Role Of Axon Guidance Molecule Sema7A In Obesity

BackgroundObesity is a chronic metabolic disease in which energy intake exceeds energy consumption or body metabolism is abnormal,resulting in a large amount of fat accumulation in the body,leading to overweight and health hazards.Obesity is a risk factor for many diseases including fatty liver,type 2 diabetes,atherosclerosis,myocardial infarction,Alzheimer's disease and so on,thus leading to the decline in the quality of life and life expectancy,and posing a serious threat to the health of people around the world,especially in China.The occurrence of obesity is regulated by many factors,such as abnormal neuromodulation,lipid metabolism,and insulin resistance.Although inflammatory cells(e.g.,macrophages)and inflammatory regulators(e.g.,chemokines)have been shown to be involved in obesity and adipose tissue inflammation,additional molecules may be involved in the regulation of adipose tissue inflammation and their regulatory mechanisms remain unknown.Semaphorin 7A(Sema7A)is a semaphorin family member of axonal guidance.molecules,which has been shown to express in neurons,endothelial cells,inflammatory cells,and so on.Sema7A regulates axon growth,osteoblast formation and osteoclast differentiation,and immune responses through its receptors integrin beta 1 and PlexinC1.Our lab recently reported that endothelial Sema7A is expressed in response to disturbed flow,promoting endothelial expression of cell adhesion molecules,leukocyte infiltration,and plaque formation via its receptor integrin beta 1 signaling pathway,suggesting that Sema7A has a cellular and molecular basis for obesity and adipose inflammation.In this study,we used Sema7A knockout mice and obesity animal models to explore the role and mechanism of Sema7A in obesity and adipose inflammation.Our results showed that Sema7A knockout promotes high fat diet-induced obesity and fatty liver,and aggravates insulin resistance,potentially by regulating the polarization of macrophages in adipose tissue.ObjectivesTo investigate the role and possible mechanism of axon guidance molecule Sema7A in obesity.Methods1.Role of Sema7A in high fat diet-induced obesity in miceSema7A knockout mice and control WT mice were fed 60%high fat diet for 8-10 weeks to establish obesity model.The changes of body weight were monitored.The changes of fat size and weight were weighed and counted.The morphological changes of adipose cells were observed by staining and sectioning adipose tissue.2.Effect of Sema7A knockout on high fat diet-induced hepatic steatosis in miceMouse livers were weighed and stained for lipid droplets and liver morphology was recorded.The liver triacylglycerol(TG)was measured.3.Effects of Sema7A on glucose tolerance and insulin sensitivityWT and Sema7A-/-mice fed high fat diet were fasted overnight for glucose tolerance test and insulin sensitivity test.To detect the role of Sema7A for insulin signaling,overnight fasted mice were injected with insulin and white adipose tissue lysate was used for Western blot.Blood glucose were measured with a One-Touch Ultra(?)Glucometer and leptin levels were measured with ELISA.4.Effects of Sema7A knockout on lipogenesis and blood lipid profile Mouse serum was prepared and the level of triglyceride,total cholesterol(TC)and low density lipoprotein cholesterol(LDL-C)were measured.RT-qPCR was used to detect the expression of genes related to lipogenesis in adipose tissue.5.Effects of Sema7A knockout on macrophage polarization in adipose tissueStromal Vascular Fraction(SVF)were isolated from adipose tissue of mice fed high fat diet by collagenase digestion.The proportion of white blood cells,total macrophages,and the percentage of Ml and M2 macrophages in total macrophages was analyzed by flow cytometry.RT-qPCR was used to detect the markers of M1 and M2 macrophages in white adipose tissue.Results:1.Sema7A knockout promotes obesity induced by high fat dietWe first observed the effect of Sema7A on obesity induced by high fat diet.It was found that the weight gain rate of Sema7A-/-mice fed high fat diet was significantly higher than that of WT mice(p<0.01 in males,P<0.05 in females).The weight of white adipose tissue in different parts of body of Sema7A-/-mice was significantly higher than that of WT mice(sWAT P<0.0001 in males,BAT P<0.001 in females,gWAT P<0.001,sWAT P<0.01,BAT P<0.001).Therefore,our results suggest that Sema7A knockout promotes obesity induced by a high fat diet.2.Sema7A knockout promotes fatty liver induced by high fat dietSince obesity increases the risk of fatty liver,we observed the effect of Sema7A on fatty liver induced by high fat diet.We found that the liver volume and weight of Sema7A-/-mice increased significantly compared with WT mice(p<0.01 in males,p<0.0001 in females).HE and oil red staining showed that more and larger red lipid droplets and more lipid vacuoles appeared in the liver of Sema7A-/-mice.The content of triglyceride in liver of Sema7A-/-mice was significantly higher than that of WT mice(p<0.05).Therefore,our data suggest that Sema7A knockout promotes fatty liver induced by a high fat diet.3.Sema7A reduces glucose tolerance and insulin sensitivityIt is believed that insulin resistance is an important contributor of obesity and type 2 diabetes.Therefore,we explored the effects of Sema7A on glucose tolerance and insulin sensitivity.We found that Sema7A-/-mice had increased glucose intolerance(P<0.01),and insulin sensitivity decreased(60 min,p<0.05;90 and 120 min,p<0.01).Western blot showed that deletion of Sema7A reduced insulin-induced AKT phosphorylation(p<0.01).Meanwhile,the serum leptin levels of the starved Sema7A-/-mice increased significantly,suggesting that there was more severe leptin resistance in knockout mice(p<0.0001).4.Sema7A deletion increased blood lipid levels and lipognesis induced by high fat dietObesity is associated with dyslipidemia.In order to investigate the effects of Sema7A on lipid metabolism and blood lipid profile,we took starved serum from two groups of mice,and found that TG,TC and LDL-C increased significantly after Sema7A was knocked out(TG,P<0.01;TC,P<0.01;LCL-C,P<0.05).Lipogenesis genes were detected by RT-qPCR.It was found that some lipogenesis genes such as FASN,ACSS1 and ACC1 were significantly increased in Sema7A-/-mice(FASN P<0.05,ACSS1 P<0.01,ACC1 P<0.05).These data suggest that Sema7A knockout promotes blood lipid levels and lipogenesis induced by a high fat diet.5.Sema7A knockout promotes macrophage polarization in adipose tissueAdipose tissue inflammation is one of the predisposing factors of obesity.To explore the effect of Sema7A on inflammation of adipose tissue,we isolated SVF from white adipose tissue.The results showed that SVF in Sema7A-/-mice in genital adipose tissue(p<0.05)and subcutaneous adipose tissue(p<0.01)increased significantly,and the percentage of M1 macrophages in genital adipose tissue macrophages increased significantly(p<0.01)?RT-qPCR was used to detect the markers of M1 and M2 macrophages in white adipose tissue.The expression of MCP-1 and other markers of M1 macrophages increased(p<0.05),while the expression of IL-10 and CD301 in M2 macrophages decreased significantly(p<0.05).Our results suggested that Sema7A affect obesity by inhibiting the polarization of macrophages in adipose tissue.Conclusions:We found that 1)Sema7A knockout promotes obesity and fatty liver in mice induced by a high fat diet;2)Sema7A knockout increases insulin resistance and lipid levels in mice induced by a high fat diet;3)Sema7A knockout affects polarization of adipose macrophages;4)Our results suggest that Sema7A participates in obesity and adipose tissue inflammation by regulating macrophage polarization,providing a potential new target for regulating obesity and related metabolic diseases.