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

Abstract

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.