高糖条件下小鼠巨噬细胞对骨骼肌细胞成肌分化和胰岛素敏感性的影响*

目的: 探讨高糖环境下小鼠巨噬细胞对骨骼肌细胞成肌分化和胰岛素敏感性的影响。方法: Transwell小室内共培养小鼠骨骼肌成肌细胞C2C12细胞和单核巨噬细胞RAW264.7细胞并给予60 mmol/L葡萄糖处理,结合培养条件随机分为单独培养对照组(SC组,n=12)、共培养对照组(CC组,n=12)、单独培养高糖组(SH组,n=12)和共培养高糖组(CH组,n=12)。相差显微镜观察细胞形态,共培养1 d和3 d后收集C2C12细胞,CCK-8检测细胞活性,免疫荧光技术检测细胞融合率和基础与胰岛素刺激的GLUT4蛋白表达,实时定量PCR检测成肌调节因子Myf5、MyoD和myogenin基因表达,2-NBDG法检测细胞基础和胰岛素刺激的糖摄取。结果: 正常糖浓度下,与RAW264.7细胞共培养促进C2C12细胞肌管形成,促进E-MHC蛋白表达(P＜0.01),促进MyoD和myogenin基因表达(P＜0.05),提高胰岛素刺激的2-NBDG摄取(P＜0.05),提高基础GLUT4水平(P＜0.05)。高糖刺激抑制C2C12细胞肌管形成,抑制成肌调节因子基因表达,抑制2-NBDG摄取,抑制GLUT4表达(P＜0.05)。高糖环境下与RAW264.7细胞共培养时未见明显肌管形成,与共培养对照组和单独培养高糖组相比,细胞活性、E-MHC蛋白水平、成肌调节因子基因水平、2-NBDG摄取和GLUT4蛋白水平均明显下降(P＜0.05)。结论: 与RAW264.7共培养促进C2C12成肌分化并提高胰岛素敏感性, 高糖条件处理这一作用可逆转,抑制C2C12成肌分化的同时诱发C2C12细胞胰岛素抵抗。

Effects of mice macrophages on skeletal muscle cells under high glucose treatment

Objective: To study the effects of mice macrophages on myogenic differentiation and insulin sensitivity of skeletal muscle cells under high glucose condition. Methods: 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, n=12), co-culture control group (CC group, n=12), single culture high glucose group (SH group, n=12) and co-culture high glucose group (CH group, n=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. Results: Under normal glucose concentration, this co-culture with RAW264. 7 promoted C2C12 myotube formation, E-MHC protein expression (P＜0. 01), MyoD and myogenin gene expressions (P＜ 0. 05), insulin-stimulated 2-NBDG uptake (P＜0. 05), and basic GLUT4 level (P＜0. 05). High glucose stimulation inhibited myotube formation, myogenic regulatory factor gene expression, 2-NBDG uptake and GLUT4 expression in C2C12 (P＜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 (P＜0. 05). Conclusion: 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.