HtrA 1基因敲除对小鼠葡萄糖代谢及胰岛功能的影响

Effect of HtrA1 gene knockout on glucose metabolism and pancreatic cell function in mice

目的探讨HtrA 1基因敲除对小鼠糖代谢及胰岛功能的影响。方法选取6周龄雄性野生型C57BL/6J小鼠(WT小鼠)和HtrA 1基因敲除小鼠(KO小鼠)按照同窝随机对照原则分组,分别给予高脂饮食(脂肪占总热量的60%)或普脂饮食(脂肪占总热量的5%),记录喂养16周时两组WT小鼠、KO小鼠的体重及喂养期间摄食量。对18周龄(普脂或高脂喂养12周)小鼠行葡萄糖耐量试验(IPGTT),并测定空腹胰岛素水平,进一步对21周龄(高脂喂养15周)小鼠行葡萄糖刺激胰岛素分泌(GSIS)试验,观察糖代谢及胰岛素分泌情况。分离小鼠胰岛在细胞水平行GSIS试验,测定胰岛素水平。取22周龄(高脂喂养16周)小鼠胰腺组织行组织切片,进行胰岛素(绿色)和胰高血糖素(红色)免疫荧光双染,以及胰岛素(绿色)和CD31(红色)免疫荧光双染。应用ZEN 2.0软件对胰岛中CD31水平进行定量,比较WT小鼠与KO小鼠胰岛中CD31的平均荧光强度。结果高脂饮食组或普脂饮食组的KO与WT小鼠的体重和摄食量的差异均无统计学意义(P值均>0.05)。普脂饮食组KO小鼠糖负荷10、20、120 min时的血糖水平均显著高于同组WT小鼠(P值均<0.05),空腹血糖及糖负荷后30、45、60、90 min时的血糖水平与同组WT小鼠的差异均无统计学意义(P值均>0.05)。高脂饮食组KO小鼠糖负荷10、20、30、45 min时血糖水平均显著高于同组WT小鼠(P值均<0.05),空腹血糖及糖负荷后60、90、120 min时血糖水平与同组WT小鼠的差异均无统计学意义(P值均>0.05)。18周龄普脂饮食组WT小鼠与KO小鼠空腹胰岛素水平的差异无统计学意义(P>0.05),高脂饮食组KO小鼠空腹胰岛素水平显著低于WT小鼠(P<0.05)。21周龄高脂饮食组空腹及注射葡萄糖后15 min时KO小鼠的胰岛素水平均显著低于WT小鼠(P值均<0.05),KO小鼠与WT小鼠注射葡萄糖后30 min时胰岛素水平的差异无统计学意义(P>0.05)。经高糖刺激(20 mmol/L葡萄糖处理)的KO小鼠胰岛细胞的胰岛素分泌能力显著低于WT小鼠(P<0.05)。高脂饮食组KO小鼠胰岛内CD31表达水平显著低于同组WT小鼠(P<0.05)。结论HtrA 1基因敲除可导致小鼠机体糖代谢紊乱及胰岛功能异常,其机制可能与胰岛细胞血管功能有关。

Objective To explore the effect of HtrA1 knockout on glucose metabolism and pancreatic cell function in mice.Methods Six-week-old male wild-type C57BL/6J mice and HtrA1 knockout mice were randomly divided into high-fat diet group(60%kcal from fat)and normal-chow diet group(5%kcal from fat).The body weight and food intake of wild-type and gene knockout mice in both groups were recorded at 16 weeks of feeding.Intrapertoneal glucose tolerance testing(IPGTT)and fasting insulin level were assessed on 18-week-old mice(12 weeks of mormal-chow or high-fat feeding).The plasma insulin secretion was assessed by glucose-stimulated insulin secretion(GSIS)assay in 21-week-old mice(15 weeks of high-fat feeding).The level of plasma insulin was determined in isolated islets by a GSIS assay.Immunofluorescence staining for insulin(green),glucagon(red)and CD31(red)was performed on pancreatic tissue from 22-week-old mice(16 weeks of high-fat feeding).The expression of CD31 in islets was quantified by ZEN 2.0 software,and the mean fluorescence intensity of CD31 in islets was compared between wild-type and gene knockout mice.Results There was no significant difference in body weight or food intake between wild-type and gene knockout mice with high-fat diet or normal-chow diet(both P>0.05).In the normal-chow diet group,the blood glucose level in gene knockout mice was significantly higher than that in wild-type mice at 10,20 and 120 min after glucose injection(all P<0.05),but there was no significant difference in the blood glucose level at 0,30,45,60 and 90 min after glucose injection between wild-type and gene knockout mice(all P>0.05).In the high-fat diet group,the blood glucose level in gene knockout mice was significantly higher than that in wild-type mice at 10,20,30 and 45 min after glucose injection(all P<0.05),but there was no significant difference in the blood glucose level at 0,60,90 and 120 min after glucose injection between wild-type and gene knockout mice(all P>0.05).In the normal-chow diet group,there was no significant difference in the fasting insulin level between 18-week-old wild-type and gene knockout mice(P>0.05).In the high-fat diet group,the fasting insulin level in the 18-week-old wild-type mice was significantly lower than that in the 18-week-old gene knockout mice(P<0.05).In the high-fat diet group,the insulin level of 21-week-old gene knockout mice was significantly lower than that of wild-type mice at fasting and 15 min after glucose injection(both P<0.05),but there was no significant difference in the insulin level at 30 min after glucose injection between 21-week-old gene knockout and wild-type mice(both P>0.05).Islet cells of gene knockout mice stimulated by high glucose(20 mmol/L glucose treatment)decreased the insulin secretion capacity compared with wild-type mice(P<0.05).The expression level of CD31 in pancreatic islets in gene knockout mice was significantly lower in the high-fat diet group than that in wild-type mice(P<0.05).Conclusion HtrA1 knockout can lead to glucose metabolism disorder and abnormal islet function in mice,and its mechanism may be related to the vascular function of islet cell.