Fig 2: Validation of a simple method for high-resolution imaging of endogenous GLUT4. (A) Theoretical resolvable volume for confocal microscopy, stimulated emission depletion (STED) and structured illumination super resolution microscopy (SIM) and the sample thinning enhanced resolution microscopy (STERM) used in the current study. D = diameter. It should be noted that these theoretical resolution values are estimates of the actual resolution of the techniques, since several factors affect the final imaging resolution. Figure modified from [16]. (B) Workflow overview for STERM. (1) Subjects underwent kicking exercise until exhaustion and (2) muscle biopsies were obtained and fixed before and after a hyperinsulinemic-euglycemic clamp. (3) Bundles of fibers were embedded in gelatin, sucrose infiltrated and mounted on cryo-pins. (4) Ultra-thin 70 nm sections were generated and (5) GLUT4 and subcellular compartment markers were labelled and (6) imaged. (C) Comparison of resolution by images obtained by confocal imaging of whole single mouse muscle fibers and 70 nm STERM sections prepared as illustrated in A. Red arrows mark vesicles which were better resolved by STERM. (D) Micrographs from transmission electron microscopy of human muscle showing GLUT4 in the perinuclear region (D1), the subsarcolemmal region (D2), the intrafibrillar region (D3), in tubule-vesicular regions (D4), in multi-vesicular endosomes (D5) and in individual vesicles (D6). Red arrowheads mark perinuclear and subsarcolemmal GLUT4, asterisks mark intrafibrillar GLUT4, red arrows mark GLUT4 in tubulovesicular structures and white arrowheads mark GLUT4 in vesicles. Scale bar = 5 µm (C) and 100 nm (D).

Fig 3: Prior exercise and insulin redistributed intramyocellular GLUT4 in humans. Human muscle biopsies from resting and prior exercised leg before and after insulin stimulation were cut in 70 nm sections at cryogenic temperature. Sections were labelled with GLUT4 and various compartment markers, and the GLUT4 content in each compartment was estimated using Mander's overlap coefficient. (A) GLUT4 overlap with vesicle associated membrane protein 2 (VAMP2) in human skeletal muscle. (B) GLUT4 overlap with trans Golgi network protein 2 (TGN46). (C) GLUT4 overlap with lysosome associated membrane protein 1 (LAMP1). (D) GLUT4 overlap with early endosome antigen 1 (EEA1). (E) GLUT4 overlap with sorting nexin 1 (SNX1). (F) GLUT4 overlap with caveolin 3 (Cav3). (G) GLUT4 overlap with dihydropyridine receptor α1 (DHPR) and Sodium–Calcium Exchanger 1 (NCX1). GSV = GLUT4 Storage Vesicles, Early Endo = Early endosomes, Late/Rec. Endo = Late/Recycling endosomes. n= (basal rest; basal exercise; insulin rest; insulin exercise) A)41; 38; 40; 29, B)32; 31; 31; 24, C)25; 30; 29; 22, D)34; 27; 33; 25, E)19; 20; 25; 19, F)61; 52; 55; 49, G)37; 36; 35; 36(DHPR) and 25; 24; 23; 23(NCX1). #/##/###p < 0.05/0.01/0.001 different from resting leg. ∗/∗∗/∗∗∗p < 0.05/0.01/0.001 effect of insulin stimulation.

Fig 4: Dex protects myocardial IRI through MIF/AMPK/GLUT4 NF-?B axis

Fig 5: Diane-35 and metformin reduced AR and IGF-1 expression and increased GLUT4 expression in the early-stage tumor tissue of PCOS patients with EC. Immunohistochemistry (A) and PCR analysis (B) for AR, GLUT4, and IGF-1 in endometrial carcinoma tissue, scale bars = 100 μm. Transmission electron microscope analysis of patients’ tissues: autophagosomes: red arrow, autolysosome: white arrow, lysosome: red asterisk, and lipid droplet: LD, scale bars = 2 μm (C,D). Western blotting analysis of LC3 I/II (E) and cleaved caspase-3 (F) expression levels from 7 different patient samples. Data are shown as the mean ± S.D. * p < 0.05 and ** p < 0.01, ANOVA.