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. 2000 Dec;11(12):4079-91.
doi: 10.1091/mbc.11.12.4079.

Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes

G Ramm  1 , J W Slot, D E James, W Stoorvogel
Affiliations
Free PMC article

Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes

G Ramm et al. Mol Biol Cell. 2000 Dec.
Free PMC article

Abstract

Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-D resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system.

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Figures

Figure 1
Figure 1
Isolation of whole mount rims from isolated rat adipocytes. (A) Basal or insulin stimulated adipocytes were sandwiched between a poly-L-lysine-coated grid and nitrocellulose membrane. (B) Cells were mechanical disrupted by pulling the grid from the nitrocellulose membrane in a cytoskeleton stabilizing buffer in the cold and fixed immediately. (C) This resulted in patches of the thin cytoplasmic rims on the grid, which were processed further for IEM.
Figure 2
Figure 2
A) Overview of a whole mount adipocyte rim obtained as described in Figure 1. The nucleus (n), small round structures (vacuoles or mitochondria, for example, see arrowheads), and cytoskeletal elements (for example see arrows) were readily discerned by morphology. Bar, 2 μm. Microtubules (B), intermediate filaments (C) or actin-filaments (D) were labeled with anti-β-tubulin, anti-vimentin, or anti-β-actin antibodies, respectively followed by 10 nm colloidal gold coupled to protein A. In B-D the arrows point to single or clusters of gold particles labeling the distinct cytoskeletal elements, the microtubules in (B) are completely covered by gold particles. Bars, 500 nm.
Figure 3
Figure 3
GLUT4 localization in whole mount preparations of isolated adipocytes. Cells were incubated for 20 min without (A-C) or with (D, E) 700 nM insulin and processed for whole mount electron microscopy. Whole mount rims were labeled for GLUT4 (10 nm gold particles). In basal cells GLUT4 is found in vesicles (A, large arrowheads), tubules (A and B, arrows), and vacuoles (A and C, v). After stimulation with insulin (D and E), GLUT4 appears at the plasma membrane (for example, see small arrowheads). GLUT4 is also shown in vesicles (large arrowheads) and tubules (arrows). Bars, 100 nm.
Figure 4
Figure 4
Characterization of intracellular GLUT4 compartments in rat adipocytes. Whole mount preparations of basal (A-F) or insulin stimulated (G, H) adipocytes were obtained as in Figure 2 and double labeled for lgp-120 (A), IRAP (B), EEA1 (C), CD-MPR (D), or VAMP2 (E-H) (10-nm gold particles) and GLUT4 (15-nm gold particles). No significant overlap was observed between lgp-120 and GLUT4 on vacuoles (A). IRAP was highly colocalized with GLUT4 in both vesicles (B) and tubules (B, inset). EEA1 was found on vacuoles, but not on small GLUT4 positive vesicles (C). CD-MPR colocalized with GLUT4 in small vesicles, vacuoles, and tubules, however some vesicles in basal cells did not contain CD-MPR (D). VAMP2 was found on intracellular compartments containing GLUT4, whereas GLUT4 was additionally found in vesicles free of VAMP2 (E, F). After stimulation with insulin both VAMP2-positive and VAMP2-negative GLUT4-vesicles were still observed (G, H). Arrowheads indicate vesicles; v, vacuoles; arrows, tubules; m, mitochondria; n, nucleus. Bar, 200 nm.
Figure 5
Figure 5
Insulin recruits GLUT4 from small vesicles. Whole mount adipocyte rims were labeled for GLUT4 as shown in Figure 3. Random parts of the rims were analyzed for the distribution of GLUT4. Three different intracellular structures were defined by morphology: vesicles (between 60 and 100 nm in diameter), vacuoles (larger spherical structures), and tubules. Plasma membrane-associated GLUT4 was represented on the sheet by gold particles that were not associated with any obvious structure. Gold particles associated with structures that could not clearly be assigned to one of the other structures were counted as nondefined. For each condition, in total 1000 gold particles were counted 9 times on samples from three independent experiments (± SEM). In basal cells, GLUT4 is present in small vesicles (61%), tubules (25%), and to a minor extent, in vacuoles (6%). Insulin caused a 4.4-fold increase of GLUT4 at the plasma membrane (p < 0.001) and to a minor extent in vacuoles (1.9-fold, p < 0.01), parallel to a 37% decrease of GLUT4-label in vesicles (p < 0.001).
Figure 6
Figure 6
Role of VAMP2 in GLUT4 translocation. Vesicles from adipocyte rims double labeled for VAMP2 and GLUT4 as shown in Figure 4, E-H were used for quantitative analysis. Counting was done in a random manner, similar to Figure 5, but now only vesicles containing ≥ 3 gold particles in total were considered. For 100 individual vesicles the number of gold particles representing VAMP2 and GLUT4 were counted (For each condition 6 × 100 vesicles were counted from two independent experiments). The distributions of all 600 counted vesicles in basal (A) or insulin treated (B) rat adipocytes are shown. Each bar represents the number of vesicles having a certain labeling characteristic. For example, the highest bar in (A) stands for 91 vesicles, each labeled with three gold particles for GLUT4 and no gold particles for VAMP2. No differences in the overall distribution of vesicles in basal (A) and insulin-stimulated (B) adipocytes were observed. Upon insulin stimulation, neither the average number of GLUT4 gold particles in VAMP2-positive vesicles (C) nor the average number of VAMP2 in GLUT4 vesicles (D) was changed. Clearly two distinct peaks representing VAMP2-negative and VAMP2-positive GLUT4-vesicles can be discerned (D). Insulin did not significantly change the percentage of VAMP2-negative GLUT4-vesicles (E). In contrast to Table 1, these quantifications include GLUT4-negative vesicles, explaining the relative lower percentage of VAMP2-negative vesicles in basal cells. The ratio of GLUT4-gold particles over VAMP2-gold particles in GLUT4-positive/VAMP2-positive vesicles remained unchanged after insulin treatment (F).
Figure 7
Figure 7
Insulin recruits GLUT4 mainly from a CD-MPR-negative GLUT4-vesicle pool, but also from CD-MPR-positive vesicles. Preparation, as shown in Figure 4D, was quantified similar to that shown in Figure 6. Compared with basal adipocytes (A) and after insulin stimulation (B), a clear change in the distribution of vesicles is visible. The average number of GLUT4 gold particles in CD-MPR-positive vesicles decreased upon insulin stimulation (C), and GLUT4 disappeared from the CD-MPR-negative vesicle pool (D). The number of vesicles containing only GLUT4 and no CD-MPR decreased upon insulin treatment (E, p < 0.005). Insulin caused a decrease of the ratio of GLUT4-gold particles over CD-MPR-gold particles in GLUT4-positive/CD-MPR-positive vesicles (F, p < 0.01).
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