Interactions between myoblasts and macrophages under high glucose milieus result in inflammatory response and impaired insulin sensitivity

Figure 1 High-fat diet fed for 12 wk induces insulin resistance in mice. A: Body weight development of control group (Con group) and insulin resistance (IR) group mice during 12 wk fed; B: Fasting blood glucose of Con and IR group mice during 12 wk fed; C: Fasting insulin levels of Con and IR group mice fed for 12 wk; D: Homeostasis model assessment of IR of Con and IR group mice fed for 12 wk; E: Triglyceride, total cholesterol, high density lipoprotein-cholesterol, and low density lipoprotein-cholesterol concentration in serum in Con and IR group mice fed for 12 wk; F: Blood glucose values during intraperitoneal glucose tolerance tests of Con and IR group mice fed for 12 wk; G: Blood glucose values during insulin tolerance tests of Con and IR group mice fed for 12 wk. Data are means ± SD, n = 6 per group. ^aP < 0.01 vs Con group. P values were calculated by two-tailed Student’s t-test. Con: Control group; IR: Insulin resistance group; ITT: Insulin tolerance test; GTT: Glucose tolerance tests.

Figure 2 Insulin resistance induced myofiber atrophy and pro-inflammatory M1 phenotype macrophages infiltration in skeletal muscle. A: Hematoxylin eosin (H&E) staining of skeletal muscle and the areas of Myofiber (Scale bar: 50 μm); B: The expression of F4/80, and the co-localization of F4/80 with CD86 and CD206 respectively in skeletal muscle detected by Immunofluorescence (Scale bar: 50 μm). Data are means ± SD, n = 6 per group. ^aP < 0.01 vs Con group. P values were calculated by two-tailed Student’s t-test. Con: Control group; IR: Insulin resistance group.

Figure 3 Myoblast under high glucose milieus resulted in macrophages polarizing to pro-inflammatory M1 phenotype. A: The viability of macrophages by CCK-8 assay; B: Representative image of macrophages by Light Microscopy, inflammatory phenotype in morphology were indicated with orange arrows (Scale bar: 50 μm); C: The ratios of F4/80⁺ cells, F4/80⁺CD86⁺ cells, and F4/80⁺CD206⁺ cells in macrophages by Flow Cytometry. Data are means ± SD, n = 6 per group. ^aP < 0.01 main effect of high glucose; ^bP < 0.01 main effect of co-cultured with myoblasts. P values were calculated by two-way analysis of variances. Mc: Macrophages; Mc + HG: Macrophages cultured in high glucose medium; McM: Macrophages co-cultured with myoblasts; McM + HG: Macrophages co-cultured with myoblasts in high glucose medium; HG: High glucose.

Figure 4 Interactions between myoblasts and macrophages under high glucose milieus resulted in chronic inflammation. The protein levels of monocyte chemoattractant protein 1, tumor necrosis factor-α, inerleukin-1β (IL-1β), IL-6 and IL-10 in culture medium by enzyme-linked immunosorbent assay. Data are means ± SD, n = 6 per group. ^aP < 0.01 main effect of high glucose; ^bP < 0.05, ^cP < 0.01 main effect of co-cultured with myoblasts. P values were calculated by two-way analysis of variances. Mc: Macrophages; Mc + HG: Macrophages cultured in high glucose medium; McM: Macrophages co-cultured with myoblasts; McM + HG: Macrophages co-cultured with myoblasts in high glucose medium; HG: High glucose.

Figure 5 Inflammatory reaction induced by M1 macrophages inhibited myogenic differentiation and led to myotube atrophy. A: Representative image of myotube by light microscopy, myotube were indicated with orange arrows (scale bar: 100 μm); B: The viability of myoblasts by CCK-8 assay; C: The ratios of E-MHC positive area in myotube by Immunofluorescence (scale bar: 50 μm). Data are means ± SD, n = 6 per group. ^aP < 0.01 main effect of high glucose; ^bP < 0.05, ^cP < 0.01 main effect of co-cultured with macrophages. P values were calculated by two-way analysis of variances. M: Myoblasts; M + HG: Myoblasts cultured in high glucose medium; MMc: Myoblasts co-cultured with macrophages; MMc + HG: Myoblasts co-cultured with macrophages in high glucose medium; HG: High glucose.

Figure 6 Inflammatory reaction induced by M1 macrophages impaired myotube insulin sensitivity. A: The GLUT4 fluorescence of myotube in the absence or presence of 100 nmo/L insulin by Immunofluorescence (scale bar: 50 μm); B: The 2-Deoxyglucose transport of myotube in the absence or presence of 100 nmo/L insulin by glucose uptake assay. Data are means ± SD, n = 6 per group. ^aP < 0.05, ^bP < 0.01 main effect of insulin; ^cP < 0.01 main effect of high glucose; ^dP < 0.01 main effect of co-cultured with macrophages. P values were calculated by two-way analysis of variances. M: Myoblasts; M + HG: Myoblasts cultured in high glucose medium; MMc: Myoblasts co-cultured with macrophages; MMc + HG: Myoblasts co-cultured with macrophages in high glucose medium; HG: High glucose.