Luo Wei, Ai Lei, Wang Bo-Fa, Wang Li-Ying, Gan Yan-Ming, Zhou Yue
Department of Sports and Health Sciences, Nanjing Sport Institute, Nanjing 210014.
Department of Exercise Physiology, Beijing Sport University, Beijing 100084.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2020 Mar;36(2):124-129. doi: 10.12047/j.cjap.5901.2020.028.
To study the effects of mice macrophages on myogenic differentiation and insulin sensitivity of skeletal muscle cells under high glucose condition. C2C12 myoblasts and RAW264. 7 macrophages were co-cultured in transwell and treated with 60 mmol/L glucose. They were randomly divided into single culture control group (SC group, =12), co-culture control group (CC group, =12), single culture high glucose group (SH group, =12) and co-culture high glucose group (CH group, =12). Cell morphology was observed by phase contrast microscope. C2C12 were collected after 1 and 3 days of co-culture. Cell viability was measured by CCK-8. Embryonic myosin heavy chain (E-MHC) and glucose transporters 4 (GLUT4) protein expressions were detected by immunofluorescence. The expressions of myogenic factor 5 (Myf5), myogenic determination gene (MyoD) and myogenin gene were detected by real-time PCR. 2-(N-(7-nitrobenz-2-oxa-13-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) assay was used to detect the cellular basis and insulin-stimulated glucose uptake. Under normal glucose concentration, this co-culture with RAW264. 7 promoted C2C12 myotube formation, E-MHC protein expression (＜0. 01), MyoD and myogenin gene expressions (＜ 0. 05), insulin-stimulated 2-NBDG uptake (＜0. 05), and basic GLUT4 level (＜0. 05). High glucose stimulation inhibited myotube formation, myogenic regulatory factor gene expression, 2-NBDG uptake and GLUT4 expression in C2C12 (＜0. 05). When co-cultured with C2C12 under high glucose treatment, compared with co-culture control group and high glucose group, cell activity, E-MHC protein expression, myogenic regulator gene expressions, 2-NBDG uptake and GLUT4 protein expression were significantly decreased (＜0. 05). Co-culture with RAW264. 7 promotes myogenic differentiation and increases insulin sensitivity in C2C12, but this effect is reversed under 60 mmol/L glucose treatment, which inhibits myogenic differentiation and induces insulin resistance.
研究高糖条件下小鼠巨噬细胞对骨骼肌细胞成肌分化及胰岛素敏感性的影响。将C2C12成肌细胞与RAW264.7巨噬细胞进行Transwell共培养，并用60 mmol/L葡萄糖处理。将其随机分为单培养对照组（SC组，n = 12）、共培养对照组（CC组，n = 12）、单培养高糖组（SH组，n = 12）和共培养高糖组（CH组，n = 12）。用相差显微镜观察细胞形态。共培养1天和3天后收集C2C12细胞。用CCK-8法检测细胞活力。通过免疫荧光检测胚胎肌球蛋白重链（E-MHC）和葡萄糖转运蛋白4（GLUT4）的蛋白表达。通过实时PCR检测生肌因子5（Myf5）、生肌决定基因（MyoD）和成肌素基因的表达。采用2-(N-(7-硝基苯并-2-恶唑-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖（2-NBDG）检测法检测细胞基础葡萄糖摄取及胰岛素刺激后的葡萄糖摄取。在正常葡萄糖浓度下，与RAW264.7共培养可促进C2C12肌管形成、E-MHC蛋白表达（P＜0.01）、MyoD和成肌素基因表达（P＜0.05）、胰岛素刺激的2-NBDG摄取（P＜0.05）以及基础GLUT4水平（P＜0.05）。高糖刺激抑制了C2C12中的肌管形成、生肌调节因子基因表达、2-NBDG摄取及GLUT4表达（P＜0.05）。在高糖处理下与C2C12共培养时，与共培养对照组和高糖组相比，细胞活性、E-MHC蛋白表达、生肌调节基因表达、2-NBDG摄取及GLUT4蛋白表达均显著降低（P＜0.05）。与RAW264.7共培养可促进C2C12的成肌分化并增加其胰岛素敏感性，但在60 mmol/L葡萄糖处理下这种作用会逆转，此时会抑制成肌分化并诱导胰岛素抵抗。
