Effect And Mechanism Of MBL On Obesity Induced By High-fat Diet

BackgroundObesity is increasingly harmful to human health,how to curb and treat obesity has become an urgent health problem.As a lysosomal-dependent self-degradation pathway,autophagy not merely take part in adipogenesis,but also plays crucial in adipocyte differentiation.The influence of immune elements on obesity and related metabolic diseases has been a hot topic in recent years.Mannan-binding lectin（MBL）has been described deed a“crux molecule in the innate immune system”.In this study,in vivo and in vitro tests were needed to proceed the regulatory effect of MBL on obese mice with high-fat diet,and preliminarily revealed the role of autophagy in MBL regulation.And a new viewpoint that MBL can affect the occurrence and development of obesity by regulating autophagy will enrich the basic theories of innate immunity and broaden the connotation of metabolic immunity,thus having important scientific significance.ObjectiveTo study the regulatory effect of MBL on lipid metabolism in obese mice induced by high-fat diet,and to explore the mechanism of MBL on adipogenic differentiation of3T3-L1 adipocytes based on autophagy.Methods1.WT and MBL^-/-male mice of C57BL/6 strain were selected to construct obesity model on high-fat diet,normal diet as control.After the induction,the metabolic cage（animal metabolism detection system）was applied to surveillance the changes of basic energy metabolism indexes for instance food ingestion,water consumption,amount of exercise,O₂ consumption and CO₂ production of mice.The degree of insulin counteractive and glucose capability in mice were appraised via insulin and glucose tolerance experiments.Mouse serum TG,TC,LDL-C and HDL-C were observed via full-automatic analysis instrument.ELISA was executed to examine the excretion levels of mouse serum INS,LEP,ADP and FFA.Epididymis adipose tissue and liver tissue were fetched,histopathological alters and lipid drops were observed by H&E staining and oil red O staining.Western blot was well used to inspection the protein expression quantity of fat lipid synthesis and decomposition related factors PPARγ,C/EBPα,p-HSL and HSL.2.3T3-L1 preadipocytes were cultivated in vitro,and imparity concentrations of MBL were treated at different stages of differentiation into mature adipocytes.Cell differentiation and lipid droplet cumulation after MBL intervention were analyzed by oil red O staining.CCK-8 was well used to test the alters of cell proliferation under disparate intervention circumstance.Western blot and q RT-PCR were to respectively examine protein and m RNA variation of autophagy key factors such as LC3B,Beclin1,p62 and others.Autophagy tool drugs Bafilomycin A1 and Chloroquine interfere with autophagy flow to further illustrate the autophagy activity.Finally,continue to use Western blot to judgment the expression and phosphorylation of signal molecules in AMPK/mTOR,a key signal pathway for autophagy.Results1.The obesity model of mice was successfully constructed at the 16th week of induction.Comparison with WT high-fat diet mice,MBL^-/-high-fat diet mice gained more weight,fasting blood glucose levels increased,the average oxygen expend and carbon dioxide output both day and night were decreased.And there was no statistical discrepancy in food intake,water consumption and exercise of mice in each group.2.Comparison with WT high-fat diet mice,MBL^-/-high-fat diet mice revealed a noticeable increase in insulin resistance and impaired glucose tolerance at 16 weeks.3.Contrast to WT high-fat diet mice,the serum content of INS,TC and LDL-C in MBL^-/-high-fat diet mice were growing,nevertheless,the content of LEP,ADP and HDL-C were lowered,which aggravated the disorder of lipid metabolism.4.Compared with WT high-fat diet mice,the liver and adipose tissue mass of MBL^-/-high-fat diet mice were augmented.The size of adipose tissue cells enlargement significantly.Liver tissue was characterized with serious steatosis,some inflammatory cells infiltration,large number of lipid droplet deposition.5.Compared with WT high-fat diet mice,the expression quantity of lipid accumulation related protein C/EBPαand PPARγwere aggrandized,while the expression quantity of catabolic related protein p-HSL（Ser563）were depressed in MBL^-/-high-fat diet mice.6.The complete differentiation of 3T3-L1 preadipocytes was achieved by the classic cocktail method at the 10th day of induction,and the lipid droplets in adipocytes at different stages of differentiation are reduced to varying degrees under the intervention of MBL（10μg/m L）.7.In the whole process of 3T3-L1 preadipocyte differentiation,comparison with the vacant group,the quantity of autophagy proteins Beclin1 and Atg7 were gradualness raised in the MBL（10μg/m L）function group,while the quantity of P62 protein was gradualness lowered,and the effect was most obvious in the precursor and 6-day adipocyte differentiation stage（middle stage of differentiation）.8.At the stage of precursor adipocytes and adipocytes differentiation for 6 days,contrast with the blank group,the expression quantity of MBL intervention group（1μg/m L-10μg/m L）autophagy-related proteins LC3B and Beclin1 were added,and the m RNA expression quantity about autophagy-related genes LC3B,Beclin1,Atg5 and Atg7 were added.The expression quantity about autophagy-related protein P62 was lessened,and the expression quantity about autophagy-related genes P62 and LAMP1were lessened,both showed a concentration-dependent relationship.9.The use of autophagy tools bafilomycin A1 and CQ confirmed that MBL enhanced the autophagy activity of adipocytes by increasing the synthesis and degradation of autophagosomes.10.Under the interpose of MBL（1μg/m L-10μg/m L）,the phosphorylation standard of AMPK（Thr172）was remarkable higher expression,while the phosphorylation standard of mTOR（Ser2481）was remarkable downgrade in adipocytes,which showing a concentration dependency relation.Conclusions1.MBL can effectively inhibit obesity in mice induced by high-fat diet,improve insulin resistance and lipid metabolism disorders,reverse the increase of adipose tissue mass and adipocyte hypertrophy,and reduce liver lipid droplets storage and steatosis.2.MBL can accelerates the autophagy process of 3T3-L1 adipocytes during differentiation through AMPK/mTOR signaling pathway,thus reducing the accumulation of lipid droplets and lipid synthesis.