Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei 050051, P.R. China.
Hebei Key Laboratory of Metabolic Diseases, Hebei General Hospital, Shijiazhuang, Hebei 050051, P.R. China.
Mol Med Rep. 2023 Oct;28(4). doi: 10.3892/mmr.2023.13068. Epub 2023 Aug 18.
The present study aimed to establish a model of palmitic acid (PA)‑induced insulin resistance (IR) in C2C12 cells and to determine the mechanism underlying how resveratrol (RSV) improves IR. C2C12 cells were divided into the control (CON), PA, PA + RSV, PA + RSV + DNA damage‑inducible transcript 4 (DDIT4)‑small interfering (si)RNA and PA + RSV + MHY1485 (mTOR agonist) groups. Glucose contents in culture medium and triglyceride contents in cells were determined. Oil red O staining was performed to observe the pathological changes in the cells. Reverse transcription‑quantitative PCR and western blotting were conducted to evaluate the mRNA and protein expression levels, respectively, of DDIT4, mTOR, p70 ribosomal protein S6 kinase (p70S6K), insulin receptor substrate (IRS)‑1, PI3K, AKT and glucose transporter 4 (GLUT4). Compared with in the CON group, glucose uptake was decreased, cellular lipid deposition was increased, phosphorylated (p)‑IRS‑1, p‑mTOR and p‑p70S6K protein expression levels were increased, and p‑PI3K, p‑AKT, GLUT4 and DDIT4 protein expression levels were decreased in the PA group. By contrast, compared with in the PA group, culture medium glucose content and cellular lipid deposition were decreased, p‑PI3K, p‑AKT, GLUT4 and DDIT4 protein expression levels were increased, p‑IRS‑1 protein expression levels were decreased, and mTOR and p70S6K mRNA and protein expression levels were decreased in the PA + RSV group. Compared with in the PA + RSV group, DDIT4 protein and mRNA expression levels were reduced in the PA + RSV + DDIT4‑siRNA group, but showed no change in the PA + RSV + MHY1485 group. Following transfection with DDIT4‑siRNA or treatment with MHY1485, the effects of RSV on improving IR and lipid metabolism were weakened, mTOR and p70S6K protein expression levels were upregulated, p‑PI3K, p‑AKT and GLUT4 protein expression levels were down‑regulated, p‑IRS‑1 protein expression levels were upregulated, and culture medium glucose content and cellular lipid deposition were increased. In conclusion, RSV may improve PA‑induced IR in C2C12 cells through the DDIT4/mTOR/IRS‑1/PI3K/AKT/GLUT4 signaling pathway, as well as via improvements in glucose and lipid metabolism.
本研究旨在建立棕榈酸（PA）诱导的 C2C12 细胞胰岛素抵抗（IR）模型，并确定白藜芦醇（RSV）改善 IR 的机制。将 C2C12 细胞分为对照组（CON）、PA 组、PA+RSV 组、PA+RSV+DNA 损伤诱导转录物 4（DDIT4）-小干扰（si）RNA 组和 PA+RSV+MHY1485（mTOR 激动剂）组。测定细胞培养液中葡萄糖含量和细胞内三酰甘油含量。油红 O 染色观察细胞的病理变化。采用逆转录-定量 PCR 和 Western blot 法分别检测 DDIT4、mTOR、p70 核糖体蛋白 S6 激酶（p70S6K）、胰岛素受体底物（IRS）-1、PI3K、AKT 和葡萄糖转运蛋白 4（GLUT4）的 mRNA 和蛋白表达水平。与 CON 组比较，PA 组细胞葡萄糖摄取减少，细胞内脂质沉积增加，磷酸化（p）-IRS-1、p-mTOR 和 p-p70S6K 蛋白表达水平升高，p-PI3K、p-AKT、GLUT4 和 DDIT4 蛋白表达水平降低。与 PA 组比较，PA+RSV 组细胞培养液中葡萄糖含量和细胞内脂质沉积减少，p-PI3K、p-AKT、GLUT4 和 DDIT4 蛋白表达水平升高，p-IRS-1 蛋白表达水平降低，mTOR 和 p70S6K mRNA 和蛋白表达水平降低。与 PA+RSV 组比较，PA+RSV+DDIT4-siRNA 组 DDIT4 蛋白和 mRNA 表达水平降低，而 PA+RSV+MHY1485 组无变化。转染 DDIT4-siRNA 或给予 MHY1485 后，RSV 改善 IR 和脂代谢的作用减弱，mTOR 和 p70S6K 蛋白表达水平上调，p-PI3K、p-AKT 和 GLUT4 蛋白表达水平下调，p-IRS-1 蛋白表达水平上调，细胞培养液中葡萄糖含量和细胞内脂质沉积增加。结论：RSV 可能通过 DDIT4/mTOR/IRS-1/PI3K/AKT/GLUT4 信号通路改善 PA 诱导的 C2C12 细胞 IR，并改善葡萄糖和脂代谢。
