Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes

Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
The Journal of Cell Biology (Impact Factor: 9.83). 08/2012; 198(4):545-560. DOI: 10.1083/jcb.201111091
Source: PubMed


Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise
steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement
of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to
the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase
(IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM.
Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B,
and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning
peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10
coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

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    • "In this context, we recently found that the processive , dimeric motor myosin Va (MyoVa) participates in GLUT4 vesicle traffic toward the cortex of L6 muscle cells (Klip et al., 2014; Sun et al., 2014). Of note, engagement of MyoVa was downstream of Rab8A activation in muscle cells and downstream of Rab10 in adipocytes (Chen et al., 2012; Sun et al., 2014). On the other hand, the effect(s) of Rab13 on insulin-dependent GLUT4 vesicle traffic remain unknown. "
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    ABSTRACT: Insulin promotes glucose uptake into skeletal muscle through recruitment of Glucose Transporter 4 (GLUT4) to the plasma membrane. Rab GTPases are molecular switches mobilizing intracellular vesicles, and Rab13 is necessary for insulin-regulated GLUT4-vesicle exocytic translocation in muscle cells. We show that Rab13 engages the scaffold protein MICAL-L2 in this process. RNAi-mediated knockdown of MICAL-L2 or truncated MICAL-L2 (MICAL-L2-CT) impaired insulin-stimulated GLUT4 translocation. Insulin increased Rab13 binding to MICAL-L2, assessed by pulldown and colocalization under confocal fluorescence and structured illumination microscopies. Association was also visualized at the cell periphery using TIRF microscopy. Insulin further increased binding of MICAL-L2 to α-actinin-4 (ACTN4), a protein involved in GLUT4 translocation. Rab13, MICAL-L2, and ACTN4 formed an insulin-dependent complex assessed by pulldown and confocal fluorescence imaging. Notably, GLUT4 associated with the complex in response to insulin, requiring the ACTN4-binding domain in MICAL-L2. This was demonstrated by pulldown with distinct fragments of MICAL-L2, and confocal and structured illumination microscopies. Finally, expression of MICAL-L2-CT abrogated the insulin-dependent colocalization of Rab13 with ACTN4 or Rab13 with GLUT4. Our findings suggest that MICAL-L2 is an effector of insulin-activated Rab13, which links to GLUT4 through ACTN4, localizing GLUT4-vesicles at the muscle cell periphery to enable their fusion with the membrane.
    Full-text · Article · Nov 2015 · Molecular biology of the cell
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    • "Vesicle traffic is then directed by Rab proteins . Indeed, Rab8A or Rab10, respectively, engage the molecular motor Myosin Va to mobilize GLUT4 vesicles from perinuclear loci to the cell periphery [33] [34]. Rab13 acts at the cell periphery potentially to promote GLUT4 vesicle tethering, docking or fusion [1] [12]. "
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    ABSTRACT: Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca(2+) mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca(2+) into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R). In skeletal muscle cells, a rapid, insulin-triggered Ca(2+) release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca(2+)]i trough L-type Ca(2+) channels activation. In both cell types, a relatively slower Ca(2+) release also occurs through IP3R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca(2+) released from IP3R of skeletal muscle also promotes mitochondrial Ca(2+) uptake. We review here these actions of insulin on intracellular Ca(2+) channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca(2+) release from the SER.
    Full-text · Article · Sep 2014 · Cell Calcium
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    • "Current models drawn from studies in 3T3-L1 adipocytes suggest that the intracellular stores of GLUT4 at steady-state include the recycling endosomes, the Trans-Golgi Network (TGN), and the GSV/IRV, the latter two compartments serving to sequester GLUT4 from recycling back to the plasma membrane (Blot and McGraw, 2008; Brewer et al., 2011; Foley et al., 2011; Stöckli et al., 2011). Although insulin-responding GLUT4-containing vesicles have been imaged within 200 nm of the plasma membrane of adipocytes (Bai et al., 2007; Chen et al., 2012; Huang et al., 2007; Lizunov et al., 2005; Xiong et al., 2010) and muscle cells (Boguslavsky et al., 2012; Sun et al., 2014), it is unknown how or where GSV/IRV are constituted to segregate away from constitutive recycling, as at steady-state GLUT4 is visualized both in the perinuclear region and in cytosolic vesicles (Boguslavsky et al., 2012; Chen et al., 2012; Foley et al., 2011; Randhawa et al., 2008; Xiong et al., 2010). "
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    ABSTRACT: GLUT4 constitutively recycles between the plasma membrane and intracellular depots. Insulin shifts this dynamic equilibrium towards the plasma membrane by recruiting GLUT4 to the plasma membrane from insulin-responsive vesicles. Muscle is the primary site for dietary glucose deposition; however, how GLUT4 sorts into insulin-responsive vesicles, and if and how insulin resistance affects this process, is unknown. In L6 myoblasts stably expressing myc-tagged GLUT4, we analyzed the intracellular itinerary of GLUT4 as it internalizes from the cell surface and examined if such sorting is perturbed by C2-ceramide, a lipid metabolite causing insulin resistance. Surface-labeled GLUT4myc that internalized for 30 min accumulated in a Syntaxin-6 (Stx6)- and Stx16-positive perinuclear sub-compartment devoid of furin or internalized transferrin, and displayed insulin-responsive re-exocytosis. C2-ceramide dispersed the Stx6-positive sub-compartment and prevented insulin-responsive re-exocytosis of internalized GLUT4myc, even under conditions not affecting insulin-stimulated signaling towards Akt. Microtubule disruption with nocodazole prevented pre-internalized GLUT4myc from reaching the Stx6-positive perinuclear sub-compartment and from undergoing insulin-responsive exocytosis. Removing nocodazole allowed both parameters to recover, suggesting that the Stx6-positive perinuclear sub-compartment was required for GLUT4 insulin-responsiveness. Accordingly, Stx6 knockdown inhibited by ∼50% the ability of internalized GLUT4myc to undergo insulin-responsive re-exocytosis without altering its overall perinuclear accumulation. We propose that Stx6 defines the insulin-responsive compartment in muscle cells. Our data are consistent with a model where ceramide could cause insulin resistance by altering intracellular GLUT4 sorting.
    Full-text · Article · Apr 2014 · Biology Open
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