Volume 23, Issue 8, Pages 2354-2364 (May 2018) Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4 Translocation and Performance in Skeletal Muscles  Masahiro Tsuchiya, Shigenori Sekiai, Hiroyasu Hatakeyama, Masashi Koide, Chayanit Chaweewannakorn, Fukie Yaoita, Koichi Tan-No, Keiichi Sasaki, Makoto Watanabe, Shunji Sugawara, Yasuo Endo, Eiji Itoi, Yoshihiro Hagiwara, Makoto Kanzaki  Cell Reports  Volume 23, Issue 8, Pages 2354-2364 (May 2018) DOI: 10.1016/j.celrep.2018.04.067 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Effects of Walking Exercise on IL-1α/β and IL-1 Signaling-Related mRNA Expressions in QFMs of WT Mice (A–C) Messenger RNA expressions of IL-1s (A), IL-1 receptors (B), and IL-1 signaling-related molecules (C) after prolonged (2 hr) walking (n = 6–8). Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Lower Endurance and Impaired Glucose Metabolism in IL-1-KO Mice during Prolonged Walking and Effects of Exogenous IL-1β (A) Rapid exhaustion in IL-1-KO mice during prolonged walking (n = 10–12). (B and C) Effects of prolonged walking on [14C]-2DG uptake (B) or glycogen content (C) in WT QFMs (left) and IL-1-KO (right) mice (n = 10–12). (D and E) Effects of intravenous injection of exogenous IL-1β (2 pg/g BW and 20 pg/g BW) on 2DG uptake (D) or IL-1β (2 pg/g BW) on mRNA expression (E) in QFMs of IL-1-KO mice with or without 2 hr of walking (n = 10–12). Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Compromised GLUT4 Translocation in QFMs of IL-1-KO Mice with Walking Exercise (A) Representative images showing QFMs (cross-sections) stained with antibodies against GLUT4 (green) and α-sarcoglycan (red), and DAPI (blue) from WT and IL-1-KO mice with or without 2 hr of walking. (B) Schema for quantification of GLUT4 localization in sarcolemma. Intensity profiles of both GLUT4 and α-sarcoglycan immunofluorescence (left) along the lines across the sarcolemma (right, white lines) were obtained, and the intensities over the background fluorescence outside myofibers were then calculated. (C) Boxplots of sarcolemma GLUT4 in WT and IL-1-KO mice with or without 2 hr of walking (n = 8 each). At least 5 different areas in each QFM section were analyzed. (D) Western blotting analysis of GLUT4 in QFMs of WT and IL-1-KO mice. Three independent experiments were performed, and representative results are presented. Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Recruitment of Neutrophils Producing IL-1β in QFMs of WT Mice with Walking Exercise (A) Representative data from FACS analysis of neutrophil (CD45+CD11b+Ly6G+F4/80− phenotype)/macrophage (CD45+CD11b+Ly6G−F4/80+ phenotype) recruitment in QFMs of WT mice (n = 6) with or without 2 hr of prolonged walking or resting. (B) Proportions of neutrophils and macrophages recruited into QFMs with or without 2 hr of walking. (C) IL-1β mRNA expression in neutrophils and macrophages sorted from QFMs of WT mice after 2 hr of walking or resting. (D) Representative images showing QFMs (cross-sections) stained with antibodies against IL-1β (green) and Gr-1 (red), and DAPI (blue) from WT mice with (upper panels) or without (lower panels) 2 hr of walking. Some immuno-positive cells are indicated by arrowheads. Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 Lower Endurance, Impaired Glucose Uptake with Compromised GLUT4 Translocation, and Aberrant Rac1 Signaling in Neutrophil-Depleted Mice (A) Rapid exhaustion in neutrophil-depleted mice (anti-Gr-1 Ab) during prolonged walking (n = 8–10). (B) Effects of neutrophil-depletion on 2DG uptake into QFMs with or without 2 hr of walking (n = 10–12). (C) Fluorescent images of GLUT4-EGFP in QFMs of control IgG–treated (upper) or anti-Gr-1 antibodies–treated (lower) GLUT4-EGFP-tg-mice without (left) or with (right) EPS-evoked in situ muscle contractions. Horizontal (xy) and longitudinal (yz) sections are shown. (D) Quantification of GLUT4-EGFP on the sarcolemma (left) and T-tubules (right) of the image shown in (C). (E) Effects of anti-Gr-1 antibody injection on EPS-evoked contraction-responsive GLUT4-EGFP translocation to the sarcolemma (left) and T-tubules (right). (F) Western blotting analysis of intracellular signaling intermediates in QFM of WT and neutrophil-depleted mice with or without EPS-evoked in situ muscle contractions. Three independent experiments were performed, and representative results are shown. Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Malfunction of Muscular Rac1 Signaling in IL-1-KO Mice (A and B) Western blotting analysis of intracellular signaling intermediates in QFM of WT and IL-1-KO mice with or without EPS-evoked in situ muscle contractions. (A) Graphs, summarizing the results obtained by analyzing western blot images. (B) Representative western blotting images are shown. Experimental values are given as means ± SEM. Three independent experiments were performed, and representative blots are shown. Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 Abnormalities in GLUT4-Containing Structures in Skeletal Muscle of IL-1-KO Mice (A) STED images of GLUT4 in soleus muscles of WT and IL-1-KO mice. (B) Boxplots of the particle size (left) and diameter (right) with or without AICAR treatment (1 mM) in soleus (upper) and EDL (lower) muscles (n = 12–21). The white line and the square represent median and mean values, respectively. (C) Boxplots of the circularity (spherical form) of particles in the soleus (upper) and EDL (lower) muscles of WT and IL-1-KO mice in the basal state (n = 12–21) are shown. Note that GLUT4-contatining structures in KO myofibers are more tubular in shape. (D and E) Western blotting analysis of intracellular GLUT4 translocation-related molecules in Soleus and EDL muscles of WT and IL-1-KO mice. (D) Representative western blotting images. (E) Graphs summarizing the results obtained by analyzing western blot images. Experimental values are given as means ± SEM. Three independent experiments were performed, and representative images are shown. Cell Reports 2018 23, 2354-2364DOI: (10.1016/j.celrep.2018.04.067) Copyright © 2018 The Author(s) Terms and Conditions