Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c

Abstract

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

Powering membrane traffic in endocytosis and recycling

“…2). In adipocytes, the return of the glucose transporter GLUT4 from a cortical tubulovesicular organelle to the cell surface after insulin stimulation is linked to myosin Ic 117 . In epithelial cells, myosins Id and Ia function in endosomal recycling 118 and basolateralapical transport 119 (an epithelium-specific form of a recycling pathway), respectively.…”

EHD2 and the Novel EH Domain Binding Protein EHBP1 Couple Endocytosis to the Actin Cytoskeleton

“…Rabbit anti-actin polyclonal antibody was a gift from Dr. Christine Chaponnier (University of Geneva, Switzerland). Mouse anti-Myc epitope (clone 9E10) monoclonal antibody was from NeoMarkers, Inc. Rabbit anti-HA polyclonal antibody was produced as described (20). Rhodamine-phalloidin, rhodamine-transferrin, goat anti-rabbit-Alexa350-conjugated and goat anti-mouse-Alexa594-conjugated antibodies were from Molecular Probes.…”

“…The EHBP1 protein was detected by immunoblotting with affinity-purified anti-EHBP1 antibody. For Northern blot analysis, total RNA from 3T3-L1 fibroblasts, differentiated adipocytes, and different mouse tissues was isolated as described (20,23). 10 g of total RNA was resolved on a 1.2% denaturing gel and was transferred to a Nytran membrane.…”

“…Electron Microscopy and Immunogold Labeling Analysis-Wholemount electron microscopy analysis was performed as described (20). Double immunogold labeling with different primary antibodies (rabbit anti-EHD2 with goat anti-GLUT4 or, as a negative control, rabbit non-immune IgG with goat non-immune IgG, all antibodies used at 10 g/ml) and specific secondary antibodies conjugated to 6 and 12 nm gold particles were carried out on parafilm sheets at room temperature.…”

Voies de signalisation de l’insuline : mécanismes affectés dans l’insulino-résistance

“…Cbl phosphorylée sur une tyrosine (pY) recrute alors l'adaptateur CrkII et le facteur d'échange nucléotidique C3G qui active la petite GTPase TC10, permettant la polymérisation de l'actine [8]. Le mouvement rapide des vésicules de GLUT4 le long des microfilaments d'actine met en jeu Myo1c, une protéine moteur moléculaire [9]. Enfin, il faut que les vésicules soient correctement ciblées vers la membrane, ce qui nécessite l'intervention de systèmes de reconnaissance vésicules/membrane cible de type v-SNARE/t-SNARE (vesicle or target SNAP (soluble NSF attachment protein) receptor): les vésicules de GLUT4 portent à leur surface des protéines VAMP 2 (vesicle associated membrane protein) 2 capables de reconnaître sur la membrane plasmique les protéines de la famille t-SNARE, syntaxine 4 et SNAP23, permettant ainsi la formation d'un complexe ternaire sous le contrôle de la protéine Munc 18c, puis la fusion avec la membrane et l'apport des transporteurs GLUT4.…”