In vitro assays,3T3-L1 preadipocytes were induced to differentiate,and the extent of cell differentiation was analyzed by oil-red O staining.The effects of MBL and LPS on cell proliferation ability during differentiation were measured by CCK-8.In vivo experiments,in order to establish normal control and high-fat obesity models,C57BL/6WT male mice and MBL^-/-male mice were fed high-fat diets and normal diets respectively.2.Cellular inflammatory factors of IL-6 and TNF-αwere detected by ELISA.The protein expression of TNF-α,IL-6 and NF-κB signaling pathway was detected by western blot.The migration of macrophages was detected by Transwell.In vivo experiments,adipose tissues were extracted from each group of mice.The expressions of TNF-αand IL-6 protein from adipose tissue were detected by western blot.3.Detection of MBL on glucose uptake capacity and insulin signalling pathway protein expression:In vitro assays,flow cytometry was used to detect the effect of MBL on glucose uptake by 3T3-L1 cells,the phosphorylation of Akt and IRS-1 try protein and the expression of GLUT4 were analyzed by western blot.In vivo experiments,insulin signaling pathway related proteins:P-Akt,P-IRS try,GLUT4 from adipose tissue were detected by Western blot.4.To detect the regulation of fatty acid oxidation by MBL:In vitro experiments,the contents of acetyl coenzyme A and ATP in cells were detected with the kit.The content of LCAD was detected by q RT-PCR.The phosphorylation of ACC and protein expression of CPT-1 were detected by Western blot.In the in vivo experiments,the phosphorylation of ACC and the protein expression of CPT-1 in the adipose tissue of each group of mice were detected by Western blot.5.The cells were intervened with AMPK inhibitor compound C to detect the role of AMPK in cellular inflammation,insulin resistance and fatty acid oxidation,and the above indicators were tested.Results1.3T3-L1 preadipocytes were successfully induced to differentiate into mature adipocytes.During the whole process of cell differentiation,the intervention of different concentrations of MBL,LPS and compound C had no significant effect on the proliferation of 3T3-L1 adipocytes.2.The regulatory role of MBL in 3T3-L1 cells in vitro（1）In the process of induced differentiation of 3T3-L1 adipocytes,ELISA and western blot showed that MBL could inhibit the expression of IL-6 and TNF-αinduced by LPS in a concentration-dependent manner,and MBL could inhibit LPS-induced activation of nuclear NF-κB,TLR4 and the phosphorylation of IKK,IκB.（2）Transwell results showed that MBL could inhibit the migration of macrophages to adipocytes.（3）Western blot confirmed that MBL increased the expression of P-IRS,P-Akt and GLUT4,key proteins of the insulin signalling pathway,and flow cytometry showed that MBL alleviated the inhibitory effect of LPS on cellular uptake of glucose.（4）Western blot and q RT-PCR experiments showed that MBL increased the levels of acetyl coenzyme A、ATP and LCAD m RNA and the expression of P-ACC and CPT-1proteins in 3T3-L1 adipocytes inhibited by LPS.（6）After the addition of the AMPK inhibitor compound C,the expression of IL-6,TNF-α,nuclear NF-κB,TLR4,P-IKK and P-IκB was increased in adipocytes,meaning that the anti-inflammatory effect of MBL on adipocytes was partially counteracted.The cellular uptake of glucose was inhibited by the intervention of compound C,and the expression of P-IRS-1,P-Akt and GLUT4,key proteins of the insulin signalling pathway,was also inhibited,impairing the ability of MBL to enhance cellular uptake of glucose.The addition of compound C inhibited the ATP content and the expression of P-ACC and CPT-1 in adipocytes,so that the stimulation of fatty acid oxidation by MBL was suppressed.3.Effects of MBL gene knockout on hyperlipid-induced obesity in mice（1）Expression of IL-6 and TNF-αwas increased in adipose tissue of MBL knockout mice compared with wild-type mice after high-fat diet.（2）Expression of P-IRS try,P-Akt,GLUT4 and CPT1 in adipose tissue of MBL knockout mice was reduced compared with wild-type mice after high-fat diet.Conclusions1.MBL attenuates LPS induced inflammation and insulin resistance in adipocytes through AMPK mediated pathway.2.MBL can effectively inhibit inflammation,reduce insulin resistance and enhance fatty acid oxidation in obese mice induced by high-fat diet.