中枢nesfatin-1表达下调对SD大鼠胰岛素信号通路的影响

Effects of central nesfatin-1 knockdown on hepatic insulin signaling pathway

目的探讨中枢nesfatin-1表达下调对SD大鼠肝胰岛素信号通路的影响。方法将雄性SD大鼠随机分为普食喂养+人工脑脊液(a CSF)输注组(NCA组,n=10),普食喂养+Ad-sh GFP输注组(NCG组,n=10),普食喂养+Ad-sh NUCB2输注组(NCN组,n=10),高脂喂养+人工脑脊液(a CSF)输注组(HFA组,n=10),高脂喂养+Ad-sh GFP输注组(HFG组,n=10),高脂喂养+Ad-sh NUCB2输注组(HFN组,n=10)。构建第三脑室微量输注系统进行第三脑室干预,留取普食组大鼠下丘脑、肝脏、脂肪和肌肉组织采用Western印迹测定nesfatin-1表达。采用Western印迹法测定各组大鼠G6Pase及PEPCK的表达水平以和IR、IRS-1、AKT、STAT3及m TOR的蛋白磷酸化水平变化。结果和NCA或NCG组分别相比,NCN组下丘脑nesfatin-1表达显著降低(P<0.05),而外周组织(肝、脂肪及肌肉)nesfatin-1表达无显著差异。和NCA组相比,HFA组的G6Pase、PEPCK表达水平均显著增高,而IR、IRS-1、AKT、STAT3、m TOR的蛋白磷酸化水平均显著降低(P<0.05)。和NCA或NCG组相比,NCN组的G6Pase、PEPCK表达水平均显著增高,而IR、IRS-1、AKT、STAT3、m TOR的蛋白磷酸化水平均显著降低(P<0.05)。和HFA或HFG组相比,HFN组的G6Pase、PEPCK表达水平均显著增高,而IR、IRS-1、AKT、STAT3、m TOR的蛋白磷酸化水平均显著降低(P<0.05)。结论中枢Ad-sh NUCB2输注可下调SD大鼠下丘脑nesfatin-1表达,同时下调机体肝胰岛素信号通路级联反应蛋白水平,可能通过抑制m TOR/STAT3信号通路从而降低机体肝脏糖异生。

Objective To investigate the effects of central nesfatin-1 knockdown on hepatic insulin signaling pathway.Methods Male Sprague-Dawley(SD) rats were randomly divided into normal-chow die (NCD) +aCSF (NCA, n=10), NCD+Ad-shGFP(NCG, n=10), NCD +Ad-shNUCB2 (NCN, n=10), high-fat diet (HFD) +aCSF(HFA, n=10), HFD +Ad-shGFP(HFG, n=10), HFD+Ad-shNUCB2 groups (HFN, n=10).After central intervention, NCD-fed rats were sacrificed and the expressions of nesfatin-1 in hypothalamic and peripheral tissues were examined.Other rats were sacrificed and the tissues were quickly taken away.The phosphorylated protein levels of IR, IRS-1, AKT, STAT3 and mTOR, and the protein levels of G6Pase, PEPCK were evaluated by Western blot.Results After central Ad-shNUCB2 intervention, hypothalamic nesfatin-1 protein levels was significantly decreased by 66% compared with rats that were given ACF or Ad-shGFP(P<0.05).However, no significant differences were found in nesfatin-1 expression in liver, muscle and adipose tissues. Compared with those of NCA group, the protein levels of G6Pase and PEPCK in HFA group were increased significantly (P<0.05).Howev-er, the phosphorylated protein levels of IR, IRS-1, AKT, STAT3 and mTOR inHFA group were decreased obviously(P<0.05).Compared with those of NCA or NCG groups, the protein levels of G6Pase and PEPCK in NCN group were increased significantly (P<0.05).However, the phosphorylated protein levels of IR, IRS-1, AKT, STAT3 and mTOR in NCN group were markedly decreased (P<0.05).Compared with HFA or HFG groups, the levels of G6Pase and PEPCK in HFN group were increased significantly (P<0.05).However, the phospho-rylated protein levels of IR, IRS-1, AKT, STAT3 and mTOR in HFN group were decreased significantly (P<0.05).Conclusions Central Ad-shNUCB2 intervention could decrease hypothalamic nesfatin-1 protein levels significantly.Knockdown of central nesfatin-1 could decrease the phosphorylation of several proteins in insulin signaling cascade.Moreover, central nesfatin-1 knockdown could lighten insulin sensitivity through inhibition of mTOR-STAT3 signaling pathway.