GLP-1-AMPK信号在DJB手术治疗T2DM中的作用及分子机制的实验研究

Gastric bypass surgery becoming a new way for type 2 diabetes (T2DM) treatment with the mechanism remains unclear.. Our previous studies showed the significantly increased secretion of GLP-1 after DJB, and increasing expression of GLP-1R in pancreas cells and increasing expression of insulin receptor in liver cells which showed the increasing the insulin sensitive of the liver. Together with the studies of the other groups that Glucagon-like peptide-1(GLP-1) reduces hepatic lipogenesis by the activation of AMP-activated protein kinase (AMPK) and antidiabetic drug metformin treating T2DM was associated with AMPK activation in duodenum, we hypothesis that foregut plays the important role in the mechanism of DJB treatment of T2DM. The increasing secretion of GLP-1 by DJB could activate AMPK in duodenum and liver.. In this study, we sought to investigate the role of AMPK activation in the proximal gut on glucose homeostasis by measuring insulin sensitivity, GLP-1 levels after DJB surgery. By in vivo duodenal intestine injections or in vitro using HepG2 insulin resistance cell model of AMPK antagonists or agonist, study the effect of AMPK activity with the ability of intestine absorption and liver glucose metabolic regulation. The Confirmation of the important role of GLP-1-AMPK signal in the treatment of T2DM, giving us the better choice of surgical procedure in T2DM treatment and providing clue for new drugs targeting AMPK for the T2DM medicine treatment.

胃肠手术治疗2型糖尿病(T2DM)疗效确切机制不清。我们前期研究发现十二指肠空肠旷置(DJB)手术后T2DM大鼠胰腺GLP-1R、肝脏InsR 的表达明显升高，血清GLP-1分泌增加等。有文献报道GLP-1激活肝组织AMPK降低其脂肪合成；二甲双胍治疗T2DM与十二指肠组织AMPK激活有关。我们提出：DJB手术治疗T2DM的关键在“前肠”，使GLP-1分泌增加，激活小肠和肝组织AMPK，减少小肠葡萄糖吸收和调节肝组织葡萄糖代谢。本研究拟采用DJB手术干预T2DM大鼠，十二指肠肠壁注射AMPK拮抗剂或激动剂，检测分析AMPK活性变化及其与GLP-1的关系；采用HepG2胰岛素抵抗细胞模型，验证GLP-1能够激活AMPK并提高肝细胞葡萄糖代谢调节能力。明确GLP-1-AMPK信号在T2DM治疗中的作用和分子机制，为选择适合的外科术式治疗T2DM和针对AMPK的降糖药物的研发提供理论依据。

本研究旨在探究DJB手术对T2DM的改善作用的分子机制。首先建立DJB手术和假手术干预T2DM大鼠的动物模型，分别在手术前1周、手术后1、2、4、6周检测不同组别大鼠空腹血糖、空腹胰岛素，计算胰岛素抵抗和胰岛素敏感指数，评价手术对T2DM大鼠血糖稳态的改善作用。实验结果显示，DJB手术能够显著改善胰岛素抵抗，调节葡萄糖稳态。.利用Westernblot、Q-PCR、免疫组化等方法，测定肝细胞胰岛素信号通路关键蛋白InsR、IRS2、PI3K、Akt的表达变化，结果显示，DJB手术明显提高了T2DM大鼠肝细胞胰岛素信号通路关键蛋白InsR和IRS2以及PI3K的表达，增加Akt磷酸化水平。我们的实验结果还显示DJB手术后肝细胞葡萄糖转运蛋白GLUT4的表达明显上调，糖异生关键酶PEPCK和G-6-Pase的表达以及肝糖元生成关键酶GSK3β的表达明显下调。DJB手术明显提高了肝葡萄糖的利用，降低了肝源性糖异生。同时DJB手术后明显下调血清中TG、TC的水平,明显改善了肝脏中乙酰辅酶A羧化酶（ACC）的表达, 降低了肝细胞脂肪酸合成的水平。.DJB手术明显减少了ZDF大鼠的BP肠袢肠壁中的炎性细胞数量，改善了炎性细胞的形态。降低了DJB手术大鼠BP肠袢部位促炎因子IL-1β、IL-6、TNF-ɑ的表达，并且显著提高了抗炎性因子IL-10的表达水平。并且免疫组化结果显示，DJB手术后大鼠BP肠袢AMPK磷酸化程度明显升高。.体外实验利用胰岛素抵抗肝细胞模型，探究GLP-1-AMPK信号在改善细胞胰岛素抵抗中的作用和分子机制。GLP-1类似物Ex-4和AMPK激动药AICAR可促进胰岛素抵抗肝细胞的葡萄糖的利用，同时使用AMPK拮抗药Compound C可阻断Ex-4引起的AMPK磷酸化水平升高，这证明GLP1是通过激活AMPK，增加AMPK磷酸化程度发挥其增强胰岛素抵抗细胞利用葡萄糖的能力的作用。

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