Liraglutide treatment promotes beta cells proliferation via regulation of microRNA expression

目的 探讨利拉鲁肽对db/db小鼠胰腺及大鼠胰岛细胞瘤细胞系INS-I中microRNA表达的影响.方法 将20只4周龄雄性db/db小鼠按随机数字表法分为对照组和利拉鲁肽组(每组10只).分别给予0.1ml生理盐水或300 ng/g利拉鲁肽皮下注射,每日2次.8周后测定糖化血红蛋白,行腹腔注射葡萄糖耐量试验(TPGTT).8周末处死小鼠,免疫组化法分析小鼠胰腺β细胞增殖水平；实时荧光定量聚合酶链反应( RT-PCR)测定小鼠胰腺microRNA(miR-375和miR-34a)的表达.INS-1细胞分别给予0.5 mmol/L棕榈酸培养0、48、72 h或100 nmol/L利拉鲁肽预处理12h后,给予0.5 mmol/L棕榈酸培养72 h,RT-PCR测定miR-375和miR-34a表达水平.采用t检验及方差分析进行组间数据比较分析.结果 与对照组相比,利拉鲁肽组小鼠糖化血红蛋白水平降低(分别为7.3％±0.3％和4.7％±0.6％,t=16.47,P＜0.01)；IPGTT血糖曲线下面积降低[分别为( 4568±197)和(1927±127) mmol·L-1·min-1,t =26.53,P＜0.05]；胰岛素曲线下面积增加[分别为(1080±247)和(2818±378)μg·L-1·min-1,t=7.73,P＜0.05].免疫组化法显示,与对照组相比,利拉鲁肽组小鼠胰岛素阳性面积增加(分别为1.40±0.30和0.37±0.09,t=19.14,P＜0.01),Brdu染色阳性细胞比例增加(分别为2.40％±0.22％和0.73％±0.10％,t=4.97,P＜0.01).利拉鲁肽组小鼠胰腺miR-375与miR-34a表达较对照组分别降低50％(分别为1.1±0.3和2.2±0.5,t=3.08,P＜0.05)和71％(分别为1.1±0.3和3.8±1.2,t=2.80,P＜0.05).棕榈酸培养使INS-1细胞miR-375表达呈剂量、时间依赖性增加,利拉鲁肽可抑制棕榈酸诱导的miR-375表达(F=7.20,P＜0.01).结论 microRNA可能是利拉鲁肽调节β细胞增殖的靶点之一.Objective To investigate the effect of glucagon-like peptide 1 analogue liraglutide on microRNA expression of db/db mice and rat insulinoma cell line( INS-l).Methods Twenty 4-week old male db/db mice were randomly assigned to receive a subcutaneous injection of liraglutide(300 ng/g bid) or normal saline (0.1 ml bid) ( control group) according to random numbers table.Glycated hemoglobin (HbAlc) was determined before and after 8 weeks of intervention.At the end of the intervention,intraperitoneal glucose tolerance test ( IPGTT) was performed Beta cells proliferation rate was determined by using immunohistochemistry methods.INS-1 cells were cultured with 0.5 mmol/L palmitic acid for 0.48,72 h or treated by 100 nmol/L liraglutide for 12 h before 72 h of palmitic acid ( 0.5 mmol/L) culture.microRNA miR-375 and miR-34a expression were examined by real-time polymerase chain reaction ( RTPCR).The t test or ANOVA analysis was used for data analysis.Results Compared with the control group,HbAle in the liraglutide group was significantly lower ( 7.3％ ± 0.3％ vs 4.7％ ± 0.6％,t =16.47,P ＜ 0.01); the glucose aera under curve in the liraglutide group was decreased significantly (( 4568 ± 197) vs ( 1927 ± 127) mmol · L-1 · min -1,t =26.53,P ＜ 0.05 ),while insulin aera under curve was increased significantly ( ( 1080 ± 247 ) vs ( 2818 4± 378 ) μg· L- 1.min - 1,t =7.73,P ＜ 0.05 ).Immunohistochemistry showed more insulin staining in single pancreas of db/db mice after liraglutide treatment than in the control group ( 1.40 ±0.30 vs 0.37 ±0.09,t =19.14,P <0.01 ).More Brdu-positive cells were observed in islets of liraglutide-treated mice than in control mice after intervention ( 2.40％ ±0.22％ vs 0.73％ ±0.10％,t =4.97,P ＜0.01 ).Compared with control mice,the miR-375 expression of pancreas in liraglutide-treated db/db mice was reduced by 50％ ( 1.1 ± 0.3 vs 2.2 ± 0.5,t =3.08,P ＜0.05 ) and miR-34a was reduced by 71％ ( 1.1 ± 0.3 vs 3.8 ± 1.2,t =2.80,P ＜ 0.05 ).In addition,the miR-375 expression of INS-1 cells was up-regulated dose- and time-dependently in response to palmitic acid;and palmitic acid-induced miR-375 expression was suppressed by liraglutide in vitro( F =7.20,P ＜ 0.01 ).Conclusion Liraglutide may preserve beta-cell proliferation in response to lipotoxicity through a regulation of miRNA.国家重点基础研究发展计划(973计划)资助项目中国科技核心期刊(ISTIC)06357-3600