(A) Phosphorylation of AktThr308 in GLUT4-GFP-expressing flexor digitorum brevis (FDB) muscle fibers cultured ± 0.5 mM palmitic acid (PA) for 24 hr or 50 µM C2 ceramide (C2) for 2 hr prior to 15 min ± 30 nM insulin (INS) stimulation. (B) Quantified microtubule-based GLUT4 trafficking assessed as the traveled distance and the displacement within the dynamic fraction of the GLUT4-GFP in basal, insulin (INS, 30 nM) or insulin and C2 ceramide (INS + C2, 30 nM + 50 µM)-treated FDB muscle fibers. (C, D) Glucose and insulin tolerance tests of mice fed a control chow or a high-fat diet (HFD) after intraperitoneal injection of glucose (2 g kg−1 body weight) or insulin (0.5 U kg−1 body weight). AUC/AOC = area under/over the curve. Analysis of variance (ANOVA) main effect of diet (¤¤¤/¤, GTT/ITT), and time (¤¤¤) and interaction (¤¤¤/¤¤, GTT/ITT). (E) Phosphorylation of AktThr308 and TBC1D4Thr642 in isolated FDB fibers from muscles from chow or HFD fed mice treated ± INS (30 nM) for 15 min. (F) Quantified microtubule-based GLUT4 trafficking assessed as the traveled distance and the displacement within the dynamic fraction of the GLUT4-GFP in basal or INS (30 nM)-stimulated FDB muscle fibers isolated from chow or HFD-fed mice. (G) Color-coded projection (first image cyan, last image red as indicated by color code bar) of 60-s live-imaging of FDB fibers expressing EB3-GFP and treated without (CTRL) or with 10 µM paclitaxel (Taxol) for 2 hr. Scale bar = 5 µm. EB3-GFP dynamics are also illustrated in Figure 5—video 1. (H) Quantification of the total and average polymerizing distance of EB3-GFP containing microtubules in fibers incubated ±50 µM C2 ceramide (C2) or 10 µM Taxol for 2 hr prior to 15–30 min of INS (30 nM) stimulation. (I) Quantification of the total and average polymerizing distance of EB3-GFP containing microtubules in fibers isolated from chow or HFD fed mice and stimulated ± INS (30 nM) for 15 min. (J) Quantification of microtubule polymerization directionality based on EB3-GFP dynamics recording as in I. For A, C–E, n = 3–4 mice, for B, F, and H–J n ≥ 13 muscle fibers from 3 to 4 mice. (G) Representative of >10 fibers from 2 different mice. Taxol-treated muscle fibers were only used as a control and not included in the statistical analysis. NA = not analysed. Data are presented as mean with individual data points or mean ± standard deviation (SD). */***p < 0.05/0.001 different from basal, #/##/###p < 0.05/0.01/0.001 different from insulin (A, B) or different from corresponding group in chow fed mice (C–F, H). ¤/¤¤/¤¤¤p < 0.05/0.01/0.001 ANOVA main effect (ME).