Male C57BL/6H mice were purchased at 5–6 wk old from Charles River Laboratories (Beijing, China). They were free fed in chamber with comfortable room temperature between 22-25 °C with 12 h day-night cycle. After one week of adaptation, animals were randomly fed with one of two diets for 12 wk: Normal standard diet (Con group, 3.42 kcal/g, 12% of energy from fat) or high-fat diet (IR group, 5.24 kcal/g, 60% of energy from fat). The contents of mineral and vitamin in these two diets was identical. This study was approved by the Institutional Animal Care and Use Committee of Nanjing Sport Institute (Protocol No. GZRDW-2022-02).

Mice were fasted for 12 h and received i.p. injection of glucose (2 g/kg)[32,33]. The blood samples were collected from the tail vein and serum glucose level was determined at time 0, 30, 60, 90, and 120 min using glucometer (ACCU-CHEK Performa Roche, Germany). For insulin tolerance test (ITT), mice were fasted for 4 h and received i.p. injection of insulin (0.75 U/kg)[33-35]. The blood samples were collected and glucose concentrations were measured at time 0, 15, 30, 60, and 120 min using glucometer.

After 12 wk of intervention, animals from Con and IR group (n = 6) were deeply anesthetized after deprived from food for 12 h (sodium amytal, i.p., 150 mg/kg). The left and right gastrocnemius muscles were then removed for further biochemical and molecular analyses.

After a 30 min standing of mice blood, serum samples were obtained by 3500xg centrifugation for 15 min at 4 °C. Biochemical parameters, including triglycerides (TG), total cholesterol (TC), high density lipoprotein-cholesterol, and low density lipoprotein-cholesterol (LDL-C), were measured with kits from SEKISUI MEDICAL (Tokyo, Japan) on an automatic biochemical analyzer from Beckman (AU5800, CA, United States). Fasting insulin (FINS) concentration was measured by enzyme-linked immunosorbent assay (ELISA) by using a kit from Millipore (EZRMI-13K, MA, United States) according to the manufacturer's instructions. Homeostasis model assessment of IR (HOMA-IR) is determined using the equations: HOMA-IR = [FINS (mU/L) × FPG (mmol/L)]/22.5.

Muscle samples were collected and fixed in isopentane for cryopreservation at -80 °C. The muscle tissue was sliced to a thickness of 8 μm in frozen microtome by cross cutting and rip cutting. The sections were subjected to the hematoxylin and eosin (H&E) and immunohistochemical staining. For H&E staining, muscle sections of cross cutting and rip cutting were used and stained with hematoxylin for 10 min, then washed and stained with eosin for 5 min. Lastly, sections were observed under light microscope (Nikon, Japan). For immunohistochemical staining, muscle sections by cross cutting were incubated with antibody mixture of F4/80 (ab111101, Abcam, United States) and CD86 (ab213044, Abcam, United States), or F4/80 and CD206 (ab8918, Abcam, United States) at 4 °C overnight. Then slides were protected from light and incubated with secondary Alexa Fluor 488- or Alexa Flior 555-labeled goat anti-rabbit or mouse antibodies (A-11034, A-21422, Molecular Probes, United States) for 2 h at room temperature. Counterstaining was followed with DAPI for 15 min in an aluminum foil-covered box. Finally, the stained tissues were examined under a fluorescence microscope (Leica, Germany). Six mice for each group were used, and more than eight visual fields were chosen and analyzed for each muscle section. The images were analyzed with Image Pro Plus 6.0 program (Media Cybernetics, United States). The H&E staining of cross cutting sections were used to analyze myofiber area. F4/80 positive area were used to analyze macrophage infiltration. Co-localization of F4/80 with CD86 or CD206 were used to quantify macrophages M1 or M2 polarizations respectively.

RAW264.7 macrophages and C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; 25 mmol/L glucose; Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, United States) under humidified atmosphere with 5% CO₂ at 37 °C. The 6-well and 24-well transwell co-culture system (Corning, United States) were employed for non-contactable co-culture of RAW264.7 macrophages and C2C12 myoblasts. For co-culture, 2 × 10⁶ and 4 × 10⁵ RAW264.7 macrophages were plated on upper chamber of 6-well and 24-well transwell plates, respectively. Then, 6.6 × 10⁶ and 13.3 × 10⁵ C2C12 myoblasts were plated on lower chamber of 6-well and 24-well transwell plates, respectively. Subsequently, the macrophages were plated at about 30% of the numbers of myoblasts, comparable to the abundance of macrophages observed in skeletal muscle from IR mice. The RAW264.7 macrophages and C2C12 myoblasts were allowed to adhere for 24 h before the co-culture was started. All co-culture treatments were performed for 5 d under DMEM containing 2% horse serum (Gibco, United States) to induce myogenic differentiation of myoblasts. As a control for the co-culture, the same cells were plated to the upper and lower chamber of the transwell co-culture system.

To observe the influence of HG on the interactions between myoblasts and macrophages, cells were treated with 60 mmol/L D-glucose in differentiation medium for 5 d from the start of co-culture[17,36,37]. A 60 mmol/L L-glucose was used in control group (Con group) for the HG as an osmotic control for 60 mmol/L D-glucose[38]. To detect the insulin sensitivity of myotube, C2C12 cells were incubated in the absence or presence of insulin (100 nmol/L) for 30 min at 37 °C before gathered[39,40]. After co-culture, the cells from the upper and lower chamber of the transwell system were collected separately for further biochemical and molecular analyses. Medium from these co-cultures were gathered for the assay of cytokines.

Cell viability was assessed by CCK-8 assay (APExBIO, United States). After 5 d of treatments, 10 μL CCK-8 solution were added to RAW264.7 macrophages or C2C12 myoblasts and cultured for another 1 h. Then the absorbance was measured at a wavelength of 450 nm using Microplate Reader (Bio-Rad, Hercules, CA, United States), and the cell viability was calculated using the formula: Cell viability (%) = (A_{experimental group} - A_blank)/(A_{control group} - A_blank) × 100%. A indicates absorbance at the wavelength of 450 nm.

Cells were observed daily using light microscope to detect the effect of co-culture and HG on myoblasts morphology. After the intervention above, myotube area and number were calculated with Image Pro Plus 6.0 program (Media Cybernetics, United States). Six dishes for each group were used, and more than eight visual fields were randomly chosen and analyzed for each dish.

After the intervention above, RAW264.7 macrophages were dissociated with trypsin-EDTA into single cell suspension. They were washed and stained with anti-CD16/32 (553142, BD Biosciences, United States) for 20 min at room temperature to block Fc receptors before incubation with PE/Cy7-labelled anti-F4/80 (ab111101, Abcam, United States), FITC-labelled anti-CD86 (ab213044, Abcam, United States), and PE-labelled anti-CD206 (ab8918, Abcam, United States) monoclonal antibodies in a dark room for 1 h. Subsequently, the cells were examined with flow cytometry (Beckman, United States). The expressions of cell markers were compared with an isotype control. The acquired flow cytometric images were analyzed with FlowJo program (version 10.0.7). The ratio of F4/80+ cells were used to quantify activated macrophage; the ratio of F4/80+CD86+CD206- cells were used to quantify M1 macrophage; the ratio of F4/80+CD206+CD86- cells were used to quantify M2 macrophage.

The protein levels of monocyte chemoattractant protein 1 (MCP1, MJE00B, R&D, United States, sensitivity: 0.666 pg/mL, specificity: Mouse), tumor necrosis factor-α (TNFα, MTA00B, R&D, United States, sensitivity: 7.21 pg/mL, specificity: Mouse), Inerleukin-1β (IL-1β, MLB00C, R&D, United States, sensitivity: 4.8 pg/mL, specificity: Mouse), IL-6 (M6000B, R&D, United States, sensitivity: 1.8 pg/mL, specificity: Mouse), and IL-10 (M1000B, R&D, United States, sensitivity: 5.22 pg/mL, specificity: Mouse) secreted into medium was quantified with ELISA kit according to the manufacturer’s instructions.

After the intervention above, myoblasts were fixed in 4% paraformaldehyde for 15 min, and subjected to immunofluorescence staining. Myoblasts were incubated with antibody mixture of E-MHC (BF-G6, Hybridoma Bank, United States) or GLUT4 (ab654, Abcam, United States), at 4 °C overnight. Then, myoblasts were incubated with Alexa Fluor 488- or Alexa Flior 555-labeled goat anti-rabbit or mouse antibodies (A-11034, A-21422, Molecular Probes, United States) for 2 h at room temperature. Finally, the stained cells were observed with fluorescence microscope (Leica, Germany). Six dishes for each group were used, and more than eight visual fields were randomly chosen and analyzed for each dish. The positive area of E-MHC and GLUT4 fluorescence were analyzed with Image-Pro plus 6.0 (Media Cybernetics, United States).

After the intervention above, myotubes were starved in DMEM containing 2% fatty acid-free bovine serum albumin for 3 h, then incubated in the absence or presence of insulin (100 nM) for 30 min. Subsequently, they were incubated with 2-NBDG (Cayman, United States) (150 μg/mL) for 40 min. Fluorescence was measured using microplate reader (Bio-Rad, Hercules, CA, United States) at an excitation wavelength of 485 nm and emission wavelength of 535 nm. The negative control and blank control were set, and then standard curve generated to calculate the glucose uptake of myotube in the absence or presence of insulin, which is an indicator to evaluate the insulin sensitivity of myotube.

The data are representative of three separate experiments performed with 6 replicates. The data are reported as mean ± SD. The Shapiro-Wilk test was used to test for normality within each data set. According to the Shapiro-Wilk test, the data in each group followed a normal distribution. The two-tailed Student’s t-test was used for comparison between two groups. The multi-group comparisons were performed with two-way ANOVA test followed by Bonferroni post hoc test to analyze the interaction between co-culture and HG. SPSS statistical software (version 20.0) was used for the statistical analyses, and the P < 0.05 was considered as statistically significant. Hedges’ g effect sizes were calculated, and effect sizes were interpreted as: g < 0.2 very small, g = 0.2-0.5 small, g = 0.5-0.8 medium, g > 0.8 large.

Throughout the 12 wk of high-fat diet, the levels of body weight and fasting blood glucose in mice of IR group were significantly higher than Con group (Figure 1A and B; P < 0.01, g > 0.8). The levels of FINS, HOMA-IR, TG, TC, LDL-C in mice of IR group were also significantly higher than Con group (Figure 1C-E; P < 0.01, g > 0.8). To separate the response to glucose or insulin challenge from glucose homeostasis, we assessed the area under the curve of both glucose tolerance test and ITT. Glucose tolerance and insulin tolerance was impaired significantly in IR group compared with Con group (Figure 1F and G, P < 0.01, g > 0.8).

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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.

To detect whether IR is relevant with macrophage infiltration and M1 phenotype polarization in skeletal muscle, H&E and immunohistochemistry staining was carried on skeletal muscle. As demonstrated by H&E, skeletal muscle from IR mice displayed more inflammatory infiltration (Figure 2A, indicated with black arrows). Furthermore, by immunohistochemical staining, the positive area of F4/80 (a surface marker of macrophages) and co-localization of F4/80 and CD86 (a surface marker of M1 phenotype macrophages) was significantly increased in skeletal muscle from IR mice (Figure 2B; P < 0.01, g > 0.8). In addition, to detect the effect of IR on skeletal muscle morphology, we analyzed the cross-sectional area of myofiber. As shown in Figure 2A, myofiber size in IR group was significantly decreased than that in Con group (P < 0.01, g = 6.26).

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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.

To understand the effect of myoblasts on macrophages polarization under HG surroundings, we researched the state of macrophages polarization in the absence or presence of myoblasts and HG. Firstly, cytotoxicity of myoblasts and HG to macrophages were tested by CCK-8 assay. As shown in Figure 3A, treatment of myoblasts and HG had no cytotoxicity on macrophages (P > 0.05, g < 0.2). By comparing the morphology of macrophages after treated without or with HG for 5 d, spindle cells and pseudopodia were observed in cells treated alone with HG (Mc + HG group) (Figure 3B, indicated with red arrows), which indicated inflammatory phenotype of macrophages in morphology. We next observed whether myoblasts also cause morphological changes of macrophages. To address this, macrophages were co-cultured with myoblasts in the presence or absence of HG. The morphology was unchanged in macrophages co-cultured with myoblasts under physiological environment (McM group), however, even more spindle cells and pseudopodia were observed in cells co-cultured with myoblasts under HG environment (McM + HG group) (Figure 3B, indicated with red arrows). Further, we examined surface markers of macrophages polarization by Flow Cytometry and expressions of related proteins in macrophages by Western Blot. Similarly, compared to Con group (Mc group), F4/80+CD86+CD206- cells were decreased and F4/80+CD206+CD86- cells were increased in McM group (P < 0.01, g > 0.8), but F4/80+CD86+CD206- cells increased and F4/80+CD206+CD86- cells were decreased in Mc + HG group (P < 0.01, g > 0.8; Figure 3C). In McM + HG group, F4/80+CD86+CD206- cells were significantly increased, and F4/80+CD206+CD86- cells were significantly decreased, compared both with Mc + HG group and McM group (P < 0.01, g > 0.8; Figure 3C).

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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.

To verify whether the increased pro-inflammatory M1 phenotype in macrophages is complemented by increasing the secretion of cytokines, to result in chronic inflammation in muscle, we tested the protein levels of MCP1, TNFα, IL-1β, IL-6 and IL-10 in culture medium treated as above. As shown in Figure 4, compared to Mc group, levels of pro-inflammatory TNFα, IL-1β and IL-6 were decreased, and level of anti-inflammatory IL-10 was increased in McM group (P < 0.01, g > 0.8); but, levels of MCP1, TNFα, IL-1β and IL-6 were increased, and level of IL-10 was decreased in Mc + HG group (P < 0.01, g > 0.8). In McM + HG group, levels of MCP1, TNFα, IL-1β and IL-6 were significantly increased, and the expressions of IL-10 was significantly decreased, compared with both Mc + HG group and McM group (P < 0.05, g > 0.8). In brief, results showed that the state of macrophages polarization described above were also reflected in secretions of cytokines in the culture medium, which resulted in chronic inflammation environment for myoblasts.

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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.

To explore the mediation of HG-related M1 phenotype macrophages to myogenesis, we researched the differentiation state of myoblast in the absence or presence of macrophages and HG. Firstly, we observed the morphology of myoblasts induced for differentiation in the presence or absence of HG and macrophages for 5 d. As shown in Figure 5A, myoblasts in Con group (M group) differentiated into multinucleated myotube after 5 d of differentiation (indicated with red arrows), and more myotube were observed when myoblasts co-cultured with macrophages under physiological environment (MMc group, P < 0.05, g > 0.8); Whereas, area of myotube in myoblasts treated alone with HG (M + HG group) decreased compered to M group (P < 0.01, g = 3.37). Both number and area of myotube in myoblasts co-cultured with macrophages under HG environment (MMc + HG group) significantly decreased when compared with M + HG group and MMc group (P < 0.01, g > 0.8), which indicated inhibition effects of HG-related M1 phenotype macrophages on myogenesis in morphology. Then, treatments of macrophages and HG had no cytotoxicity on myoblasts by CCK-8 assay (P > 0.05, g < 0.2; Figure 5B). Further, we examined expression of E-MHC (marker of myotube formation) in myotube. Compared to M group, E-MHC positive area was increased in MMC group (P < 0.01, g = 2.44); but E-MHC positive area was decreased in M + HG group (P < 0.01, g = 5.35). In MMc + HG group, E-MHC positive area was significantly decreased compared both with M + HG group and MMc group (P < 0.01, g = 4.19; Figure 5C).

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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.

To further probe the mediation of HG-related M1 phenotype macrophages to IR in myotube, we studied the insulin action to stimulate glucose uptake and expressions of related proteins of myoblast in the absence or presence of macrophages and HG. As shown in Figure 6, insulin-stimulated GLUT4 fluorescence (Figure 6A) and glucose uptake (Figure 6B) were increased significantly in the M and MMc groups (P < 0.01, g > 0.8), which displayed fair insulin sensitivity of control myotube. Whereas, these insulin action in myotube were blocked by the exposure of HG (M + HG group and MMc + HG group) (P > 0.05, g < 0.2). Compared to M + HG group, both basal and the insulin-stimulated GLUT4 fluorescence and glucose uptake were decreased significantly in MMc + HG group (P < 0.01, g > 0.8).

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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.