Characterization of Insulin-Responsive GLUT4 Storage Vesicles Isolated from 3T3-L1 Adipocytes

Abstract

Insulin regulates glucose transport in muscle and adipose tissue by triggering the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.

Perinuclear Localization and Insulin Responsiveness of GLUT4 Requires Cytoskeletal Integrity in 3T3-L1 Adipocytes

“…The great advantage of the present technique is that it allows for the first time the purification of membranes that can be observed to be insulin-sensitive without the use of reagents that can contaminate the preparations such as immunoglobulin polypeptides. This contrasts with other methods where membranes enriched with GLUT4 are obtained but do not seem to be reduced in abundance when cells are treated with insulin (16,67). The GLUT4-containing membranes purified by the method described here thus appear to be particularly excellent for the application of microsequencing techniques to identify resident proteins.…”

“…4), and this receptor is thought to be present mostly in recycling endosomes that apparently show only modest sensitivity to insulin (12,16). However, this problem also applies to GLUT4-containing membrane preparations obtained by immunoadsorption with anti-GLUT4 antibodies because GLUT4 is also present in the recycling endosome compartment (12,(15)(16)(17). Furthermore, immunoelectron microscopy shows that virtually all of the membrane vesicles obtained by the method described here do actually contain GLUT4 (Fig.…”

“…These latter proteins are present at the cell surface in higher abundance than GLUT4 in unstimulated cells and move through a rapid constitutive endosomal recycling pathway (13, 14). Complicating our understanding of the interrelationships between these trafficking systems are findings suggesting that GLUT4 also partially co-localizes with endosomal membranes containing the transferrin receptor and GLUT1 (12,15,16). Because normal glucose homeostasis in humans is dependent upon the dynamics of GLUT4 membrane trafficking, intensive efforts have been directed at understanding the basis for the unique recycling characteristics and regulation of GLUT4.…”

NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages

“…This distribution is similar to that of glucose transporter-4 (GLUT-4), which in muscle and adipose cells traffics in an insulin-regulated manner between the cell surface and exocytic storage vesicles, but it is also partially contained in early (recycling) endosomes (50,51).…”

Insulin Recruits GLUT4 from Distinct Compartments via Distinct Traffic Pathways with Differential Microtubule Dependence in Rat Adipocytes

“…1). Additionally, more recent works have suggested that insulin recruits GLUT4 to the plasma membrane from the postendosomal vesicle-associated membrane protein (VAMP) 1 -2-positive compartment as well as from the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-positive compartment that is dynamically shuttling between endosomes and the trans-Golgi network (TGN) (6,7). While such a diversity of the subcellular localization of GLUT4 would hamper our understanding of the insulin-regulated trafficking of GLUT4, it implies that the transit of GLUT4 to and from such distinct subcellular compartments would be regulated by different signals and trafficking machinery (e.g.…”