Human Adipocytes Induce Inflammation and Atrophy in Muscle Cells During Obesity

Gene expression profile of myocytes cocultured with human adipocytes. Muscle cells were differentiated in the 3D hydrogel for 3 days, and then VAT adipocytes in the 3D hydrogel were added for an additional period of 1 day. Cells were then collected for RNA extraction obtained after reverse transcription cDNAs. A: cDNAs were analyzed using a human myogenesis and myopathy PCR array. The graph represents the percentage of decreased gene expression in myocytes cocultured with adipocytes (MYO+AD) compared with myocytes alone (control, MYO). Data are presented as the mean ± SEM of five independent experiments. **P < 0.01, ***P < 0.001, MYO+AD vs. MYO. B and C: Cells were also fixed and stained using antibody directed against titin (green, Alexa Fluor 488–conjugated anti-mouse IgG) and phalloidin (red, Alexa Fluor 546 phalloidin). A representative photomicrograph from confocal microscopy is presented. Scale bars: C, 20 µm; D, 10 µm. D–F: Muscle cells were differentiated for 3 days, and then 3D adipocytes from SAT or VAT from paired biopsy samples of obese subjects were added for an additional period of 3 days. Cells were fixed and stained using antibody directed against desmin, troponin, or titin (green, Alexa Fluor 488–conjugated anti-mouse IgG). Graphs represent quantifications of myotube thickness (D), troponin staining (E), and titin staining (F). Data are the mean ± SEM of six to eight independent experiments. *P < 0.05, MYO+VAT AD vs. MYO. CAV-3, caveolin-3; MuSK, muscle-specific kinase; NEB, nebulin; SGCA, α-sarcoglycan; TTN, titin.

Inflammatory profile of cocultured human adipocytes and myocytes: IL-6 and IL-1β as key factors. Muscle cells were differentiated in the 3D hydrogel for 3 days, and then SAT or VAT adipocytes from obese subjects, cultured in the 3D hydrogel, were added for an additional period of 3 days. Media were then collected for the measurement of cytokine and chemokine secretion by a multiplex assay. A: Significant changes in the secretory profile in the 3D cultures of muscle cells and obese VAT adipocytes (AD+MYO) compared with AD as control and MYO are presented in the graph. Data are expressed as fold variations between AD (white bars), AD+MYO (black bars) and MYO (gray bars) to take into account human interindividual variations. Data are presented as the mean ± SEM of seven independent experiments. *P < 0.05, **P < 0.01 AD+MYO vs. AD. B: Significant changes in the secretory profile in the 3D cultures of muscle cells and obese SAT (MYO+SAT AD) or obese VAT adipocytes (MYO+VAT AD) are presented in the graph. Data are expressed as fold variations between MYO (control) and MYO+SAT AD (white bars) or MYO+VAT AD (black bars). Data are presented as the mean ± SEM of eight independent experiments. C: Gene expression of IL-6, IL-8, and IL-1β in muscle cells cultured alone (control, MYO, white bars) or exposed to adipocytes from paired biopsy samples of obese SAT (MYO+SAT AD, gray bars) or VAT adipocytes (MYO+VAT AD, black bars), estimated by real-time PCR. Data (fold over control, MYO) are presented as the mean ± SEM of six independent experiments performed with different adipocyte preparations. *P < 0.05, **P < 0.01 MYO+VAT AD vs. MYO. #P < 0.05, MYO+SAT AD vs. MYO+VAT AD. D: 3D VAT adipocytes were added for an additional period of 3 days and were treated with neutralizing antibodies of IL-6 (2.5 µg/mL) and IL-1β (0.5 µg/mL) (MYO+AD+abIL-6/IL-1β) IgG1 (control MYO+AD IgG) in the 3D setting. Media were then collected for multiplex assay. Black bars represent the percentage decrease in secretions in MYO+AD+abIL-6/IL-1β cocultures compared with control MYO+AD IgG cocultures. Data are presented as the mean ± SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, MYO+AD IgG vs. MYO+AD+abIL-6/IL-1β. E: Multiplex assay of the inflammatory secretome of myocytes treated (MYO IL-6/IL-1β, black bars) or not (MYO, white bars) with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) over 3 days. Data are presented as the mean ± SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 MYO IL-6/IL-1β vs. MYO. ab, antibodies; FRK, fractalkine; IP-10, interferon-γ–induced protein-10; MIP1α, macrophage inflammatory protein 1α; ns, nonsignificant; VEGF, vascular endothelial growth factor.

IGF-II and IGFBP-5 rescue the atrophy of myotubes induced by adipocytes. A–D: Muscle cells were differentiated in the 3D hydrogel for 3 days without (MYO) or with 3D VAT adipocytes (from obese subjects) (MYO+AD) added for an additional period of 3 days in the presence or absence of IGF-II (50 ng/mL) and IGFBP-5 (200 ng/mL) (MYO+AD+IGF-II/IGFBP-5). Cells were fixed and stained with the corresponding antibodies. A: Myotube thickness was quantified using ImageJ software measuring the intensity of the desmin staining in 10 random fields (×20) in six independent cultures. Quantification of immunofluorescence was performed using ImageJ software measuring intensity of the MF20 (B) and titin (C) staining in five random fields (×20). D: Immunostaining of titin (green, Alexa Fluor 488–conjugated anti-mouse IgG) and nuclei (blue, DAPI). A representative photomicrograph of titin staining is presented (scale bar = 50 µm). Data are presented as the mean ± SEM of six independent experiments. **P < 0.01, MYO+AD vs. MYO or MYO+AD+IGF-II/IGFBP-5. E and F: Differentiated muscle cells exposed or not to obese VAT adipocytes were stimulated with IGF-II (50 ng/mL) and IGFBP-5 (200 ng/mL) for 10 min at 37°C. Serine 473 phosphorylation of Akt (pS 473 Akt, 56 kDa), serine 65 of 4E-BP1 (pS65 4E-BP1, 21 kDa), and serine 240 and 244 phosphorylation of S6 ribosomal protein (pS 240/244 S6 ribosomal protein, 32 kDa) were detected using the corresponding antibodies. E: A representative Western blot is presented among the five different experiments. F: The graph represents quantifications of the immunoblots in IGF-II/IGFBP-5–stimulated conditions normalized to total proteins. Data are presented as the mean ± SEM of five to seven independent experiments. *P < 0.05, **P < 0.01, MYO+AD vs. MYO. #P < 0.05, ##P < 0.01, without vs. with IGF-II/IGFBP-5 treatment.

Skeletal muscle characteristics of HFD-induced obese mice. Serum cytokine level (A) and gastrocnemius gene expression (B) evaluated in 20-week-old mice after 12 weeks of being fed a standard diet (chow, white bars) or an HFD (black bars). n = 8 mice per experimental group. C–E: Histological analysis of the gastrocnemius muscle obtained from 24-week-old mice (n = 9 mice per experimental group) after 16 weeks of being fed a standard diet (chow) or an HFD. C: Representative microphotographs of a cross-section (right panel) and a longitudinal section (left panel). Scale bar = 100 μm. D: Measurement of variance coefficients of the fiber size in the cross-sectional samples of chow-fed and HFD-fed mice using Feret’s diameter as the geometrical parameter. n = 61 (minimum) to 201 (maximum) fibers were analyzed for each sample (three random fields/mouse, ×10). E: Quantification of adipocyte spots in the longitudinal section samples of chow-fed and HFD-fed mice (total biopsy sample). *P < 0.05, ***P < 0.001, chow-fed vs. HFD-fed mice. A.U., arbitrary units; CV, coefficient of variation; ns, nonsignificant.