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• W4382238996 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking. Editor's evaluation This manuscript reveals localization of Glut4 glucose transporters at microtubules in mouse and human muscle fibers and shows that disruption of microtubules or a kinesin-1 motor alters Glut4 trafficking. Evidence is also provided supporting the idea that insulin resistance disrupts Glut4 dynamics at microtubules. Overall, these studies provide compelling evidence that Glut4 and its regulation by insulin involves Glut4 movements that require microtubule function. https://doi.org/10.7554/eLife.83338.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Skeletal muscle is quantitatively the largest site of glucose disposal, a process facilitated by insulin and contraction-responsive translocation and insertion of glucose transporter 4 (GLUT4) into the surface membrane of muscle fibers (Jaldin-Fincati et al., 2017; Klip et al., 2019). Insulin-resistant human and rodent muscle exhibit impaired insulin-stimulated GLUT4 translocation (Zierath et al., 1996; King et al., 1992; Etgen et al., 1997; Garvey et al., 1998) and muscle-specific deletion of GLUT4 is sufficient to cause systemic insulin resistance and glucose intolerance (Zisman et al., 2000). However, the details of GLUT4 regulation – particularly in adult skeletal muscle – and the causes of skeletal muscle insulin resistance remain unclear. In L6 myoblasts and 3T3-L1 adipocytes, insulin resistance not only decreases insulin-stimulated GLUT4 recruitment to the surface membrane, but also affects the distribution of GLUT4 between intracellular compartments (Foley and Klip, 2014; Xiong et al., 2010). This suggests that disturbed intracellular sorting of GLUT4 contributes to peripheral insulin resistance. Motor protein-mediated trafficking on the microtubule cytoskeleton is well established to allow long-range transport of a diverse assortment of molecules and to position intracellular organelles and membrane structures in various cell types (de Forges et al., 2012). For GLUT4, long-range microtubule-dependent GLUT4 movement beneath the plasma membrane has been observed in adipocyte cell culture (Lizunov et al., 2005) and similar long-range movement was also seen in adult rodent skeletal muscle (Lizunov et al., 2012). A requirement for microtubule-based protein trafficking is supported by several observations in cultured cells. Microtubule disruption dispersed perinuclear GLUT4 in 3T3-L1 adipocytes (Guilherme et al., 2000; Fletcher et al., 2000) as well as L6 myoblasts (Foley and Klip, 2014) and impaired GLUT4 membrane insertion in some (Foley and Klip, 2014; Fletcher et al., 2000; Chen et al., 2008; Emoto et al., 2001) but not all studies (Molero et al., 2001; Shigematsu et al., 2002). Neither the requirement of microtubules for intracellular GLUT4 positioning and trafficking nor the influence of insulin resistance on microtubules and/or microtubule-based GLUT4 trafficking have been investigated in adult skeletal muscle fibers. Therefore, we presently characterized various aspects of microtubule-based GLUT4 trafficking in predominantly adult human and mouse skeletal muscle. Our findings suggest that an intact microtubule network is required for KIF5B-mediated intracellular GLUT4 movement and maintaining insulin-responsive glucose uptake, and that impaired microtubule-based GLUT4 trafficking is a feature of skeletal muscle insulin resistance. Results GLUT4 was enriched at microtubule nucleation sites and traveled on microtubule filaments in adult mouse and human muscle To study the involvement of microtubules in GLUT4 trafficking, we first used structured illumination microscopy to image the subsarcolemmal (up to 4 µm into the muscle fiber) microtubule network and GLUT4 in mouse and human skeletal muscle at super-resolution. Due to amenability for live fiber isolation we used flexor digitorum brevis (FDB), a muscle consisting predominantly of type IIa and IIx fibers (Tarpey et al., 2018), from mice and vastus lateralis, a highly mixed muscle (Staron, 1991; Horwath et al., 2021), from humans. In both mouse (Figure 1A) and human (Figure 1B) muscle, we observed GLUT4 to be localized on microtubule filaments and to be enriched at microtubule filament intersections, previously identified as microtubule nucleation sites (Oddoux et al., 2013). Next, to study GLUT4 movement in live muscle fibers, we overexpressed GLUT4-7myc-GFP (GLUT4-GFP) (Bogan et al., 2001) alone or together with mCherry-Tubulin in mouse FDB muscle fibers (Figure 1C). GLUT4-GFP was localized in the same pattern as endogenous GLUT4 and observed along the microtubule network, including on the more stable subpopulation (Bulinski and Gundersen, 1991) of detyrosinated microtubules (Figure 1—figure supplement 1A) implicated in trafficking of lipid droplets, mitochondria, and autophagosomes in other cells types (Mohan et al., 2019; Herms et al., 2015), as well as on mCherry-Tubulin-labeled microtubules (Figure 1—figure supplement 1B). Live-imaging revealed long-range lateral directional movement of GLUT4 along filamentous tracks (Figure 1—figure supplement 1C), corresponding to mCherry-Tubulin containing microtubule filaments (Figure 1D and Figure 1—video 1). The GLUT4 structures occasionally exhibited long tubular morphology (>2 µm) but were mostly minor tubular structures or spheres (size varying from ~0.4 μm2 down to the unresolvable) observed to undergo budding and fusion events on the microtubule tracks (Figure 1—figure supplement 1D, E). Live-imaging, including fluorescence recovery after photobleaching experiments, revealed particularly dynamic and bidirectional movement at the microtubule nucleation sites (Figure 1—figure supplement 1F–H and Figure 1—video 2). Collectively, a portion of GLUT4 localized to microtubule nucleation sites and on microtubule filaments in adult mouse and human skeletal muscle. Furthermore, GLUT4 underwent continuous movement, budding and fusion along the microtubule tracks in live mouse skeletal muscle. Figure 1 with 3 supplements see all Download asset Open asset 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 of GLUT4 (green) and α-tubulin (white) (right panel). Arrows indicate GLUT4 at microtubule nucleation sites, arrowheads mark GLUT4 vesicles along the microtubule filaments. (C) Overview of workflow for live-imaging of fluorescently conjugated proteins in adult mouse FDB muscle fibers. (D) Live-imaging of FDB-expressing GLUT4-GFP and mCherry-Tubulin. Yellow projection of mCherry-Tubulin outlines the microtubule filaments (top panel left). Movement of GLUT4-GFP was visualized by color-coded projection (first image cyan, last image red, top panel right). The merged projection (bottom), demonstrated movement of GLUT4-GFP along the mCherry-Tubulin containing microtubule filaments indicated by color-coded projections on top of the microtubule filaments. The movement of GLUT4-GFP is shown in Figure 1—video 1. (A, B) Images are representative of >5 fibers from ≥3 different mice in A + D and 3 different fibers from 3 different subjects in B. Scale bar = 5 µm (A, B, D) and 2 µm (inserts in B, D). GLUT4 trafficking and localization required intact microtubules Next, we tested if microtubule-based GLUT4 trafficking was insulin responsive and dependent on an intact microtubule network. Insulin (30 nM) stimulation increased insulin signaling at the level of Akt Thr308 as expected (Figure 2—figure supplement 1A), and the microtubule depolymerizing compound Nocodazole (Noco) (13 µM) significantly reduced both the total and the Noco-resistant (Khawaja et al., 1988) detyrosinated pool of polymerized microtubules by ~90% and ~50%, respectively (Figure 2—figure supplement 1B, C). We did not observe any significant increase in GLUT4 movement on microtubules upon insulin stimulation but GLUT4 movement was completely prevented by microtubule depolymerization (Figure 2A, B, Figure 2—figure supplement 1D, Figure 2—video 1). Having established that GLUT4 trafficking was dependent on the microtubule network, we next tested if microtubule disruption affected the overall GLUT4 localization and distribution between different compartments. For quantification, we divided the GLUT4 structures into size categories corresponding to (1) large structures at the microtubule nucleation sites (>4 µm2), (2) intermediate endomembrane structures (0.4–4 µm2) (Gruenberg, 2001; Huotari and Helenius, 2011), and (3) the smallest resolvable endomembrane structures (<0.4 µm2) including presumably insulin-responsive GLUT4 storage vesicles (GSVs) (Figure 2—figure supplement 1E). Microtubule disruption by Noco (13 µM) drained the GLUT4 structures at the microtubule nucleation sites and reduced the amount of the smallest structures, while causing an increase in the intermediate structures (Figure 2C, D). These changes were reversed within 9 hr after removal of Noco (Figure 2C, D). Within the smallest category, there was a shift toward fewer but larger area GLUT4 membrane structures (Figure 2—figure supplement 1F). The total number and area of GLUT4 structures did not differ between conditions (Figure 2—figure supplement 1G). In a previous study in L6 myoblasts, microtubule disruption prevented pre-internalized GLUT4 from reaching a Syntaxin6-positive perinuclear subcompartment involved in GSV biogenesis and from undergoing insulin-responsive exocytosis (Foley and Klip, 2014). We therefore tested in adult muscle, if microtubule disruption similarly prevented accumulation in a perinuclear Syntaxin6-positive subcompartment. However, we observed a limited and Noco-insensitive (in mice) co-localization of Syntaxin6 with either endogenous GLUT4 in human and mouse skeletal muscle, or fluorescent GLUT4-EOS (Lizunov et al., 2013) in mouse skeletal muscle (Figure 2E, F, Figure 2—figure supplement 1H). Altogether, our data demonstrate that GLUT4 trafficking and distribution is disrupted by pharmacological microtubule network depolymerization in a fully reversible manner. Figure 2 with 2 supplements see all Download asset Open asset 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) for 4 hr prior to insulin. The dynamics of GLUT4-GFP in the different conditions are also exemplified in Figure 2—video 1. (B) Quantified microtubule-based GLUT4 trafficking. (C) Representative images of muscle fibers ± pre-treatment with Noco 13 µM, for 15 hr, followed by recovery in Noco-free medium for 9 hr. (D) Quantification of GLUT4 distribution between the microtubule nucleation sites (structures sized >4 µm2), intermediate-sized structures (0.4–4 µm2) and the smallest resolvable structures (<0.4 µm2) in fibers treated as in C. Compartment identification is described in Figure 2—figure supplement 1E. (E) GLUT4 and Syntaxin6 (Stx6) in muscle fiber from human vastus lateralis muscle. (F) GLUT4-Stx6 overlap in perinuclear region of mouse flexor digitorum brevis muscle fibers in Dimethylsulfoxide (DMSO) medium with and without Noco (13 µM) treatment. For A, B, n ≥ 14 muscle fibers from 5 different mice. For C, D, n = 9–11 muscle fibers from 3 different mice. For E, n = 3 subjects. Data are presented as mean with individual data points. ***p < 0.001 different from basal, ###p < 0.001 different from INS, ##p < 0.01 different from Noco recovery. ¤¤¤p < 0.001 analysis of variance (ANOVA) effect. Scale bar = 5 µm (A–C) and 2 µm (E). 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 Download elife-83338-fig2-data1-v2.zip Prolonged, but not short-term, microtubule disruption blocked insulin-induced muscle glucose uptake To investigate the requirement of microtubule-based GLUT4 trafficking and localization for insulin-induced muscle glucose uptake, we assessed muscle glucose uptake ± insulin and ± microtubule disruption in isolated incubated intact mouse soleus and extensor digitorum longus (EDL) muscles. When mouse soleus and EDL muscles were incubated ex vivo ± insulin and ± Noco (13 µM) for 15 min and up to 2 hr, an interaction between insulin and Noco was observed and the insulin-induced glucose uptake was gradually impaired over time and completely disrupted after 2 hr in both muscles (Figure 3A). The increase by insulin stimulation was significantly impaired after 40 min in soleus and 2 hr in EDL muscle (Figure 3B). Insulin-stimulated phospho-signaling via Akt and TBC1D4 was unaffected by Noco treatment (Figure 3—figure supplement 1A–C). Figure 3 with 1 supplement see all Download asset Open asset 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) Insulin-stimulated 2-DG transport (insulin minus basal) from muscles shown in A. (C) Experimental setup for muscle-on-a-chip system with glucose sensor. (D) Microtubules imaged with α-tubulin in glucose transporter 4 (GLUT4)-GFP-expressing mouse flexor digitorum brevis (FDB) fibers treated ± Noco (13 µM) for 5 min or 2 hr. (E) 180-s measurements of glucose concentration in perifusate from basal and insulin-treated FDB muscle fibers in muscle chips pre-incubated with DMSO, Noco (13 µM, 5 min or 2 hr) or colchicine (25 µM, 2 hr). (F) Insulin-stimulated glucose uptake into FDB muscle fibers in muscle chips calculated from the last 20 s of the concentration curves in E. (G) Representative GLUT4 images from isolated mouse FDB muscle fibers treated ± Noco (13 µM) for 5 min or 2 hr. (H) Quantification of GLUT4 in large, intermediate- and small-sized membrane structures in FDB fibers treated ± Noco (13 µM) for 5 min or 2 hr. The membrane compartment division by size is shown in Figure 2E. For A, B, n = 6–7 muscles from 6 to 7 mice. For D, G, H, n = 8–10 muscle fibers from 3 different mice. For E, F, n ≥ 3 muscle chips from 3 to 4 mice. Data are presented as mean with individual data points. Paired observations from the same mouse are indicated by a connecting line. */**/***p < 0.05/0.01/0.001 different from basal/DMSO, #/##/###p < 0.05/0.01/0.001 different from DMSO. ¤/¤¤/¤¤¤p < 0.05/0.01/0.001 analysis of variance (ANOVA) effect. Scale bar = 5 µm. 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 Download elife-83338-fig3-data1-v2.zip To understand the temporal resolution of microtubule network disruption and its effect on insulin-induced glucose uptake, we investigated glucose uptake adult isolated single fibers in real time using a custom-made perifused organ-on-chip system (Gowers et al., 2015; Trouillon and Gijs, 2016) featuring a glucose-sensing electrode for glucose uptake measurements (Figure 3C). In brief, this chip continuously measures glucose concentration in perifusate post muscle fiber exposure, allowing the estimation of glucose uptake over time. We confirmed the ability of the chip to measure glucose uptake in skeletal muscle fibers (Figure 3—figure supplement 1D–F). Specifically, the chip measured glucose uptake in isolated FDB fibers at ~5 µM glucose concentration sensitivity (defined as a registered fluctuation of thrice the standard deviation [SD] of the baseline measurements) and a temporal resolution of <4 s (Figure 3—figure supplement 1G, H). Noco (13 µM) caused complete microtubule disruption in FDB fibers within 5 min similar to a 2-hr treatment (Figure 3D). Interestingly, acute microtubule disruption (5 min Noco) affected neither basal (Figure 3—figure supplement 1I) nor insulin-induced muscle glucose uptake, whereas 2 hr treatment by Noco or colchicine (25 µM), another microtubule network disrupter, completely blocked insulin-induced muscle glucose uptake (Figure 3E, F). Notably, GLUT4-containing large membrane structures corresponding mainly to microtubule nucleation sites were already reduced after 5 min of Noco exposure, whereas accumulation of GLUT4 in intermediate- and small-sized membrane structures was only observed after 2 hr of Noco exposure (Figure 3G, H). Thus, an intact microtubule network is not required for the immediate insulin-induced GLUT4 translocation response in adult skeletal muscle fibers. However, prolonged disruption of the microtubule network causes a more pronounced missorting of GLUT4 and renders skeletal muscle unresponsive to insulin. KIF5B-containing kinesin-1 motor proteins regulate muscle GLUT4 trafficking Next we investigated which motor protein(s) mediate microtubule-dependent GLUT4 trafficking in skeletal muscle. The kinesin-1 heavy chain protein KIF5B has been implicated in GLUT4 trafficking in adipocytes (Semiz et al., 2003; Habtemichael et al., 2018). Thus, we studied the effect of kinesore, a small molecule modulator which both inhibits kinesin-1 interaction with specific cargo adaptors but also stimulates Kinesin motor function (Randall et al., 2017), in incubated soleus and EDL muscles and differentiated primary human myotubes as well as the effect of Kif5b short hairpin (sh) RNA knockdown and kinesore in L6 skeletal muscle cells overexpressing exofacially tagged GLUT4 (Kishi et al., 1998; Wang et al., 1998; Figure 4A). In both soleus and EDL muscle, 2 hr of kinesore (50 μM) exposure strongly impaired insulin-stimulated glucose uptake (Figure 4B) without affecting p-Akt Ser473 and slightly increased basal and insulin-stimulated p-TBC1D4 Thr642 (Figure 4—figure supplement 1A). AMPK is an insulin-independent stimulator of GLUT4 translocation which may indirectly stimulate TBC1D4 Thr642 phosphorylation (Kjøbsted et al., 2015). Notably, phosphorylation of AMPK and downstream ACC2 were stimulated by kinesore in both basal and insulin-stimulated soleus and EDL muscles (Figure 4—figure supplement 1B). In primary human myotubes differentiated for 7 days kinesore (50 μM) and Noco (13 μM) reduced the glucose uptake and completely blocked the insulin response (Figure 4C). In L6 myoblasts, we lowered KIF5B expression using shRNA by ~70% in L6 myoblasts (Figure 4—figure supplement 1C). This did not affect GLUT4 expression (Figure 4—figure supplement 1D) but impaired insulin-stimulated GLUT4 translocation (Figure 4D). Unlike the inhibitory effect in incubated mouse muscle and primary human myotubes, kinesore-stimulated GLUT4 translocation and glucose uptake additively to insulin in L6 muscle cells, and modestly potentiated insulin-stimulated Akt Thr308 without affecting AMPK signaling (Figure 4—figure supplement 1E–J). Collectively, although kinesore surprisingly had a stimulatory and seemingly MT-independent effect on GLUT4 translocation in L6 muscle cells, our shRNA data in L6 myoblasts and kinesore data in adult muscle and primary human myotubes support the requirement of KIF5B-containing kinesin-1 motor proteins in GLUT4 translocation in skeletal muscle. Figure 4 with 1 supplement see all Download asset Open asset 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) muscles pretreated with kinesore (50 µM) for 2 hr. (C) Deoxyglucose (2-DG) transport in basal and insulin-stimulated primary human myotubes pretreated ± kinesore (50 µM) or Noco (13 µM) for 2 hr. (D) Exofacial GLUT4 signal in serum starved (4 hr) basal and insulin-stimulated (100 nM, 15 min) L6 myoblasts (left) and insulin response (insulin minus basal, right) in GLUT4 surface content. L6 myoblasts were transfected with short hairpin scramble RNA (shScramble) or shRNA targeting Kif5b 72 hr prior to the experiment. Analysis of variance (ANOVA) main effect of insulin (¤¤¤) and shKif5b (¤¤¤) and interaction (¤). (B) n = 8 muscles in each group, lines indicate muscles from same mouse. (C) Each data point represents the average of 3 replicates and originate from at least 3 independent experiments. Data are presented as mean with individual data points. */**/***p < 0.01/0.001 effect of insulin. ##/###p < 0.01/0.001 different from DMSO/Scramble. ¤/¤¤/¤¤¤p < 0.05/0.01/0.001 ANOVA effect. 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 Download elife-83338-fig4-data1-v2.zip Insulin resistance induced by C2 ceramide and high-fat diet impaired microtubule-based GLUT4 trafficking Having established an essential role for the microtubule network in GLUT4 trafficking and muscle glucose uptake, we proceeded to test if microtubule-based GLUT4 trafficking was impaired in insulin-resistant states. We induced insulin resistance in adult mouse skeletal muscle both in vitro using short-chain C2 ceramide and in vivo using diet-induced obesity (Figure 5A). In vitro, treatment of isolated FDB muscle fibers with C2 ceramide (50 µM) impaired Akt Thr308 phosphorylation (Figure 5—figure supplement 1A) and in insulin-stimulated fibers markedly reduced microtubule-based GLUT4 trafficking defined as the number of moving GLUT4 structures (Figure 5B) and the total microtubule-based traveling of GLUT4 structures (Figure 5—figure supplement 1B). In vivo, mice fed a 60% high-fat diet (HFD) for 10 weeks exhibited impaired tolerance to insulin and glucose as well as reduced insulin-stimulated phosphorylation of Akt Thr308 and Akt substrate TBC1D4 Thr642 in isolated FDB fibers (Figure 5—figure supplement 1C–E), confirming whole-body and skeletal muscle insulin resistance. Similar to C2 ceramide-treated fibers, HFD-exposed FDB muscle fibers exhibited impaired microtubule-based GLUT4 trafficking (Figure 5C, Figure 5—figure supplement 1F). This prompted us to investigate whether the microtubule polymerization was itself insulin responsive and/or affected by insulin resistance. To test this, we transfected mouse FDB muscle fibers with the microtubule plus-end-binding protein EB3-GFP, which binds the tip of growing microtubules via its calponin homology domain and has previously been used for live-cell characterization of microtubule polymerization (Stepanova et al., 2003). As previously reported (Oddoux et al., 2013), EB3-GFP transfection allowed visualization of growing microtubules as a dynamic comet tail-like appearance, an effect completely prevented by the microtubule stabilizer taxol (10 µM) (Figure 5—figure supplement 1G, Figure 5—video 1). In our datasets, we analyzed the microtubule polymerization frequency (by counting EB3-GFP puncta; Komarova et al., 2009), the average polymerization distance, the total polymerization distance and the polymerization directionality following C2 ceramide exposure or HFD. Insulin tended (p = 0.095) to increase the number of polymerizing microtubules by an average of 28% compared to basal fibers while C2 ceramide treatment reduced the amount of polymerizing microtubules significantly and taxol almost abolished microtubule polymerization (Figure 5D, E). C2 ceramide treatment also reduced the total polymerization distance while the average polymerization was unaffected (Figure 5—figure supplement 1H). Figure 5 with 2 supplements see all Download asset Open asset 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 + 50 µM) treated flexor digitorum brevis (FDB) muscle fibers. (C) Quantified microtubule-based GLUT4 trafficking in basal or INS (30 nM) treated FDB fibers from chow or high-fat diet (HFD) fed mice. (D) Representative images of polymerizing microtubules in EB3-GFP-expressing FDB muscle fibers treated ± C2 (50 µM), paclitaxel (Taxol, 10 µM) for 2 hr prior to 15–30 min of INS (30 nM) stimulation. Red circles highlight microtubule tip-bound EB3-GFP. (E) Quantification of polymerizing microtubules based on EB3-GFP in FDB fibers treated as in D. (F) Quantification of polymerizing microtubules based on EB3-GFP in FDB fibers isolated from chow or 60% HFD fed mice and treated ± INS (30 nM) for 15–30 min. For B–F, n ≥ 13 muscle fibers from 3 to 4 mice. Taxol-treated muscle fibers were only used as a control and not included in the statistical analysis. NA = not statistically analysed. Data are presented as mean with individual data points. #/##/###p < 0.05/0.01/0.001 different from INS (B) or different from corresponding group in chow fed mice (C) or control fibers (E). ¤/¤¤/¤¤¤p < 0.05/0.01/0.001 main effect (ME) of diet/C2. 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 Download elife-83338-fig5-data1-v2.zip In contrast to the effect of C2 ceramide on microtubule dynamics, HFD-induced insulin resistance was not associated with alterations in the number of polymerizing microtubules, average polymerization distance or total polymerization distance (Figure 5F, Figure 5—figure supplement 1I). The polymerization directionality was also not affected by HFD (Figure 5—figure supplement 1J). Altogether, different models of insulin resistance impaired microtubule-based GLUT4 trafficking in adult muscle fibers, suggesting a role in adult skeletal muscle insulin resistance. In contrast, defective microtubule polymerization was observed with C2 ceramide but not with the presumably more physiologically relevant HFD insulin resistance model. Discussion In the present study, we provide translational evidence in adult human and mouse skeletal muscle, showing that the microtubule network is crucial for long-range directional GLUT4 trafficking via motor proteins, likely including KIF5B. Microtubule polymerization in isolated mouse muscle fibers could be abolished pharmacologically within minutes without affecting insulin-stimulated glucose uptake whereas longer pharmacological inhibition progressively caused GLUT4 mislocalization and lowered insulin responsiveness. These data are consistent with a model where microtubules are required for correct intramyocellular GLUT4 compartmentalization but not the ultimate insulin-stimulated GLUT4 translocation to the cell surface from these compartments. Importantly, microtubule-based GLUT4 movement was impaired in two classical mouse insulin resistance models, short-chain ceramide treatment and diet-induced obesity. These data implicate dysregulation of microtubule-mediated GLUT4 trafficking and their localization in the etiology of adult skeletal muscle insulin resistance. What may cause the reduced number of GLUT4 moving on microtubule" @default.
• W4382238996 created "2023-06-28" @default.
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• W4382238996 date "2022-11-15" @default.
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• W4382238996 title "Decision letter: Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle" @default.
• W4382238996 doi "https://doi.org/10.7554/elife.83338.sa1" @default.
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