肌源性IL-6对骨骼肌细胞胰岛素抵抗的影响及其机制*

doi:10.12047/j.cjap.6310.2022.099

摘要/Abstract

摘要： 目的: 研究外源性钙负荷促进离体细胞肌源性IL-6释放,调节AMPK、p38MAPK等信号通路以改善胰岛素抵抗的效应。方法: 以正常培养的C2C12细胞系和棕榈酸诱导形成胰岛素抵抗C2C12细胞系为实验对象。预实验通过不同浓度钙培养肌细胞24 h后,检测培养液葡萄糖浓度并在显微镜下观察其收缩的情况。正式试验1将细胞分为4组:A组为Control组(正常培养液培养),B组为IR组(0.6 mmol/L棕榈酸哺育细胞24 h后备用),C组为1 000 ng/ml IL-6哺育IR细胞48 h组(IL-6+IR组),D组为IL-6shRNA哺育正常细胞48 h组(IL-6shRNA组)。正式试验2将细胞分为3组:A组为IR组,B组为100 μmol/L CaCl₂哺育IR细胞48 h组(钙哺育组,CaCl₂+IR组),C组为100 μmol/L CaCl2和IL-6shRNA共哺育IR细胞48 h组(共哺育组,CaCl₂+IL-6shRNA+IR组),采用Real-time PCR方法检测IL-6 mRNA 、GLUT mRNA表达水平,采用Western blot方法检测AMPK、p38MAPK、IRS-1和PI-3K蛋白表达水平。结果: 预实验结果表明,与不加CaCl₂组比较,加入不同浓度CaCl₂24 h后,培养液上清葡萄糖浓度均显著降低(P＜0.05或P＜0.01),显微镜下观察可见肌细胞有明显收缩,且以100 μmol/L CaCl₂最为明显。正式实验1结果表明,与IR组比较,IL-6+IR组p-AMPK、p-IRS-1、p-PI-3K蛋白表达水平、GLUT4mRNA水平、葡萄糖摄取能力均显著升高(P＜0.05或P＜0.01),p38MAPK蛋白表达水平显著降低(P＜0.01);与Control组比较,IL-6shRNA组p-AMPK、p-IRS-1、p-PI-3K蛋白表达水平、IL-6、GLUT4mRNA表达水平均显著降低(P＜0.05或P＜ 0.01),p38MAPK蛋白表达水平均显著升高(P＜0.01)。正式试验2结果表明,与IR组比较,CaCl₂+IR组p-AMPK、p-IRS-1、p-PI-3K蛋白表达水平和GLUT4mRNA表达水平均显著升高(P＜0.05或P＜0.01),p38MAPK蛋白表达水平显著降低(P＜0.01);与CaCl₂+IR组比较,CaCl₂+IL-6shRNA+IR组p-AMPK、p-IRS-1、p-PI-3K蛋白表达水平和GLUT4mRNA表达水平均显著降低(P＜0.05或P＜0.01),p38MAPK蛋白表达水平显著升高(P＜0.01)。结论: 外源性钙负荷可以引发肌细胞收缩,并且肌源性IL-6通过激活AMPK、PI-3K等信号通路与抑制p38MAPK信号通路而改善骨骼肌细胞胰岛素抵抗。

关键词: 肌源性白细胞介素6, 钙负荷, C2C12 细胞系, 胰岛素抵抗

Abstract: Objective: To study the effects of exogenous calcium-load on promoting muscle-derived IL-6 secretion, and regulating AMPK and p38MAPK signal pathway to improve insulin resistance. Methods: C2C12 cell lines and palmitic acid-induced insulin resistance C2C12 cell lines were selected as the experimental objects. Preliminary experiment was aimed to determinate the glucose concentrations of culture solutions and observe contraction status of cells under microscope following different calcium concentrations culture 24 h. In the first official experiment, cells were divided into four groups: control group (A group, normal culture solution), IR group(B group, 0.6 mmol/L palmitic acid culture cells 24 h), 1 000 ng/ml IL-6 culture IR B group cells 48 h(IL-6+IR group) and IL-6 shRNA culture A group cells (IL-6shRNA group). In the second official experiment, cells were divided into three groups: IR group(A group), 100 μmol/L CaCl₂ culture IR group cells 48 h(CaCl₂+IR group) and 100 μmol/L CaCl₂ and IL-6shRNA co- culture IR group cells 48 h(CaCl₂+IL-6shRNA+IR group). The expression levels of GLUT4 mRNA and IL-6 mRNA were measured by real-time PCR, the protein expression levels of p-AMPK, p-p38MAPK, p-IRS-1 and p-PI-3K were measured by Western blot. Results: Preliminary experiment results showed that compared with 0 μmol/L CaCl₂ group, the glucose concentrations were decreased significantly after cells treated with CaCl₂, at different concentrations. The cell contractions were observed under microscope and the cell contraction was most obvious treated with 100 μmol/L CaCl₂. The first official experiment results showed that compared with IR group, the contents of p-AMP-activated protein kinase(p-AMPK), p-insulin receptor substrate 1(p-IRS-1), p-phosphoinositide-3 kinase(p-PI-3K), the expression level of glucose transporter 4(GLUT4) mRNA and the glucose uptake of IL-6+IR group were increased significantly(P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was decreased significantly (P＜0.01) ; Compared with control group, the expression levels of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of IL-6shRNA group were decreased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was increased significantly (P＜0.01). The second official experiment results showed that compared with IR group, the expression levels of p-AMPK, P-IRS-1, p-PI-3K, the level of GLUT4 mRNA of CaCl₂+IR group were increased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was decreased significantly (P＜0.01); Compared with CaCl₂+IR group, the contents of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of CaCl₂+IL-6 shRNA+IR group were decreased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was increased significantly (P＜0.01). Conclusion: Exogenous Ca-load can stimulate muscle cells contraction, and exercise-induced IL-6 improves insulin resistance by activating AMPK, PI-3Kand inhibiting p38MAPK signal pathway.

Key words: muscle-derived IL-6, Calcuim-load, C2C12 cell lines, insulin resistant

中图分类号:

R587.1

唐晖, 赵一平, 黄丹, 蔡建光, 汪毅. 肌源性IL-6对骨骼肌细胞胰岛素抵抗的影响及其机制^*[J]. 中国应用生理学杂志, 2022, 38(5): 530-536.

TANG Hui, ZHAO Yi-ping, HUANG Dan, CAI Jian-guang, WANG Yi. Effects of muscle derived-IL-6 on insulin resistance of skeletal muscle cells and its mechanisms[J]. CJAP, 2022, 38(5): 530-536.

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肌源性IL-6对骨骼肌细胞胰岛素抵抗的影响及其机制^*

Effects of muscle derived-IL-6 on insulin resistance of skeletal muscle cells and its mechanisms

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