Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle
- August Krogh Section for Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark
- Heart and Skeletal Muscle Biology, Global Drug Discovery, Novo Nordisk, Denmark
- Exercise Science Laboratory, Faculty of Medicine, Universidad Finis Terrae, Chile
- College of Physical Education, Chongqing University, China
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
- Clinical Drug Development, Novo Nordisk, Denmark
- Department of Electrical Engineering, Polytechnique Montréal, Canada
- Laboratory of Integrative Systems Physiology, Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Switzerland
- Microsystems Laboratory 2, Institute of Electrical and Micro Engineering, École Polytechnique Fédérale de Lausanne, Switzerland
Figures
Figure 1 with 3 supplements
Glucose transporter 4 (GLUT4) was enriched at microtubule nucleation sites and traveled on microtubule filaments in mouse and human muscle.
Structured illumination microscopy (SIM) in mouse flexor digitorum brevis (FDB) muscle (A) and human vastus lateralis muscle (B) of endogenous α-tubulin and GLUT4 (left panel) and 3D reconstruction …
Figure 1—figure supplement 1
Glucose transporter 4 (GLUT4) undergoes budding and fusion on the microtubules and moves bidirectionally to and from the microtubule nucleation sites.
(A) Confocal imaging of α-tubulin (top panel), detyrosinated tubulin (De-Tubulin, bottom panel) and GLUT4-GFP in flexor digitorum brevis (FDB) fibers from muscles expressing GLUT4-GFP. (B) …
Figure 1—video 1
Glucose transporter 4 (GLUT4) traveled on microtubules.
Video of live flexor digitorum brevis (FDB) fibers electroporated with GLUT4-GFP (green) and mCherry-Tubulin (magenta) 6 days earlier. The video is played at 10 frames per second with one frame …
Figure 1—video 2
Glucose transporter 4 (GLUT4) traveled to and from GLUT4-enriched regions at microtubule nucleation sites.
Video of GLUT4-GFP at GLUT4-enriched/large structure in live flexor digitorum brevis (FDB) fibers showing spherical and tubular GLUT4-GFP structures that fuse and bud off from the GLUT4-enriched …
Figure 2 with 2 supplements
Glucose transporter 4 (GLUT4) trafficking and localization was dependent on an intact microtubule network.
(A) Representative time-lapse traces of GLUT4-GFP vesicle tracking in muscle fibers ± insulin (INS, 30 nM) for 15–30 min with or without microtubule network disruption by Nocodazole (Noco, 13 µM) …
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Figure 2—source data 1
Data used for quantification of GLUT4-Stx6 overlap in perinuclear region of mouse flexor digitorum brevis muscle fibers in DMSO medium with and without Noco (13 µM) treatment.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig2-data1-v2.zip
Figure 2—figure supplement 1
Glucose transporter 4 (GLUT4) trafficking and localization was dependent on an intact microtubule network.
(A) Phosphorylation of Akt Thr308 in GLUT4-GFP-expressing flexor digitorum brevis (FDB) fibers treated ± 30 nM insulin (INS) for 15 min. (B) Polymerized microtubules in GLUT4-GFP-expressing fibers …
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Figure 2—figure supplement 1—source data 1
Data used for quantification of Figure 2—figure supplement 1A–D, G and raw unedited blots for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig2-figsupp1-data1-v2.zip
Figure 2—video 1
Glucose transporter 4 (GLUT4) movement was disrupted by Nocodazole treatment.
Video of live flexor digitorum brevis (FDB) fibers electroporated with GLUT4-GFP 6 days earlier. To facilitate visualization, the video was generated so that moving GLUT4 appears green-red flashing …
Figure 3 with 1 supplement
Time-dependent effect of microtubule disruption on insulin-induced muscle glucose uptake.
(A) 2-Deoxyglucose (2-DG) transport in basal and insulin-stimulated mouse soleus and extensor digitorum longus (EDL) muscles pretreated with Nocodazole (Noco, 13 µM) for the indicated time. (B) …
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Figure 3—source data 1
Data used for quantification of 2-DG transport and glucose clearance and uptake in Figure 3A, B, E, F, polymerized microtubules in Figure 3D and glucose transporter 4 (GLUT4) localization in Figure 3H.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig3-data1-v2.zip
Figure 3—figure supplement 1
Time-dependent effect of microtubule disruption on insulin-induced muscle glucose uptake.
Quantification of protein expression in soleus (A) and extensor digitorum longus (EDL) (B) muscles in the basal and insulin-stimulated state ± Nocodazole (Noco, 13 µM) as indicated. (C) …
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Figure 3—figure supplement 1—source data 1
Data used for quantification of protein expression and electrochemical glucose sensing in Figure 3—figure supplement 1.
Data used for quantification of Figure 3—figure supplement 1A, B, F, I and raw unedited blots for Figure 2—figure supplement 1A, B.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig3-figsupp1-data1-v2.zip
Figure 4 with 1 supplement
Kinesin-1 containing KIF5B-regulated glucose transporter 4 (GLUT4) localization and translocation.
(A) Schematic overview of L6 muscle cell system to assess GLUT4 surface content. (B) 2-Deoxyglucose (2-DG) transport in basal and insulin-stimulated mouse soleus and extensor digitorum longus (EDL) …
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Figure 4—source data 1
Data used for quantification of glucose transporter 4 (GLUT4) localization and GLUT4 surface content in Figure 4.
Data used for quantification of 2-DG transport and GLUT4 translocation in Figure 4B–D.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig4-data1-v2.zip
Figure 4—figure supplement 1
Kinesin-1 containing KIF5B-regulated glucose transporter 4 (GLUT4) localization and translocation.
(A, B) Quantification and representative immunoblots of protein expression in soleus and extensor digitorum longus (EDL) muscles in the basal and insulin-stimulated state ± 2 hr kinesore (50 µM) …
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Figure 4—figure supplement 1—source data 1
Data used for quantification of glucose transporter 4 (GLUT4) surface content, protein expression, and glucose uptake in Figure 4—figure supplement 1.
Data used for quantification of Figure 4—figure supplement 1A–C, E–I and raw unedited blots for Figure 4—figure supplement 1A, B, D, I.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig4-figsupp1-data1-v2.zip
Figure 5 with 2 supplements
Insulin resistance impairs microtubule-based glucose transporter 4 (GLUT4) trafficking.
(A) Overview of in vitro and in vivo insulin resistance models used. (B) Quantified microtubule-based GLUT4 trafficking in basal, insulin (INS, 30 nM) and insulin + C2 ceramide (C2) (INS + C2, 30 nM …
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Figure 5—source data 1
Data used for quantification of glucose transporter 4 (GLUT4) trafficking and microtubule polymerization in Figure 5B, C, E, F.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig5-data1-v2.zip
Figure 5—figure supplement 1
Insulin resistance impairs microtubule-based glucose transporter 4 (GLUT4) trafficking.
(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 …
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Figure 5—figure supplement 1—source data 1
Data used for quantification of protein expression, glucose transporter 4 (GLUT4) trafficking, glucose and insulin tolerance, microtubule polymerization and polymerization directionality in Figure 5—figure supplement 1.
Data used for quantification of Figure 5—figure supplement 1A–F and H–J and raw unedited blots for Figure 5—figure supplement 1A, D.
- https://cdn.elifesciences.org/articles/83338/elife-83338-fig5-figsupp1-data1-v2.zip
Figure 5—video 1
Polymerizing microtubules in muscle fibers.
Video of polymerizing microtubule (MT) tips detected by EB3-GFP in live flexor digitorum brevis (FDB) fibers electroporated with EB3-GFP 6 days earlier. To facilitate visualization, the video was …
Figure 6
Overview of muscle chip for glucose sensing.
(A) Overview of the different poly(dimethylsiloxane) (PDMS) layers for the tissue chamber unit. Scale bar = 5 mm. (B) Microfluidic system for the glucose-sensing chamber. The electrode was placed in …
