| Background and objective:Glucose and lipid metabolism is the main source of energy supply.A number of studies have shown that long-chain non-coding RNAs(lncRNAs)play important roles in glucose and lipid metabolism,and abnormal expression of lncRNA is closely related to the disorder glucose and lipid metabolism.LncPrep+96kb is new lncRNA,which is highly expressed in skeletal muscle,adipose tissue,liver and other organs of mice.In previous study,we successfully constructed LncPrep+96kb knockout mice in this study,we will comprehensively evaluate the glucose and lipid metabolism and insulin sensitivity of LncPrep+96kb gene knockout mice induced by high fat diet.We will further explore the possible mechanism of the effects of LncPrep+96kb on glucose and lipid metabolism and insulin sensitivity from adipose tissue.Methods:6-week-old wild-type and LncPrep+96kb knockout male mice were selected.After one week for adaptive feeding,the mice were randomly divided into 4 groups:normal diet control group(WT-CD,n=6),normal diet knockout group(KO-CD,n=6),high-fat diet control group(WT-HFD,n=6)and high-fat diet knockout group(KO-HFD,n=6).The mice were fed for 18 weeks and weighed once a week.Two weeks before the end of modeling,the glucose tolerance level of mice was measured by intraperitoneal glucose tolerance test(IPGTT).The insulin sensitivity was measured by insulin tolerance test(ITT)one week before the end of modeling.After 18 weeks,the mice were killed,and the serum was collected to detect the levels of TG,TC,LDL-C and HDL-C.The serum insulin,leptin,adiponectin,IL-6 and TNF-αlevel were detected by Elisa.The weight of epididymal adipose tissue,liver,heart and kidney were recorded and the organ index was calculated.Western blotting was used to detect the expression of p-AKT in epididymal adipose tissue,liver and skeletal muscle after basic state and insulin stimulation.All these methods were used to comprehensively evaluate the changes of glucose and lipid metabolism and insulin sensitivity in mice.The adipose tissue of mouse epididymis was collected,and the morphological changes of adipose tissue were observed by HE staining.F4/80 immunohistochemical staining was used to evaluate the infiltration of macrophages in adipose tissue.Flow cytometry was used to detect the proportion of macrophages in adipose tissues.Real-time quantitative PCR and Western blotting were used to detect the expression of key genes of lipid synthesis,lipolysis and inflammatory factors in adipose tissues,including PPARγ,CD36,C/EBPα,SREBF1,Fasn,ATGL,MCAD,LCAD,IL-6,TNF-α,MCP-1.Proadipocyte were isolated and cultured from LncPrep+96kb knockout mice.Proadipocyte 3T3-L1 was transfected of LncPrep+96kb overexpre-ssion plasmid.These cells were induced into adipocytes in vitro to evaluate the effect of LncPrep+96kb on triglyceride accumulation in adipocytes.At the same time,western blotting was performed to detect the changes of p-AKT stimulated by insulin in LncPrep+96kb knockout adipocytes.These in vivo and in vitro methods were employed to comprehensively evaluate the pathological changes of adipose tissue in high-fat diet-induced LncPrep+96kb knockout mice and explore the possible mechanism of LncPrep+96kb on lipid synthesis,lipolysis and insulin sensitivity in adipose tissues.ResultsAfter 18 weeks of feeding with different diets,the body weight of mice in the high-fat diet group was significantly higher than that in the normal diet group.There was no significant difference in body weight between KO-CD and WT-CD,but the weight of KO-HFD was significantly higher than that of WT-HFD.IPGTT showed that the impaired glucose tolerance in KO-HFD was more severe than that in WT-HFD.After 2 hours of glucose injection,the blood glucose concentration remained in high level.At the same time,we detected the level of serum insulin and found that the level of insulin was significantly increased,it suggested that the mice developed insulin resistance.ITT experiment showed that there was no significant change among groups.Compared with the normal diet group,the serum levels of TG,TC and LDL-C in the high fat group increased significantly,while the level of HDL decreased.The level of TG in KO-HFD was higher than that in WT-HFD.We further examined the changes of p-AKT in adipose tissue around epididymis,skeletal muscle and liver in the basic state and found that p-AKT in adipose tissue and skeletal muscle was significantly lower in KO-HFD than that in WT-HFD.After stimulation of insulin,the p-AKT in adipose tissue and skeletal muscle tissue was also significantly lower in KO-HFD than that in WT-HFD.In order to further explore the possible mechanism of abnormal glucose and lipid metabolism induced by high fat diet in LncPrep+96kb knockout mice from adipose tissue,we examined the pathological changes of adipose tissue around epididyma.We found that the weight of epididymal adipose tissue in KO-HFD was significantly higher than that in WT-HFD,and the organ index of periepididymal adipose tissue in KO-HFD was also higher than that in WT-HFD.The leptin level in KO-HFD was significantly higher than that in WT-HFD,while the level of adiponectin decreased.HE staining showed adipocytes in KO-HFD was further enlarged and irregular.Real-time quantitative PCR showed that the mRNA expression of adipogenic genes,including PPARy,C/EBPα and CD36 significantly increased in KO-HFD,while the mRNA expression of lipolysis gene ATGL and MCAD significantly decreased.Western blotting showed that the protein level of PPAR γ and CD36 increased significantly and the protein level of ATGL decreased significantly in adipose tissues of KO-HFD.In vitro experiments were conducted to further evaluate the effects of knockout and overexpression of LncPrep+96kb on adipogenesis of adipocytes.Oil red O staining showed that triglyceride accumulation increased significantly after knockout of LncPrep+96kb and decreased significantly after overexpression of lncPrep+96kb.LncPrep+96kb knockout also inhibited insulin-induced AKT phosph-orylation.Elisa was employed to detect the changes of serum inflammatory factors.The levels of serum IL-6 and TNF-α in KO-HFD were significantly higher than those in WT-HFD.Immunohistochemical staining showed that macrophages in adipose tissues increased significantly and formed a characteristic coronal structure in KO-HFD group.Flow cytometry showed a significant increase in the proportion of M1 macrophages in adipose tissue of KO-HFD.Compared with WT-HFD,mRNA level of inflammatory factors IL-6 and TNF-α were significantly increased in KO-HFD group.Conclusion:1.High fat diet induced more severe abnormal glucose tolerance and lipid metabolism in LncPrep+96kb knockout mice.2.LncPrep+96kb knockout promoted lipid synthesis and inhibited lipolysis in adipocytes,resulting in excessive fat accumulation around the epididymis after induction of high-fat diet.3.LncPrep+96kb knockout promoted macrophage infiltration and inflammatory changes in adipose tissues after induction of high-fat diet. |
