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...
Aims/hypothesis. Vascular adhesion protein-1 (VAP-1), which is identical to semicarbazide-sensitive amine oxidase (SSAO), is a dual-function membrane protein with adhesion properties and amine oxidase activity. A soluble form of VAP-1 is found in serum, where concentrations are enhanced in diabetes and obesity. In vitro, soluble VAP-1 enhances lymphocyte adhesion to endothelial cells, thus possibly participating in the enhanced lymphocyte adhesion capacity that is implicated in the cardiovascular complications associated with diabetes or obesity. In both, the tissue origin of the soluble VAP-1/SSAO is unknown. We examined whether adipose tissue, which has abundant expression of VAP-1/SSAO, is a source of soluble VAP-1. Methods. We detected VAP-1/SSAO in plasma of diabetic animals, with or without VAP-1 immunoprecipitation, and in culture medium from 3T3-L1 adipocytes and human adipose tissue explants. VAP-1 protein glycosylation was measured.Results. Diabetic and obese animals have increased plasma SSAO activity associated with VAP-1 protein.We also found that 3T3-L1 adipocytes and human adipose tissue explants release a soluble form of VAP-1/SSAO, which derives from the membrane. The release of soluble VAP-1 was enhanced by exposure of murine and human adipocytes to TNF-α and blocked by batimastat, a metalloprotease inhibitor. Partial ablation of adipose tissue reduced plasma SSAO activity in normal and diabetic rats.
Plasma level of the protein VAP-1/SSAO (Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFalpha in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the VAP-1/SSAO membrane form. While TNFalpha produced a decrease on VAP-1/SSAO membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma VAP-1/SSAO levels observed in diabetes.
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