高糖条件下腺苷酸活化蛋白激酶对大鼠胃平滑肌细胞能量代谢调控机制的研究

目的探讨高糖条件下腺苷酸活化蛋白激酶(AMPK)对大鼠胃平滑肌细胞能量代谢调控机制的影响。方法制备并确定糖尿病胃轻瘫(DGP)大鼠模型,实验分为正常对照(NC)组和DGP组,抗体芯片观察AMPK磷酸化通路的变化,确定能量代谢相关功能蛋白;SeahorseXFe细胞能量代谢分析系统观察2-脱氧-D-葡萄糖(2-DG)与化合物C(Compound C)对高糖条件下大鼠胃平滑肌细胞耗氧率(OCR)与细胞外酸化率(ECAR)的影响;沉默AMPK后,观察高糖条件下大鼠胃平滑肌细胞能量代谢相关功能蛋白变化。结果高糖条件下AMPK抑制大鼠胃平滑肌细胞OCR的基础呼吸、最大呼吸值、三磷酸腺苷产量(P<0.05),促进大鼠胃平滑肌细胞ECAR的糖酵解水平和能力(P<0.01)。抗体蛋白芯片发现18个磷酸化差异抗体,涉及与能量代谢相关蛋白包括:p53、Ca²⁺/CaM依赖性蛋白激酶Ⅱ(CaMKⅡ)、Ca²⁺/CaM依赖性蛋白激酶Ⅳ(CaMKⅣ)、磷脂酰肌醇特异性磷酯酶C-β3(PLC-β3)、蛋白激酶A(PKA)、乙酰CoA羧化酶1(ACC1)、真核延伸因子2(eEF2)、真核延伸因子激酶2(eEF2K)。与HG(24 h)组比较,HG(48 h)组p53、ACC1、eEF2表达升高(P<0.05或P<0.01),PLC-β3表达降低(P<0.01)。与HG(48 h)组比较,HG(48 h)+siRNA组p53、ACC1表达降低(P<0.01)。与HG(24 h)组比较,HG(48 h)组p-CaMKⅡThr³⁰⁵/CaMKⅡ与p-CaMKⅣThr^196/200/CaMKⅣ比值升高(P<0.01)。与HG(48 h)组比较,HG(48 h)+siRNA组p-CaMKⅡThr³⁰⁵/CaMKⅡ比值降低(P<0.01)。HG(48 h)组PKA活性高于HG(24 h)组(P<0.01)。结论高糖条件下AMPK通过调控p53、ACC1、eEF2、CaMKⅡ、CaMKⅣ、PLC-β3及PKA生物学作用,抑制大鼠胃平滑肌细胞线粒体代谢途径及促进糖酵解途径。

Study on the mechanism of AMPK on energy metabolism of rat gastric smooth muscle cells under high glucose conditions

Objective To investigate the effect and mechanism of AMPK on energy metabolism of rat gastric smooth muscle cells.Methods The DGP rat model was prepared and determined. The experiment was divided into NC group and DGP group. The antibody chip was used to observe the changes of AMPK phosphorylation pathway and determine the functional proteins related to energy metabolism. It was observed the effect of 2-DG and compound C on OCR and ECAR of rat gastric smooth muscle cells under high glucose by the Seahorse XFecell energy metabolism analysis system. After silencing AMPK,the changes of functional proteins related to energy metabolism were observed in rat gastric smooth muscle cells under high glucose.Results In rat gastric smooth muscle cells under high glucose,the activated AMPK inhibited the basic respiration,maximum respiration value,and ATP production of OCR(P<0. 05);and promoted glycolysis and capacity of glycolysis of ECAR(P<0. 01). Eighteen differentially phosphorylated antibodies were found on the antibody protein chip,and proteins involved in energy metabolism include:p53,CaMKⅡ,CaMKⅣ,PLC-β3,PKA,ACC1,eEF2 and eEF2K. Compared with HG(24 h)group,the expression of p53,ACC1,eEF2 and the ratio of p-CaMKⅡThr³⁰⁵/CaMKⅡ to p-CaMKⅣThr^196/200/CaMKⅣ in HG(48 h)group increased(P<0. 05 or P<0. 01),while the expression of PLC-β3 decreased(P<0. 01).Compared with HG(48 h)group,the expressions of p53 and ACC1 and the ratio of p-Ca MKⅡThr³⁰⁵/CaMKⅡin HG(48 h)+siRNA group decreased(P<0. 01). The activity of PKA in HG(48 h)group was higher than that in HG(24 h)group(P<0. 01).Conclusion The activated AMPK inhibits mitochondrial metabolism pathway and promotes glycolysis pathway in rat gastric smooth muscle cells by regulating the biological effects of p53,ACC1,eEF2,CaMKⅡ,Ca MKⅣ,PLC-β3 and PKA in high glucose condition.