Tang Hui, Zhao Yi-Ping, Huang Dan, Cai Jian-Guang, Wang Yi
Department of P.E, Hunan University of Science and Technology, Xiangtan 411201.
Department of P.E, Renmin University of China, Beijing 100872, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2022 Sep;38(5):530-536. doi: 10.12047/j.cjap.6310.2022.099.
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.
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 CaCl culture IR group cells 48 h(CaCl+IR group) and 100 μmol/L CaCl and IL-6shRNA co- culture IR group cells 48 h(CaCl+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.
Preliminary experiment results showed that compared with 0 μmol/L CaCl group, the glucose concentrations were decreased significantly after cells treated with CaCl, at different concentrations. The cell contractions were observed under microscope and the cell contraction was most obvious treated with 100 μmol/L CaCl. 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(＜0.05 or ＜0.01), the p-p38MAPK protein expression level was decreased significantly (＜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 (＜0.05 or ＜0.01), the p-p38MAPK protein expression level was increased significantly (＜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 CaCl+IR group were increased significantly (＜0.05 or ＜0.01), the p-p38MAPK protein expression level was decreased significantly (＜0.01); Compared with CaCl+IR group, the contents of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of CaCl+IL-6 shRNA+IR group were decreased significantly (＜0.05 or ＜0.01), the p-p38MAPK protein expression level was increased significantly (＜0.01).
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.
研究外源性钙负荷对促进肌肉来源的白细胞介素-6（IL-6）分泌、调节腺苷酸活化蛋白激酶（AMPK）和p38丝裂原活化蛋白激酶（p38MAPK）信号通路以改善胰岛素抵抗的影响。
选取C2C12细胞系和棕榈酸诱导的胰岛素抵抗C2C12细胞系作为实验对象。预实验旨在测定不同钙浓度培养24 h后培养液中的葡萄糖浓度，并在显微镜下观察细胞的收缩状态。在第一次正式实验中，细胞分为四组：对照组（A组，正常培养液）、胰岛素抵抗组（B组，用0.6 mmol/L棕榈酸培养细胞24 h）、用1 000 ng/ml IL-6培养胰岛素抵抗B组细胞48 h（IL-6+胰岛素抵抗组）和用IL-6短发夹RNA（shRNA）培养A组细胞（IL-6shRNA组）。在第二次正式实验中，细胞分为三组：胰岛素抵抗组（A组）、用100 μmol/L氯化钙培养胰岛素抵抗组细胞48 h（氯化钙+胰岛素抵抗组）和用100 μmol/L氯化钙与IL-6shRNA共同培养胰岛素抵抗组细胞48 h（氯化钙+IL-6shRNA+胰岛素抵抗组）。采用实时荧光定量聚合酶链反应（PCR）检测葡萄糖转运蛋白4（GLUT4）mRNA和IL-6 mRNA的表达水平，采用蛋白质免疫印迹法检测磷酸化AMPK（p-AMPK）、磷酸化p38MAPK（p-p38MAPK）、磷酸化胰岛素受体底物1（p-IRS-1）和磷酸化磷脂酰肌醇-3激酶（p-PI-3K）的蛋白表达水平。
预实验结果显示，与0 μmol/L氯化钙组相比，不同浓度氯化钙处理细胞后培养液中的葡萄糖浓度显著降低。在显微镜下观察细胞收缩情况，100 μmol/L氯化钙处理组细胞收缩最明显。第一次正式实验结果显示，与胰岛素抵抗组相比，IL-6+胰岛素抵抗组的p-AMPK、p-IRS-1、p-PI-3K含量、GLUT4 mRNA表达水平及葡萄糖摄取量均显著增加（P＜0.05或P＜0.01），p-p38MAPK蛋白表达水平显著降低（P＜0.01）；与对照组相比，IL-6shRNA组的p-AMPK、p-IRS-1、p-PI-3K表达水平、GLUT4 mRNA表达水平及葡萄糖摄取量均显著降低（P＜0.05或P＜0.01），p-p38MAPK蛋白表达水平显著增加（P＜0.01）。第二次正式实验结果显示，与胰岛素抵抗组相比，氯化钙+胰岛素抵抗组的p-AMPK、p-IRS-1、p-PI-3K表达水平、GLUT4 mRNA水平均显著增加（P＜0.05或P＜0.01），p-p38MAPK蛋白表达水平显著降低（P＜0.01）；与氯化钙+胰岛素抵抗组相比，氯化钙+IL-6 shRNA+胰岛素抵抗组的p-AMPK、p-IRS-1、p-PI-3K含量、GLUT4 mRNA表达水平及葡萄糖摄取量均显著降低（P＜0.05或P＜0.01），p-p38MAPK蛋白表达水平显著增加（P＜0.01）。
外源性钙负荷可刺激肌肉细胞收缩，运动诱导的IL-6通过激活AMPK、PI-3K并抑制p38MAPK信号通路改善胰岛素抵抗。
