In adipocytes, insulin triggers the redistribution of Glut4 from intracellular compartments to the plasma membrane. Two models have been proposed to explain the effect of insulin on Glut4 localization. In the first, termed dynamic exchange, Glut4 continually cycles between the plasma membrane and intracellular compartments in basal cells, and the major effect of insulin is through changes in the exocytic and endocytic rate constants, k ex and k en . In the second model, termed static retention, Glut4 is packaged in specialized storage vesicles (GSVs) in basal cells and does not traffic through the plasma membrane or endosomes. Insulin triggers GSV exocytosis, increasing the amount of Glut4 in the actively cycling pool. Using a flow cytometrybased assay, we found that Glut4 is regulated by both static and dynamic retention mechanisms. In basal cells, 75-80% of the Glut4 is packaged in noncycling GSVs. Insulin increased the amount of Glut4 in the actively cycling pool 4 -5-fold. Insulin also increased k ex in the cycling pool 3-fold. After insulin withdrawal, Glut4 is rapidly cleared from the plasma membrane (t1 ⁄ 2 of 20 min) by rapid adjustments in k ex and k en and recycled into static compartments. Complete recovery of the static pool required more than 3 h, however. We conclude that in fully differentiated confluent adipocytes, both the dynamic and static retention mechanisms are important for the regulation of plasma membrane Glut4 content. However, cell culture conditions affect Glut4 trafficking. For example, replating after differentiation inhibited the static retention of Glut4, which may explain differences in previous reports.

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulinmimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k ex ). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k ex ), endocytosis (k en ), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k ex or k en . Insulin increased k ex in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k ex and release from retention in control cells but only k ex in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulinregulated step.Glucose uptake in muscle and adipose tissue is rate-limited by the number of facilitative glucose transport proteins present in the plasma membrane (1). In adipocytes, the majority of glucose uptake occurs via the insulin-responsive glucose transporter 4 (Glut4). Insulin regulates glucose uptake by changing the steady state distribution of Glut4 from predominantly an intracellular, perinuclear localization to the plasma membrane, a process known as Glut4 translocation. Under basal conditions, less than 5% of the total Glut4 is found at the PM, 2 whereas after insulin stimulation 30 -50% is localized to the PM. In basal cells, most of the Glut4 is sequestered into a noncycling/slowly cycling pool in specialized Glut4 storage vesicles (GSVs). A small fraction of the Glut4 is found in a more rapidly cycling pool that is distributed between the PM and endosomal compartments. Insulin increases cell surface Glut4 by two main mechanisms as follows: insulin releases Glut4 from retention in the sequestered GSVs into the actively cycling pool (2-5), and insulin also increases the rate constant of exocytosis, resulting in a further increase in surface Glut4 (2-6).Although the trafficking pathways followed by Glut4 are well characterized, the mechanism of regulation of Glut4 trafficking by ...

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and ␣ 2 -macroglobulin (␣-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k ex ) for both Glut4 and Tf. However, the k ex of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k en ) of Glut4 was also five times slower than Tf. Insulin did not affect the k en of either protein. In basal cells, the k en for ␣-2-M/ LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k en for ␣-2-M/LRP-1 by 30%. In contrast, the k ex for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k en of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k en can be entirely accounted for by the well established increase in Glut4 k ex .

Background: Cell surface levels of glucose transporter Glut4 are tightly controlled in adipocytes. Results: The effects of insulin and differentiation on the trafficking kinetics of Glut4, the transferrin receptor, and LRP1 were measured to identify regulatory steps. Conclusion: Six independent steps determine cell surface Glut4; insulin stimulates three of these. Significance: These results provide a framework for functionally mapping treatments/proteins that affect Glut4 translocation.

Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptor-related protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin.