^(13)C-葡萄糖钳夹实验评估小鼠胰岛素敏感性  

Evaluation of insulin sensitivity in mice using ^(13)C-glucose labeled clamp technique

作　　者：曹可欣 刘卓航 姜懿珅 孙旭 陈洁 CAO Kexin;LIU Zhuohang;JIANG Yishen;SUN Xu;CHEN Jie(State Key Laboratory of Common Mechanism Research for Major Diseases,Department of Immunology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院,重大疾病共性机制研究全国重点实验室免疫学系,北京100005

出　　处：《基础医学与临床》2025年第4期442-449,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(82401014)。

摘　　要：目的建立^(13)C同位素标记的高胰岛素正血糖钳夹模型用以评估小鼠胰岛素敏感性。方法高脂喂养建立小鼠胰岛素抵抗模型,检测胰岛素下游信号蛋白激酶,即Akt的磷酸化水平;使用葡萄糖耐量试验,胰岛素耐量试验和丙酮酸耐量试验检测小鼠血糖的变化,计算三者曲线下面积以量化血糖水平在2 h内的总体变化;通过小鼠颈静脉插管手术和^(13)C同位素标记的高胰岛素正血糖钳夹实验,监测小鼠血糖并计算葡萄糖灌注速率;收集小鼠尾静脉血清进行液相色谱串联质谱分析,计算葡萄糖清除速率和肝脏糖异生速率;抑制小鼠糖原分解后,检测Akt的磷酸化水平以评估胰岛素信号传导;进行钳夹实验并计算葡萄糖灌注速率,收集小鼠尾静脉血清进行液相色谱串联质谱分析,计算葡萄糖清除速率和肝脏糖异生速率。结果高脂喂养小鼠血糖水平升高(P<0.001),并伴随肝脏,肌肉中的p-Akt水平减少。葡萄糖耐量试验、胰岛素耐量试验和丙酮酸耐量试验显示,高脂喂养的小鼠血糖明显增高(P<0.05),曲线下面积升高(P<0.001)。高脂喂养的小鼠在^(13)C标记的钳夹实验后血糖趋于稳定,与对照组相比,其葡萄糖灌注速率下降(P<0.001),葡萄糖清除速率下降(P<0.0001),肝脏糖异生速率上升(P<0.01)。用药物抑制糖原分解后,与对照组相比,小鼠血糖水平升高(P<0.001),并伴随肝脏、肌肉中的p-Akt水平减少,胰岛素信号通路受损。^(13)C标记的钳夹实验表明,处理组小鼠葡萄糖灌注速率下降(P<0.01),葡萄糖清除速率下降(P<0.0001),肝脏糖异生速率上升(P<0.01)。结论建立了一种改良的高胰岛素正血糖钳夹实验模型,可以评估小鼠体内胰岛素的敏感性。Objective To establish a ^(13)C isotope-labeled hyperinsulinemic-euglycemic clamp model for the assessment of insulin sensitivity in mice.Methods The mouse model of insulin resistance was established by high-fat diet feeding.The phosphorylation level of downstream insulin signaling protein,Protein Kinase,also known as Akt was assessed.Glucose metabolism was evaluated using glucose tolerance test,insulin tolerance test,and pyruvate tolerance tests and the area under the curve of the respective testes over 2 hours was calculated to quantify overall blood glucose level.Mice underwent jugular vein catheterization surgery,and a ^(13)C isotope-labeled hyperinsulinemic-euglycemic clamp experiment was conducted to monitor blood glucose levels and to calculate the glucose infusion rate(GIR).Tail vein serum was collected for liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis to determine glucose disposal rate(GDR)and hepatic gluconeogenesis rate(HGP).Following the inhibition of glycogenolysis in mice,Akt phosphorylation level was measured to evaluate insulin signaling.The clamp test was repeated to calculate GIR,and tail vein blood serum was analyzed by LC-MS/MS to determine GDR and HGP.Results After one week of high-fat diet feeding,mice exhibited significantly elevated blood glucose level(P<0.001)accompanied by reduced p-Akt level in liver and muscle.Glucose tolerance tests,insulin tolerance tests and pyruvate tolerance test demonstrated a significant increase in blood glucose level(P<0.05)and a higher area under curves(AUC)(P<0.001)in high-fat-fed mice.During the ^(13)C-labeled hyperinsulinemic-euglycemic clamp experiment,after the blood glucose levels were stable,the GIR of high-fat-fed mice was significantly reduced(P<0.001),GDR was decreased(P<0.0001)and hepatic gluconeogenesis rate was increased(P<0.01).After pharmacological inhibition of glycogenolysis,mice showed elevated blood glucose level(P<0.001)and further reductions in p-Akt level in liver and muscle.The ^(13)C-labeled clamp experiment revealed

关 键 词：胰岛素抵抗 葡萄糖钳夹实验 颈静脉插管手术 D-葡萄糖-^(13)C6 

分 类 号：R33[医药卫生—人体生理学]