Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625-635), suggesting that it may play a role distinct from endosomal v-SNAREs (soluble N-ethylmaleimidesensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathione S-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by ϳ35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H 2 O 2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.

Insulin and guanosine-5Ј-O-(3-thiotriphosphate) (GTP␥S) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms.In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (ϳ30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTP␥S response was significantly attenuated (ϳ85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTP␥S-stimulated GLUT4 translocation by ϳ40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTP␥S-stimulated GLUT4 translocation but inhibited the insulin response by ϳ40%. GTP␥S-and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (ϳ50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin Opermeabilized 3T3-L1 adipocytes with GTP␥S caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.

The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.

ADP-ribosylation factors (ARFs) play important roles in both constitutive and regulated membrane trafficking to the plasma membrane in other cells. Here we have examined their role in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These cells express ARF5 and ARF6. ARF5 was identified in the soluble protein and intracellular membranes; in response to insulin some ARF5 was observed to re-locate to the plasma membrane. In contrast, ARF6 was predominantly localized to the plasma membrane and did not redistribute in response to insulin. We employed myristoylated peptides corresponding to the NH 2 termini of ARF5 and ARF6 to investigate the function of these proteins. Myr-ARF6 peptide inhibited insulin-stimulated glucose transport and GLUT4 translocation by ϳ50% in permeabilized adipocytes. In contrast, myr-ARF1 and myr-ARF5 peptides were without effect. Myr-ARF5 peptide also inhibited the insulin stimulated increase in cell surface levels of GLUT1 and transferrin receptors. Myr-ARF6 peptide significantly decreased cell surface levels of these proteins in both basal and insulin-stimulated states, but did not inhibit the fold increase in response to insulin. These data suggest an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and argue for a role for both ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane.Insulin stimulates glucose disposal in peripheral tissues by virtue of the expression of the GLUT4 glucose transporter isoform (1-3). In the absence of insulin, this transporter is intracellularly sequestered within the elements of the endosomal system, the trans Golgi network and a specialized storage compartment (4 -7). Upon insulin stimulation or muscle contraction, GLUT4 is re-distributed from these intracellular locations to the plasma membrane, resulting in a dramatic increase in the rate of glucose entry into these tissues (4 -7). Several studies have suggested that the insulin-stimulated translocation of GLUT4 to the plasma membrane is mechanistically akin to the fusion of small synaptic vesicles with the neuronal plasma membrane (reviewed in Ref.2). This has been supported by the identification of a morphologically similar GLUT4 storage compartment within adipocytes, and by the identification of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) 1 located in GLUT4 vesicles (the v-SNAREs cellubrevin and vesicle-associated membrane protein 2) (8 -11) which bind in a highly specific manner to t-SNAREs located in the adipocyte plasma membrane (Syntaxin 4 and Syndet) (8,10,(12)(13)(14). Vesicle-associated membrane protein 2 has been shown to be the predominant v-SNARE that targets small synaptic vesicles to the pre-synaptic plasma membrane by interacting with the cognate t-SNAREs, syntaxin1, and synaptosome-associated protein of 25 kDa (SNAP-25) found on the target membrane (15). Recent studies implicating vesicle-associated membrane protein 2 in insulinstimulated GLUT4 translocation further strengthen the me...

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-1-ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.