CHL1在高糖高脂诱导的胰岛素抵抗细胞和小鼠模型中的作用及其机制探讨

Impact and mechanism of CHL1 in insulin resistant adipocytes and insulin resistant mouse model induced by high glucose and high fat

目的探讨细胞黏附分子CHL1在高糖高脂诱导的胰岛素抵抗细胞和小鼠模型中的作用及其机制。方法将前脂肪细胞系3T3-L1通过完全随机法分为对照组和CHL1过表达组,分别转染空载体和CHL1过表达载体,随后通过胰岛素诱导分化为成熟脂肪细胞,再通过高糖高脂诱导胰岛素抵抗。葡萄糖消耗实验检测成熟脂肪细胞胰岛素抵抗效应。Western blot法检测两组细胞CHL1和葡萄糖转运蛋白4(GLUT4)的表达水平以及丝氨酸/苏氨酸激酶(AKT)磷酸化水平,实时定量PCR(RT-PCR)法检测肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-6的mRNA表达水平。然后,选取6~8周龄的C57BL/6雄性小鼠24只,体重20~25 g,通过完全随机法分为常规饲料组(常规组)、高脂饲料组(高脂组)、过表达对照+高脂组和CHL1过表达+高脂组,每组6只。通过高脂饲料饲喂小鼠构建胰岛素抵抗小鼠模型,通过尾静脉注射慢病毒过表达CHL1。各组小鼠饲养4个月后称重,然后处死收集附睾白色脂肪并称重,苏木素-伊红染色观察小鼠附睾白色脂肪病理情况,免疫组织化学染色观察其CHL1表达情况,RT-PCR法检测小鼠附睾白色脂肪组织中CHL1、TNF-α、IL-6 mRNA的表达水平。结果(1)细胞实验结果:Western blot结果示CHL1过表达组细胞中CHL1蛋白表达高于对照组(P<0.01);葡萄糖消耗实验结果示,CHL1过表达组细胞葡萄糖剩余量低于对照组(P<0.05);RT-qPCR结果示,CHL1过表达组细胞TNF-α和IL-6 mRNA表达水平均低于对照组(P均<0.01);Western blot结果示,CHL1过表达组细胞GLUT4和p-AKT/AKT蛋白表达均高于对照组(P均<0.01)。(2)动物实验结果:饲养4个月后高脂组和过表达对照+高脂组小鼠体重和附睾白色脂肪质量均高于常规组(P均<0.01),CHL1过表达+高脂组则均低于过表达对照+高脂组(P均<0.01);苏木素-伊红染色结果示,高脂组和过表达对照+高脂组小鼠附睾白色脂肪细胞体积大于常规组,CHL1过表达+高脂组则小于过表达对照+高脂组(P均<0.01);RT-qPCR结果示,高脂组和过表达对照+高脂组小鼠附睾白色脂肪组织中IL-6和TNF-αmRNA表达水平均高于常规组(P均<0.01),CHL1过表达+高脂组则均低于过表达对照+高脂组(P均<0.05);免疫组织化学染色半定量结果示,高脂组和过表达对照+高脂组小鼠附睾白色脂肪组织中CHL1蛋白表达水平均低于常规组(以常规组的表达量为1,高脂组、过表达对照+高脂组的表达量分别为0.344、0.427),CHL1过表达+高脂组(表达量1.465)则高于过表达对照+高脂组;RT-qPCR结果示,高脂组和过表达对照+高脂组小鼠附睾白色脂肪组织中CHL1 mRNA表达水平均低于常规组(P均<0.01),CHL1过表达+高脂组则高于过表达对照+高脂组(P<0.01)。结论CHL1可改善高糖高脂诱导的细胞和小鼠模型的胰岛素抵抗,其机制可能与抑制AKT激活和相关炎症反应有关。

Objective To investigate the role and mechanism of cell adhesion molecule L1 like(CHL1)in insulin resistant adipocytes and insulin resistant mouse model induced by high glucose and high fat.Methods The 3T3-L1 preadipocytes were randomly divided into control group(transfected with empty vector)and CHL1 overexpression group(transfected with CHL1 vector),cells were then induced to mature adipocytes by insulin,and insulin resistance was then induced by high sugar and high fat.The glucose content was measured to determine the glucose consumption of cells from the two groups.Protein expression levels of CHL1 and glucose transporter 4(GLUT4),serine/threonine protein kinase(AKT)phosphorylation levels were detected by Western blot(WB),the mRNA expression levels of TNF-αand IL-6 were detected by real-time quantitative PCR(RT-qPCR).24 C57BL/6 adult male mouse were randomly divided into conventional diet group(regular group),high-fat diet group(high-fat group),empty vector overexpression+high-fat group and CHL1 overexpression+high-fat group(n=6 each group).CHL1 overexpression was induced by tail vein injection of lentivirus.Four months later,mice were sacrificed,body weight was determined,and the epididymal white adipose tissue was collect.Hematoxylin-eosin staining(HE)was used to observe the pathology of mouse epididymal white adipose tissue,the expression of CHL1 was evaluated by immunohistochemical staining(IHC),RT-qPCR was used to detect the mRNA expression levels of CHL1,TNF-αand IL-6 in mouse epididymal white adipose tissue.Results In vitro,glucose consumption was significantly higher in the CHL1 overexpression group than in the control group(P<0.05),and the protein expressions of CHL1 and GLUT4 were higher in the CHL1 overexpression group than those in the control group(P<0.01),and the mRNA expressions levels of TNF-αand IL-6 were lower in the CHL1 overexpression group than those in the control group(P<0.01).In vivo,the body weight and epididymal white adipose tissue of mouse were higher in the high-fat group and the empty vector overexpression+high-fat group than those in the conventional group(P<0.01),which were lower in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group(P<0.01).HE results showed that the volume of epididymal white adipocytes was larger in the high-fat group and the overexpression control+high-fat group than that in the conventional group,which was smaller in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group(P<0.01).The mRNA expression levels of IL-6 and TNF-αin epididymal white adipose tissue of mice were higher in the high-fat group and the empty vector overexpression+high-fat group than those in the conventional group(P<0.01),which were lower in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group(P<0.05).IHC results showed that protein expression of CHL1 in epididymal white adipose tissue was lower in the high-fat group and the empty vector overexpression+high-fat group than in regular group,which was upregulated in the CHL1 overexpression+high fat group than in the empty vector overexpression+high-fat group(P<0.01).RT-qPCR results showed that mRNA expression of CHL1 in epididymal white adipose tissue was lower in the high-fat group and the empty vector overexpression+high-fat group than in regular group(P<0.01),which was higher in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group(P<0.01).Conclusion Overexpression of CHL1 can improve insulin resistance in adipocytes and mouse insulin resistance model induced by high glucose and high fat,and the beneficial effects might be mediated by the inhibition of AKT activation and the reduction of related inflammatory responses.