Lipid rafts are required for GLUT4 internalization in adipose cells

Abstract

We have assessed the role of lipid rafts on GLUT4 traffic in adipose cells. High GLUT4 levels were detected in caveolae from adipocytes by two approaches, the mechanical isolation of purified caveolae from plasma membrane lawns and the immunogold analysis of plasma membrane lawns followed by freeze-drying. The role of lipid rafts in GLUT4 trafficking was studied by adding nystatin or filipin at concentrations that specifically disrupt caveolae morphology and inhibit caveolae function without altering clathrin-mediated endocytosis. These caveolae inhibitors did not affect the insulin-stimulated glucose transport. However, they blocked both the GLUT4 internalization and the down-regulation of glucose transport triggered by insulin removal in 3T3-L1 adipocytes. Our data indicate that lipid rafts are crucial for GLUT4 internalization after insulin removal. Given that high levels of GLUT4 were detected in caveolae from insulin-treated adipose cells, this transporter may be internalized from caveolae or caveolae may operate as an obligatory transition station before internalization.caveolae ͉ nystatin ͉ filipin L ipid rafts are lateral assemblies of sphingolipids and cholesterol that form a separate liquid-ordered phase in the liquid-disordered matrix of the lipid bilayer (1). Rafts function as platforms to which distinct classes of proteins are associated, such as glycosylphosphatidylinositol-anchored proteins, transmembrane proteins, and diacylated proteins. Caveolae are a specialized type of raft arranged in the form of f lask-shaped invaginations. They are abundant in adipose cells, where they account for 20% of the plasma membrane surface area (2). Caveolae participate in receptor-mediated potocytosis, signal transduction, transcytosis, endocytosis independent of clathrin, and bacterial entry (3, 4). They are dynamic structures (5, 6), they can bud from the plasma membrane (PM) and their internalization is regulated by the general molecular transport machinery of vesicle budding, fission, docking, and fusion. Purified endothelial caveolae hold the elements needed for intracellular vesicular transport including members of the VAMP, GTPase, and annexin families, along with SNAPs and NSF (7). Moreover, they can undergo fission in vitro, which is mediated by dynamin (8).Insulin stimulates glucose uptake by recruiting GLUT4 to the surface. After insulin removal, GLUT4 is rapidly internalized from the plasma membrane and is effectively sequestered within the cell. It has been suggested that GLUT4 is internalized through a clathrin-mediated pathway and disruption of clathrincoated vesicles results in the accumulation of GLUT4 at the cell surface in adipocytes (9, 10). Morphological analyses in 3T3-L1 adipocytes have revealed the association between GLUT4 and clathrin-coated pits (11) and partial overlap between the itinerary of GLUT4 and that of the transferrin receptor (12). However, in isolated rat adipocytes GLUT4 is associated with smooth, non-clathrin-coated plasma membrane invaginations reminiscent of caveolae (13...

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

“…A number of transporters appear to use lipid rafts and lipid raft localized t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and v-SNARE proteins as a means to direct their trafficking to the correct cellular compartment (29,30). In a study of GLUT4 trafficking in 3T3-L1 adipocytes, SNAP23 and syntaxin 4 were associated with plasma membrane rafts while VAMP2 was primarily in intracellular lipid raft membranes.…”

Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells

“…Caveolar and noncaveolar rafts together with their constituent proteins coisolate by isopycnic floatation as low-buoyant density membrane fractions (10) obtained after cold detergent extraction (8,12) or detergent-free extraction and sonication (13)(14)(15), and both are disrupted by cholesterol depletion (16,17), currently achieved by cyclodextrins (18). Cholesterol depletion experiments have suggested that raft integrity is needed for protein trafficking along certain steps of the exocytic (12,19) and endocytic (20)(21)(22)(23) pathways as well as for signal transduction (10,17).…”

Compartmentalization of the Exocyst Complex in Lipid Rafts Controls Glut4 Vesicle Tethering

“…Components of insulin signaling are constitutively localized in lipid rafts, including some or all of the insulin receptor (Gustavsson et al, 1999;Kimura et al, 2002), flotillin (Baumann et al, 2000;Liu et al, 2005), and TC10 , which is specifically activated by insulin in these microdomains due to the recruitment of the exchange factor C3G via the tyrosine phosphorylation of c-Cbl and Cbl-b (Liu et al, 2003). Disruption of lipid rafts with cholesterol-extracting drugs such as beta-cyclodextrin or by overexpression of dominant-negative forms of caveolin block the actions of insulin, without disrupting other cellular functions (Gustavsson et al, 1999;Parpal et al, 2001;Ros-Baro et al, 2001;Shigematsu et al, 2003). Moreover, introduction of a modification of TC10 that prevents lipid raft association blocks the activation of the G protein by insulin, as well as the stimulation of glucose uptake in fat cells .…”