Akt Activation Is Required at a Late Stage of Insulin-Induced GLUT4 Translocation to the Plasma Membrane
Ellen M. van Dam,
Roland Govers and
David E. James
Garvan Institute of Medical Research, St. Vincents Hospital, Darlinghurst, 2010 New South Wales, Australia
Address all correspondence and requests for reprints to: David E. James, Garvan Institute of Medical Research, St. Vincents Hospital, 384 Victoria Street, Darlinghurst, 2010 New South Wales, Australia. E-mail: D.James{at}garvan.org.au.
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ABSTRACT
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Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortexthis process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PMthis process may be the major Akt-dependent step in the insulin regulation of glucose transport.
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INTRODUCTION
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INSULIN STIMULATES GLUCOSE uptake in muscle and fat cells by triggering translocation of the glucose transporter GLUT4 from an intracellular compartment to the cell surface (1). In the nonstimulated state, GLUT4 is present in various intracellular compartments, including the endosomal system, the trans-Golgi network (TGN), and cytoplasmic tubulovesicular elements, suggesting a complex intracellular trafficking itinerary (2, 3). Previous studies have indicated a role for endosomes and the TGN in GLUT4 trafficking (2, 4, 5). Upon insulin stimulation, GLUT4 is recruited to the plasma membrane (PM) from vesicle-associated membrane protein-2-carrying vesicles, the so-called GLUT4 storage vesicles (GSVs), as well as from the dynamic endosomal-TGN system (6). A major question in this field is how does the insulin signaling pathway converge these trafficking steps to orchestrate the translocation mechanism. Binding of insulin to its receptor (IR) activates its intrinsic protein tyrosine kinase activity, resulting in autophosphorylation and subsequent phosphorylation of several interacting proteins, such as the IR substrate (IRS) proteins. The tyrosyl phosphorylated IRS proteins, IRS-1 and IRS-2, recruit phosphatidylinositol 3 kinase (PI-3K), resulting in increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels at the PM. This leads to the recruitment of phosphoinositide-dependent protein kinase 1 (PDK1) and Akt, also named protein kinase B, from the cytosol to the cell surface. This is accompanied by phosphorylation and activation of Akt, which has been shown to play an essential role in insulin-stimulated GLUT4 translocation (7, 8, 9, 10, 11, 12). However, the precise mechanisms of Akt action on GLUT4 translocation remain to be determined.
Previously, it was shown that insulin-induced GLUT4 translocation to the PM is blocked at 19 C (13). Intracellular transport between several organelles has been reported to be temperature-sensitive. For example, endosome-lysosome fusion is blocked at lower temperatures (14), as is transport out of the TGN (15, 16, 17). Because GLUT4 traverses many different organelles in both the basal and insulin-stimulated condition, pinpointing the nature of the defect associated with GLUT4 trafficking at 19 C could provide clues concerning the mechanism of GLUT4 trafficking.
In the present study, we have found that upon incubation of adipocytes at 19 C there is an insulin-dependent accumulation of GLUT4 vesicles just beneath the cell surface, whereas insertion of GLUT4 vesicles into the membrane is blocked at this reduced temperature. Strikingly, insulin-stimulated Akt phosphorylation at both S473 and T308 was inhibited at 19 C. These data suggest that the trafficking of GLUT4 to the cell surface in response to insulin is comprised of two separate regulatory steps: one involving movement of vesicles out to the cortex of the cell, and the second involving docking and/or fusion of GLUT4-containing vesicles with the cell surface. It would appear to be the latter step that is preferentially regulated by Akt.
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RESULTS
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We have previously shown that insulin-stimulated GLUT4 translocation to the PM is blocked in 3T3-L1 adipocytes preincubated at 19 C (13). To study this translocation block in detail, we made use of a 96-well plate quantitative translocation assay recently developed in our laboratory (18). For this study, hemagglutinin (HA)-tagged GLUT4 (HA-GLUT4) was expressed in adipocytes. Cells were incubated at either 37 C or 19 C, and insulin was added for the indicated times. The amount of HA-GLUT4 at the PM was determined and expressed as percentage of total cellular HA-GLUT4 (Fig. 1
). Insulin caused a rapid and robust 10-fold increase in cell surface levels of HA-GLUT4 at 37 C. Consistent with our previous study (13), we observed a slight (3-fold) increase in cell surface levels of GLUT4 when cells were incubated at 19 C. However, insulin-stimulated GLUT4 translocation was completely inhibited at this temperature (Fig. 1
). Therefore, the exocytosis of the insulin-regulatable pool is likely to be blocked at 19 C.

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Fig. 1. Insulin-Induced GLUT4 Translocation to the PM Is Impaired at 19 C
3T3-L1 adipocytes expressing HA-GLUT4 were incubated for 2 h at 37 or 19 C and stimulated for the indicated times with 200 nM insulin. Amount of HA-GLUT4 at the PM was determined by fluorescence immunolabeling of nonpermeabilized cells and expressed as percentage of total cellular HA-GLUT4, determined by labeling of permeabilized cells.
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We next set out to establish the time course for these temperature-induced changes in GLUT4 trafficking. Cells expressing HA-GLUT4 were serum starved for 2 h at 37 C, after which they were shifted to 19 C in the continuous absence of insulin for indicated times (Fig. 2A
). Intriguingly, the increase in cell surface GLUT4 was achieved within 15 min at 19 C, after which there was no further increase in GLUT4 at the PM even in cells incubated for prolonged periods at 19 C. We next examined the time course for the block in insulin action at 19 C. We used a similar protocol to that described above where HA-GLUT4-expressing cells were incubated for different times at 19 C before addition of insulin for 20 min. As shown in Fig. 2B
, the block in insulin action was rapid, occurring within 20 min after switching to 19 C (time point 0 and 15 min). In our previous studies, we observed that despite the block in insulin-stimulated GLUT4 translocation to the cell surface, we still observed an insulin-dependent redistribution of GLUT4 within intracellular membranes at 19 C (13). To examine this further, we next examined the effect of reduced temperature on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes by immunofluorescence microscopy. Three separate intracellular pools of GLUT4 in adipocytes can be identified based on morphology (6). This includes a pool that overlaps with endosomal markers such as cellubrevin, a pool that overlaps with TGN markers including Syntaxin 16, and a separate more diffuse vesicular pool that only overlaps with insulin-responsive aminopeptidase and vesicle-associated membrane protein-2. Labeling of GLUT4 in cells that have been incubated at 37 C can be seen in Fig. 3
where GLUT4 overlaps with Syntaxin 16 in the perinuclear area and where a considerable amount of GLUT4 is present in the periphery of the cell. Intriguingly, in cells incubated at 19 C, GLUT4 moved out of the perinuclear area, whereas TGN markers like Syntaxin 16 (Fig. 3
) and cation-independent mannose 6-phosphate receptor (data not shown) retained their perinuclear localization. Hence, it is possible that at 19 C retrograde transport of GLUT4 from endosomes to the TGN is blocked, and this may account for the rapid increase in cell surface levels of HA-GLUT4 in cells incubated at the reduced temperature. This interpretation is consistent with our previous proposal that GLUT4 rapidly recycles between endosomes and the TGN in the basal state, and this cycle may serve to sequester GLUT4 within the cell. The fact that only a finite pool of GLUT4 exchanges with the cell surface at 19 C suggests that the insulin-responsive pool does not communicate with the endosomal pool in the absence of insulin, and indeed this is consistent with our recently published data (18, 19). Strikingly, stimulation with insulin at 19 C resulted in an accumulation of GLUT4 beneath the cell surface (Fig. 3
). Because HA-GLUT4 does not fuse with the PM under these conditions (Fig. 1
), these data suggest that at 19 C the effect of insulin to stimulate the movement of GLUT4 vesicles to the periphery of the cell is maintained, whereas the engagement of vesicles with the cell surface is blocked at this reduced temperature. One possibility to account for these data is that reduced temperature may alter the composition of the PM or the underlying cytoskeleton, resulting in reduced docking and/or fusion of all exocytic/recycling vesicles. To test this, we measured recycling of transferrin (Tf), a prototype recycling protein, in the presence or absence of insulin in adipocytes overexpressing the human Tf receptor (hTfR) at either 37 C or 19 C. At 37 C, Tf was efficiently recycled into the medium (Fig. 4
). Importantly, there was still considerable recycling of Tf at 19 C, although the rate of recycling was reduced compared with 37 C, as expected. These results argue against a general block in fusion of recycling vesicles with the PM at 19 C. As previously described (20, 21, 22), insulin increased the rate of Tf recycling at 37 C. Interestingly, although we observed a significant increase in Tf recycling with insulin at 37 C, there was only a slight effect of insulin at 19 C. However, in view of the relatively small effect of insulin on Tf recycling even at 37 C, we cannot exclude the possibility that a partial insulin effect on Tf recycling may still be present at 19 C.

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Fig. 2. 19 C Incubation Rapidly Alters GLUT4 Trafficking to the PM
A, 3T3-L1 adipocytes expressing HA-GLUT4 were incubated for the indicated times at 37 C or 19 C in the absence of insulin, and the amount of GLUT4 at the PM was determined. B, HA-GLUT4-expressing adipocytes were incubated for the indicated periods of time at 37 C or 19 C and then stimulated for 20 min with 200 nM insulin; for both 20-min time points, cells were stimulated with insulin at 37 C. Cell surface GLUT4 levels were determined by immunolabeling.
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Fig. 3. 19 C Incubation Impairs Retrograde Transport of GLUT4 to the TGN
3T3-L1 adipocytes were incubated for 2 h at 37 C or 19 C in the absence of insulin. Cells were then either fixed, or stimulated with insulin for 20 min before fixation. Cells were immunolabeled for GLUT4 and Syntaxin 16.
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Fig. 4. 19 C Incubation Slows Down But Does Not Inhibit Tf Recycling
Serum-starved 3T3-L1 adipocytes expressing the hTfR were loaded with 125I-Tf for 2 h at 16 C. Cell surface associated 125I-Tf was removed at 4 C, after which recycling of endocytosed 125I-Tf was measured at either 19 C (circles) or 37 C (squares) in the presence (filled symbols) or absence (open symbols) of 200 nM insulin. Intracellular 125I-Tf is plotted as a percentage of total endocytosed 125I-Tf (n = 2; mean ± SD).
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To resolve the molecular basis for the impaired engagement of GLUT4 vesicles with the cell surface, we next examined elements of the signal transduction pathway. Considerable evidence points to an important role for PI-3K and its downstream target Akt in GLUT4 trafficking (1, 23, 24, 25). We therefore measured Akt activity indirectly by determining the phosphorylation of Akt on S473 and on T308 (Fig. 5
). At 37 C, insulin rapidly triggered Akt phosphorylation on S473 and T308, consistent with many previous studies. Strikingly, insulin-stimulated Akt phosphorylation at S473 and T308 was almost completely blocked at 19 C. To determine the locus of the signaling block at 19 C, we next examined elements of the insulin-signaling pathway upstream of Akt. As shown in Fig. 5
, there was robust insulin-stimulated tyrosine phosphorylation of IR and IRS-1 at 19 C, similar to that observed at 37 C. Because phosphorylated IRS-1 recruits PI-3K, we next investigated whether PI-3K is activated at 19 C by indirectly measuring the accumulation of PIP3 at the PM in intact cells. To accomplish this, we introduced the PH domain of ARNO (EGFP-PH/ARNO), which binds with high specificity to PIP3 (26, 27), into adipocytes and examined its localization upon insulin stimulation at both 37 C and 19 C, similar to that described previously (28). In the basal situation, EGFP-PH/ARNO was mainly present in the cytosol (Fig. 6A
), consistent with previous studies showing low levels of PM PIP3 under these conditions (29). As expected, at 37 C insulin caused a redistribution of EGFP-PH/ARNO to the cell surface, consistent with increased PI-3K activity (Fig. 6B
). Interestingly, a similar insulin-dependent recruitment of EGFP-PH/ARNO to the cell surface was observed in cells treated with insulin at 19 C (Fig. 6C
). These data indicate that the insulin signaling block at 19 C is located between PIP3 production and Akt activation and likely involves either activation of PDK1 or PDK2 or the recruitment of Akt to the cell surface. These findings are of considerable interest, raising two important points about the insulin regulation of GLUT4 translocation. First, they suggest that insulin activates a signaling pathway that does not involve Akt to trigger the movement of GLUT4 vesicles out to the cell cortex. Second, these data implicate an important role for Akt at a late stage in the translocation mechanism, probably involving the docking and/or fusion of GLUT4 with the cell surface.

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Fig. 5. Akt Phosphorylation Is Severely Impaired at 19 C 3T3-L1 adipocytes were incubated for 2 h at 37 C, followed by an incubation in the presence of absence of insulin for the indicated times, followed by a shift to 19 C in the presence of insulin for the indicated times. Cells were homogenized, and an aliquot of the postnuclear supernatant was subjected to SDS-PAGE and immunoblotting using anti-Akt, antiphospho-Akt T308, antiphospho-Akt S473, or antiphosphotyrosine antibodies.
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Fig. 6. PI-3K Activity Is Not Impaired at 19 C
3T3-L1 adipocytes expressing EGFP-PH/ARNO were incubated for 2 h at 37 C in the absence of serum (A); incubated for 2 h in the absence of serum at 37 C followed by a 30-min incubation in the presence of 200 nM insulin at 37 C (B); or incubated for 2 h at 37 C in the absence of serum followed by a 30-min incubation in the presence of 200 nM insulin at 19 C (C). Cells were fixed and immunolabeled for green fluorescent protein to enhance the signal. A representative cell is shown.
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We and others have performed detailed kinetic analysis of GLUT4 trafficking in 3T3-L1 adipocytes and have made the observation that there is a significant difference in the half-time for GLUT4 exocytosis in the immediate response phase after insulin addition, compared with that observed under steady-state conditions (18, 19, 30). One interpretation of these data is that the molecular mechanism(s) governing these two different processes may be quite distinct. To determine whether Akt plays a role in both processes, we next incubated cells with insulin for 30 min at 37 C to establish steady-state redistribution of GLUT4 and then moved the cells to 19 C in the continuous presence of insulin. As observed in Fig. 5
(preinsulin), there was a rapid decline in Akt phosphorylation such that after 30 min at 19 C phosphorylation of S473 and T308 had returned to basal levels. In contrast, cell surface levels of GLUT4 did not begin to decline until after 60 min of incubation at 19 C, and they had returned to basal levels by 120 min (Fig. 7
). These data raise the intriguing possibility that Akt is only involved in the initial discharge mechanism of GLUT4 and that in the continuous presence of insulin, GLUT4 may continue to cycle via the PM in an Akt-independent manner. Additional studies are, however, required to validate this observation because the reversal studies described here are somewhat complicated by the effects of reduced temperature on GLUT4 trafficking per se. For example, the delayed reversal of GLUT4 trafficking may be due to slowed GLUT4 endocytosis at 19 C. In addition, these data clearly establish that the inability to activate Akt at 19 C does not simply reflect a kinetic block but rather a modification in either the upstream kinase(s) or regulatory phosphatases. In support of a role of reduced temperature on the kinase(s) regulating Akt, we have found that Calyculin A, an inhibitor of protein phosphatase types 1 and 2A, did not increase Akt phosphorylation at 19 C in the presence of insulin (Fig. 8B
). To ensure that Calyculin A inhibits Akt dephosphorylation in adipocytes, we treated adipocytes with insulin at 37 C before shifting cells to 19 C in the continuous presence of insulin and in the presence or absence of Calyculin A. As shown in Fig. 8C
, Akt remains phosphorylated only in the presence of Calyculin A, indicating that the phosphatase inhibitor is functional.

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Fig. 7. GLUT4 Remains at the PM upon Priming with Insulin at 37 C before a 19 C Incubation
3T3-L1 adipocytes expressing HA-GLUT4 were incubated for 2 h, followed by an incubation in the presence or absence of 200 nM insulin for 20 min. Cells were transferred to 19 C in the presence of 200 nM insulin for indicated times. The amount of HA-GLUT4 at the PM was determined by fluorescence immunolabeling of nonpermeabilized cells and expressed as a percentage of total cellular HA-GLUT4, determined by labeling of permeabilized cells.
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Fig. 8. Calyculin A and Wortmannin Have No Effect on Insulin-Stimulated Akt Phosphorylation at 19 C
A, Serum-starved 3T3-L1 adipocytes were incubated for 2 h at 37 C and stimulated with 200 nM insulin for the indicated times in the presence or absence of 100 nM Calyculin A or 100 nM wortmannin. B, Serum-starved 3T3-L1 adipocytes were incubated for 2 h at 37 C, followed by an incubation at 19 C for the indicated times in the presence of 200 nM insulin and in the presence or absence of 100 nM Calyculin A or 100 nM wortmannin. C, 3T3-L1 adipocytes were incubated for 2 h at 37 C and stimulated with 200 nM insulin for 30 min. Cells were then incubated at 19 C in the continuous presence of insulin and in the presence or absence of 100 nM Calyculin A for the indicated times (AC). Cells were homogenized, and an aliquot of the postnuclear supernatant was subjected to SDS-PAGE and immunoblotting using either anti-Akt or antiphospho-Akt S473 antibodies.
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Because we observed an effect of insulin on the intracellular localization of GLUT4, we next tested whether insulin addition at 19 C, before shifting cells to 37 C, would enhance the rate of GLUT4 translocation to the cell surface. We therefore incubated cells at 19 C for 2, h followed by an incubation at 19 C in the presence or absence of insulin for 20 min, after which cells were shifted to 37 C in the presence of insulin and 2-deoxy-[3H]glucose (2-DOG) uptake was measured as an index of GLUT4 translocation. Figure 9A
shows that preincubation with insulin at 19 C, before shifting the cells in the continuous presence of insulin to 37 C, enhanced the rate of glucose uptake at 37 C. This priming effect was not due to an increased rate of Akt phosphorylation under these conditions, as shown in Fig. 9B
. The time course of insulin-induced Akt phosphorylation at 37 C was indistinguishable from that observed after adding insulin at 19 C before shifting to 37 C in the presence of insulin. Collectively, these data suggest that Akt phosphorylation is required at a late stage of GLUT4 translocation to the cell surface.

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Fig. 9. Priming Adipocytes with Insulin at 19 C Enhances Glucose Uptake, But Not Akt Phosphorylation at 37 C
A, Serum-starved 3T3-L1 adipocytes were incubated for 2 h at 19 C, followed by an incubation in the presence (preinsulin) or absence (insulin) of insulin for 20 min. Cells were then transferred to 37 C in the presence of insulin for the indicated times, and 2-DOG uptake was measured. Glucose uptake is expressed as percentage of maximum uptake. Means and SEM of four different experiments are shown. *, P < 0.05. B, 3T3-L1 adipocytes were either incubated for 2 h at 19 C or incubated for 2 h at 19 C followed by a stimulation of 200 nM insulin for 20 min at 19 C. Cells were then transferred to 37 C in the presence of 200 nM insulin for the indicated times. Cells were homogenized, and an aliquot of the postnuclear supernatant was subjected to SDS-PAGE and immunoblotting using either anti-Akt or antiphospho-Akt S473 antibodies.
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DISCUSSION
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In this study, we have used a low-temperature block to unveil two separate insulin-regulated steps in the GLUT4 translocation process. One involves the transport of GLUT4 vesicles to the cell surface, and the other involves docking and/or fusion of the vesicles with the PM. These studies are completely consistent with several other studies showing that insulin may affect at least two distinct processes in GLUT4 trafficking (31, 32, 33, 34). Importantly, we made the novel observation that whereas insulin-stimulated insertion of GLUT4 into the PM was inhibited at 19 C, a significant insulin-dependent redistribution of GLUT4 to the cell cortex was still evident at this reduced temperature. Because we did observe PI-3K activity at 19 C, but no significant increase in Akt phosphorylation, these data suggest that Akt probably does not participate in the initial step, but rather in a more distal step likely involving docking of GLUT4 vesicles with the PM.
There is a growing body of evidence supporting a model in which insulin regulates two steps in GLUT4 translocation, only one being dependent on PI-3K and its downstream effector Akt. A recent study by Czech and colleagues (31) showed that GLUT4 vesicles accumulated just beneath the PM upon incubation of adipocytes with insulin and LY294002, a PI-3K inhibitor. These data implicate an insulin-dependent, but PI-3K-independent, movement of GLUT4 toward the cell surface and a PI-3K-dependent step at a later stage of the translocation process, probably at the cell surface. Furthermore, Pessin and colleagues (32, 35) observed an insulin-dependent accumulation of vesicles beneath the PM at 23 C in 3T3-L1 adipocytes, further suggesting a different regulatory mechanism for the trafficking and fusion of GLUT4 vesicles. In addition, it was recently reported that incubation of either adipocytes or muscle cells with phosphatidylinositol-3-phosphate (PI3P) triggered GLUT4 translocation to the PM. Interestingly, the insulin-dependent formation of PI3P at the cell surface was relatively resistant to the PI-3K inhibitors wortmannin and LY294002, whereas GLUT4 translocation under these conditions was reduced (33). Hence, these data are also consistent with a multistep process and implicate a role for PI3P in the wortmannin-independent step. It is intriguing that the wortmannin-resistant PI-3K isoform, PI-3KC2
(36), has been shown to be activated by insulin in both adipocytes and muscle cells (37).
The initial step in insulin-dependent GLUT4 translocation involving accumulation of vesicles in the cell cortex likely involves movement of GLUT4 vesicles along microtubules. The movement of GLUT4 toward the cell surface has been shown to be dependent on the kinesin KIF5B (34). Interestingly, this KIF5B-dependent movement of GLUT4 along microtubules was independent of PI-3K, further strengthening the notion that there is a PI-3K/Akt-independent GLUT4 trafficking step. However, the effect of nocodazole, a microtubule depolymerizing agent, on GLUT4 translocation has been controversial (38, 39, 40, 41). As described by Czech and colleagues (34), this may be because, at least under some circumstances, depolymerization of microtubules mimics this first effect of insulin to disperse the GLUT4 pool to the periphery of the cell.
Our data suggest that the second insulin-dependent step in GLUT4 translocation to the PM, perhaps involving docking and/or fusion with the PM, is likely Akt-dependent. These data are consistent with other studies suggesting a function for PI-3K at the cell surface (31, 42). Bose et al. (31) found an insulin-dependent accumulation of GLUT4 vesicles at the cell cortex in the presence of PI-3K inhibitors. Furthermore, Pessin and colleagues (42) have shown a PI-3K-dependent regulation of the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Syntaxin 4 that regulates the docking and fusion of GLUT4 vesicles with the PM. Intriguingly, it has been shown that expression of either constitutively active PI-3K (43) or Akt (9, 10) in adipocytes is sufficient to stimulate GLUT4 translocation and insertion into the PM to an extent that is comparable to that observed with insulin alone. Similarly, a recent study found that incubation of adipocytes and muscle cells with PIP3 was sufficient to cause translocation of GLUT4 to the cell surface (44). These data suggest that the second step in GLUT4 translocation involving docking and fusion of vesicles with the cell surface is likely to play a dominant role in the translocation process. In contrast, the first step, likely involving microtubules, may play a more permissive role perhaps acting to facilitate the efficient delivery of vesicles close to their site of insertion. Consistent with such a kinetic effect, we found that insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C (Fig. 9
).
The potential role of Akt at the PM is interesting in lieu of one of its downstream effectors, AS160, a RabGAP that has recently been implicated in insulin-dependent GLUT4 trafficking (12, 45). Rab proteins are thought to play a regulatory role in docking of membranes (46), and so AS160 may play an important role in catalyzing the GTP exchange on the relevant Rab in an Akt-dependent manner. The identification of the responsible Rab protein involved in GLUT4 translocation has not been elucidated yet, although both Rab4 and Rab11 have been implicated in GLUT4 trafficking (47, 48, 49). Alternatively, Akt could be involved in phosphorylation of the SNARE complex at the PM. Many SNARE proteins and SNARE regulatory proteins are phosphorylated, and their interactions may be regulated by phosphorylation (50). However, to date, SNARE proteins and their regulators involved in GLUT4 translocation have not been identified as Akt substrates.
An unexpected observation from this study is that upon incubation of adipocytes at 19 C, there was a slight but significant increase in cell surface GLUT4 levels in the absence of insulin. Interestingly, this is commensurate with decreased localization of GLUT4 in the TGN area. We previously proposed that in the absence of insulin, GLUT4 continuously cycles between endosomes and the TGN (cycle 2), and this may play an important role in intracellular sequestration (1, 5). It is possible therefore that the retrograde transport of GLUT4 from endosomes to the TGN is blocked at 19 C, resulting in default movement to the PM. Another possibility is that incubation of cells at 19 C activates a signal transduction pathway that selectively recruits GLUT4 from the TGN region. Irrespective of the explanation, our data suggest that exocytosis from cycle 2, most likely endosomes/TGN, is not blocked at 19 C. This is intriguing because stimulation of GLUT4 translocation by osmotic shock is unaffected by lowering the temperature to 19 C (data not shown). Klip and colleagues (51) have recently shown that there are two separate routes to the PM, one activated by insulin and the other by osmotic shock. One interpretation of these data is that adipocytes possess at least two regulated pathways by which GLUT4 can transiently move to the cell surface. One of these pathways is insulin-dependent and involves Akt-dependent recruitment of specialized GLUT4 containing vesicles to the cell surface. The second pathway is insulin-independent and likely involves the recruitment from endosomes and the TGN, and this pathway may be activated by agonists including low temperature, osmotic shock, endothelin 1/Arf6, and GTP
S (52, 53, 54, 55). Intriguingly, the effect of each of these agonists to stimulate GLUT4 translocation is PI-3K-independent.
In summary, the present study demonstrates that lowering the temperature to 19 C results in a constitutive trafficking of GLUT4 to the PM. Although the insulin-dependent GLUT4 compartment remains intact at 19 C, it fails to fuse with the PM upon insulin stimulation. This impairment is caused by an impairment in Akt phosphorylation at this temperature. Because GLUT4 translocation can be primed by stimulation of insulin at 19 C, these data indicate a role for Akt at a late stage, like tethering or docking of vesicles with the PM. These data support a revision of previous models of GLUT4 trafficking. In the basal state, the majority of GLUT4 is stored in GSVs, and these structures are discharged to the cell surface in response to insulin. Insulin activates at least two steps to accelerate the discharge of these structures. The first involves movement of vesicles out to the cell cortex in an Akt-independent manner, and the second involves docking and fusion of these vesicles with the PM. This latter process is an Akt-dependent process. Once discharged, we imagine that GLUT4 can now continue to cycle between the PM and intracellular structures, but now in a process that may not rely upon either GSVs or Akt activation.
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MATERIALS AND METHODS
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Materials and Antibodies
3T3-L1 murine fibroblasts were purchased from the American Type Culture Collection (ATCC, Rockville, MD). DMEM and newborn calf serum were obtained from Invitrogen (Carlsbad, CA), Myoclone-Plus fetal calf serum from Trace Scientific (Melbourne, Australia), and antibiotics from Life Technologies (Paisley, UK). Normal swine serum was from Dako (Carpinteria, CA), and paraformaldehyde was from ProSciTech (Thuringowa Central, Australia). Insulin was obtained from Calbiochem (San Diego, CA), and BSA was from USB (Cleveland, OH). Tissue culture plastics and black clear-bottom 96-well plates were from BD Falcon (Bedford, MA). Bicinchoninic acid reagent and enhanced chemiluminescence reagents were from Pierce (Rockford, IL). All other materials were obtained from Sigma (St. Louis, MO).
Polyclonal rabbit antibodies raised against Akt or phospho-Akt were purchased from Cell Signaling (Beverly, MA). The monoclonal antibody raised against phosphotyrosine was obtained from Upstate (Lake Placid, NY). Rabbit antibody raised against Syntaxin 16 was the generous gift of Dr. Wanjin Hong (Institute of Molecular and Cell Biology, Singapore). Mouse IgG1-
MOPC21 antibody was from Sigma. The monoclonal antibody 16B12, which recognizes the influenza HA epitope, was purchased from Covance (Berkeley, CA). The polyclonal rabbit antibody raised against green fluorescent protein was purchased from Molecular Probes (Leiden, The Netherlands). Antibodies against GLUT4 have been described previously (56). ALEXA488 and Cy3-conjugated secondary antibodies were obtained from Molecular Probes or Jackson ImmunoResearch (West Grove, PA), respectively. Horseradish peroxidase-conjugated secondary antibodies were from Amersham (Buckinghamshire, UK).
Cell Culture, Transient Transfection, and Retroviral Transfection
3T3-L1 fibroblasts were cultured in DMEM supplemented with 10% newborn calf serum, 2 mM L-glutamine, 100 U/liter penicillin, and 100 µg/liter streptomycin at 37 C in 10% CO2. Confluent cells were then differentiated into adipocytes as previously described (18). Cells were used between d 6 and 12 post differentiation and between passages 7 and 20. For culturing of fibroblasts in gelatin-coated 96-well plates, cells were seeded at a 1:1 cell surface ratio, and differentiation was initiated 4 d post seeding. To establish basal conditions before use, cells were incubated in serum-free DMEM for 2 h unless otherwise indicated. To generate 3T3-L1 adipocytes expressing HA-GLUT4 or hTfR, fibroblasts were infected with retrovirus as previously described (5, 57) and differentiated into adipocytes. To generate 3T3-L1 adipocytes expressing the PH domain of ARNO, fully differentiated adipocytes were electroporated (0.16 kV and 950 µF) with 100 µg of the EGFP-PH/ARNO plasmid (28). After electroporation, the adipocytes were replated on glass coverslips and allowed to recover for 30 h before use.
Quantitative GLUT4 Translocation Assay
HA-GLUT4 translocation to the PM was measured as previously described (18) with minor modifications. Briefly, cells were grown in black clear-bottom 96-well plates and starved for 2 h in serum- and bicarbonate-free DMEM containing 20 mM HEPES (pH 7.4) and 0.2% BSA (DMEM/BSA) for 2 h at 37 C before starting the experiment. In some conditions, insulin was added at 37 C at a final concentration of 200 nM. Plates were then transferred to 19 C, and vehicle or 200 nM insulin was added. At given time points, paraformaldehyde was added to the wells to a concentration of 3%. After 15 min, the paraformaldehyde was quenched by the addition of glycine (final concentration, 50 mM). The cells were washed extensively and incubated for 20 min with 5% normal swine serum (NSS) in the absence or presence of 0.1% saponin to analyze the amount of HA-GLUT4 at the PM or the total HA-GLUT4 content, respectively. Cells were incubated for 60 min with anti-HA or, as a control, a nonrelevant antibody (mouse IgG1-
MOPC21) in PBS containing 2% NSS. Cells were extensively washed and incubated for 20 min in 5% NSS in the presence or absence of 0.1% saponin. Cells were then incubated with ALEXA488-conjugated goat-antimouse in PBS containing 2% NSS. After washing, fluorescence was measured using the bottom-reading mode in a fluorescence microtiter plate reader (FLUOstar Galaxy, BMG Labtechnologies, Offenburg, Germany).
Glucose Uptake Assay
3T3-L1 adipocytes cultured in 12-well plates were serum starved in Krebs-Ringer phosphate (KRP) buffer [12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.4 mM NaH2PO4, 0.6 mM Na2HPO4 (pH7.4)] containing 0.2% BSA (KRP/BSA) for 2 h at 37 C. Cells were then incubated for 2 h at 19 C. Vehicle or 100 nM insulin was added for 20 min, after which the cells were transferred to ice. Cells were then incubated at 37 C in the presence or absence of 100 nM insulin for the indicated times. 2-DOG uptake was measured as described previously (39). Briefly, the assay was initiated by the addition of 50 µl of 1 mM 2-DOG (20 µCi/mmol). After 2 min, the assay was terminated by rapidly washing the cells three times with ice-cold PBS. Cells were subsequently solubilized in 1% Triton X-100, and 3H content was determined by scintillation counting. Nonspecific 2-DOG uptake was determined in the presence of 50 µM cytochalasin B.
Confocal Laser Scanning Microscopy
3T3-L1 adipocytes were cultured as described above on glass coverslips. The cells were serum depleted for 2 h at either 19 C or 37 C, after which they were either kept basal or stimulated with 200 nM insulin for 20 min at either 19 C or 37 C. Cells were then fixed with 3% paraformaldehyde in PBS. Fixed cells were washed with PBS, and free aldehyde groups were quenched with 50 mM glycine in PBS. The cells were then processed for immunolabeling by permeabilization and labeling in PBS containing 0.1% saponin and 2% BSA, using standard procedures. Primary antibodies were detected with ALEXA-488 or Cy3 conjugated secondary antibodies. Optical sections were analyzed by confocal laser scanning microscopy using a Leica TCS SP system (Leica, Mannheim, Germany).
Akt Phosphorylation
3T3-L1 adipocytes were serum starved for 2 h at 37 C, after which they were either stimulated with 200 nM insulin in the presence or absence of 100 nM wortmannin or 100 nM Calyculin A at 37 C, or they were shifted to 19 C in the presence or absence of 200 nM insulin and 100 nM wortmannin or 100 nm Calyculin A for the indicated times. Cells were then quickly washed with 20 mM HEPES, 250 mM sucrose, 1 mM EDTA (pH 7.4) (HES buffer) at 4 C. Cells were scraped in HES buffer containing phosphatase inhibitors (2 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM ammonium molybdate, and 10 mM sodium fluoride), then homogenized by subsequent passage through 22- and 27-gauge needles. Postnuclear supernatant was prepared, and protein concentration was measured using bicinchoninic acid reagent. Equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. Membranes were incubated with antibodies against Akt, phospho-Akt, or phosphotyrosine.
Tf Recycling Assay
Tf was saturated with Fe3+ and labeled with 125I using iodo-beads (Pierce) according to standard procedures. Recycling of 125I-Tf was basically measured as described (58). In short, adipocytes expressing hTfR were serum starved for 2 h at 37 C before loading cells with 2 µg/ml 125I-Tf in KRP/BSA at 16 C for 2 h. The cells were washed, and PM-bound 125I-Tf was removed at 4 C by subsequent washes at pH 5 and 7.4. Cells were then incubated at either 19 C or 37 C in the presence or absence of 200 nM insulin to allow recycling, and the release of 125I-Tf was determined. The data were corrected for nonspecific 125I-Tf (<10%) that was determined in parallel experiments in which excess (200 µg/ml) of nonlabeled Tf was present during loading.
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ACKNOWLEDGMENTS
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We thank Hala Bazzi for excellent technical assistance, Drs. Will Hughes and Roger Daly for critical review of this manuscript, and Dr. Tamas Balla for generously providing the EGFP-PH/ARNO construct.
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FOOTNOTES
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Current address for R.G.: Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Free University (VU), 1081 HV Amsterdam, The Netherlands.
This work was supported by a fellowship from the European Molecular Biology Organization (to R.G.) and grants from the National Health and Research Council of Australia and Diabetes Australia (to D.E.J.). Microscopic equipment at the Garvan Institute of Medical Research was acquired with the generous assistance of many individuals and corporations and, in particular, Pieter Huveneers and Lady Mary Fairfax.
First Published Online January 13, 2005
Abbreviations: 2-DOG, 2-Deoxy-[3H]glucose; GLUT, glucose transporter; GSV, GLUT4 storage vesicle; HA, hemagglutinin; hTfR, human Tf receptor; IR, insulin receptor; IRS, insulin receptor substrate; KRP, Krebs-Ringer phosphate; NSS, normal swine serum; PDK1, phosphoinositide-dependent protein kinase 1; PI-3K, phosphatidylinositol 3 kinase; PI3P, phosphatidylinositol 3 phosphate; PIP3, phosphatidylinositol 3,4,5 trisphosphate; PM, plasma membrane; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Tf, transferrin; TGN, trans-Golgi network.
Received for publication October 14, 2004.
Accepted for publication January 3, 2005.
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REFERENCES
|
---|
- Bryant NJ, Govers R, James DE 2002 Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 3:267277[CrossRef][Medline]
- Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE 1991 Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 113:123135[Abstract]
- Martin S, Millar CA, Lyttle CT, Meerloo T, Marsh BJ, Gould GW, James DE 2000 Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidence for a secretory mode of regulation. J Cell Sci 19:34273438
- Livingstone C, James DE, Rice JE, Hanpeter D, Gould GW 1996 Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3L1 adipocytes. Biochem J 315:487495[Medline]
- Shewan AM, Van Dam EM, Martin S, Luen TB, Hong W, Bryant NJ, James DE 2003 GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif. Mol Biol Cell 14:973986[Abstract/Free Full Text]
- Ramm G, Slot JW, James DE, Stoorvogel W 2000 Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes. Mol Biol Cell 11:40794091[Abstract/Free Full Text]
- Tanti JF, Grillo S, Gremeaux T, Coffer PJ, Van Obberghen E, Le Marchand-Brustel Y 1997 Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology 138:20052010[Abstract/Free Full Text]
- Hill MM, Clark SF, Tucker DF, Birnbaum MJ, James DE, Macaulay SL 1999 A role for protein kinase Bß/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol 19:77717781[Abstract/Free Full Text]
- Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a constitutively active Akt Ser/Thr kinase in 3T3L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:3137231378[Abstract/Free Full Text]
- Cong LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:18811890[Abstract/Free Full Text]
- Ducluzeau PH, Fletcher LM, Welsh GI, Tavare JM 2002 Functional consequence of targeting protein kinase B/Akt to GLUT4 vesicles. J Cell Sci 115:28572866[Abstract/Free Full Text]
- Sano H, Kane S, Sano E, Miinea CP, Asara JM, Lane WS, Garner CW, Lienhard GE 2003 Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J Biol Chem 278:1459914602[Abstract/Free Full Text]
- Robinson LJ, James DE 1992 Insulin-regulated sorting of glucose transporters in 3T3L1 adipocytes. Am J Physiol 263:E383E393
- Haylett T, Thilo L 1991 Endosome-lysosome fusion at low temperature. J Biol Chem 266:83228327[Abstract/Free Full Text]
- Griffiths G, Pfeiffer S, Simons K, Matlin K 1985 Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J Cell Biol 101:949964[Abstract]
- Matlin KS, Simons K 1983 Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34:233243[Medline]
- Saraste J, Palade GE, Farquhar MG 1986 Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci USA 83:64256429[Abstract]
- Govers R, Coster AC, James DE 2004 Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway. Mol Cell Biol 24:64566466[Abstract/Free Full Text]
- Coster AC, Govers R, James DE 2004 Insulin stimulates the entry of GLUT4 into the endosomal recycling pathway by a quantal mechanism. Traffic 5:763771[CrossRef][Medline]
- Shepherd PR, Soos MA, Siddle K 1995 Inhibitors of phosphoinositide 3-kinase block exocytosis but not endocytosis of transferrin receptors in 3T3L1 adipocytes. Biochem Biophys Res Commun 211:535539[CrossRef][Medline]
- Ko KW, Avramoglu RK, McLeod RS, Vukmirica J, Yao Z 2001 The insulin-stimulated cell surface presentation of low density lipoprotein receptor-related protein in 3T3L1 adipocytes is sensitive to phosphatidylinositide 3-kinase inhibition. Biochemistry 40:752759[CrossRef][Medline]
- Foran PG, Fletcher LM, Oatey PB, Mohammed N, Dolly JO, Tavare JM 1999 Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin. J Biol Chem 274:2808728095[Abstract/Free Full Text]
- Simpson F, Whitehead JP, James DE 2001 GLUT4at the cross roads between membrane trafficking and signal transduction. Traffic 2:211[CrossRef][Medline]
- Litherland GJ, Hajduch E, Hundal HS 2001 Intracellular signalling mechanisms regulating glucose transport in insulin-sensitive tissues (review). Mol Membr Biol 18:195204[CrossRef][Medline]
- Elmendorf JS 2002 Signals that regulate GLUT4 translocation. J Membr Biol 190:167174[CrossRef][Medline]
- Venkateswarlu K, Oatey PB, Tavare JM, Cullen PJ 1998 Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr Biol 8:463466[Medline]
- Klarlund JK, Tsiaras W, Holik JJ, Chawla A, Czech MP 2000 Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs. J Biol Chem 275:3281632821[Abstract/Free Full Text]
- Balla T, Varnai P 2002 Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci STKE 125:26
- Oatey PB, Venkateswarlu K, Williams AG, Fletcher LM, Foulstone EJ, Cullen PJ, Tavare JM 1999 Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3L1 adipocytes. Biochem J 344:511518[CrossRef][Medline]
- Holman GD, Lo Leggio L, Cushman SW 1994 Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools. J Biol Chem 269:1751617524[Abstract/Free Full Text]
- Bose A, Robida S, Furcinitti PS, Chawla A, Fogarty K, Corvera S, Czech MP 2004 Unconventional myosin Myo1c promotes membrane fusion in a regulated exocytic pathway. Mol Cell Biol 24:54475458[Abstract/Free Full Text]
- Thurmond DC, Pessin JE 2000 Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant. EMBO J 19:35653575[Abstract/Free Full Text]
- Maffucci T, Brancaccio A, Piccolo E, Stein RC, Falasca M 2003 Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation. EMBO J 22:41784189[Abstract/Free Full Text]
- Semiz S, Park JG, Nicoloro SM, Furcinitti P, Zhang C, Chawla A, Leszyk J, Czech MP 2003 Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules. EMBO J 22:23872399[Abstract/Free Full Text]
- Elmendorf JS, Boeglin DJ, Pessin JE 1999 Temporal separation of insulin-stimulated GLUT4/IRAP vesicle plasma membrane docking and fusion in 3T3L1 adipocytes. J Biol Chem 274:3735737361[Abstract/Free Full Text]
- Domin J, Pages F, Volinia S, Rittenhouse SE, Zvelebil MJ, Stein RC, Waterfield MD 1997 Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin. Biochem J 326:139147[Medline]
- Brown RA, Domin J, Arcaro A, Waterfield MD, Shepherd PR 1999 Insulin activates the
isoform of class II phosphoinositide 3-kinase. J Biol Chem 274:1452914532[Abstract/Free Full Text]
- Shigematsu S, Khan AH, Kanzaki M, Pessin JE 2002 Intracellular insulin-responsive glucose transporter (GLUT4) distribution but not insulin-stimulated GLUT4 exocytosis and recycling are microtubule dependent. Mol Endocrinol 16:10601068[Abstract/Free Full Text]
- Molero JC, Whitehead JP, Meerloo T, James DE 2001 Nocodazole inhibits insulin-stimulated glucose transport in 3T3L1 adipocytes via a microtubule-independent mechanism. J Biol Chem 276:4382943835[Abstract/Free Full Text]
- Fletcher LM, Welsh GI, Oatey PB, Tavare JM 2000 Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake. Biochem J 352:267276[CrossRef][Medline]
- Emoto M, Langille SE, Czech MP 2001 A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3L1 adipocytes. J Biol Chem 276:1067710682[Abstract/Free Full Text]
- Min J, Okada S, Kanzaki M, Elmendorf JS, Coker KJ, Ceresa BP, Syu LJ, Noda Y, Saltiel AR, Pessin JE 1999 Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes. Mol Cell 3:751760[CrossRef][Medline]
- Tengholm A, Meyer T 2002 A PI3-kinase signaling code for insulin-triggered insertion of glucose transporters into the plasma membrane. Curr Biol 12:18711876[CrossRef][Medline]
- Sweeney G, Garg RR, Ceddia RB, Li D, Ishiki M, Somwar R, Foster LJ, Neilsen PO, Prestwich GD, Rudich A, Klip A 2004 Intracellular delivery of phosphatidylinositol (3,4,5)-trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake. J Biol Chem 279:3223332242[Abstract/Free Full Text]
- Kane S, Sano H, Liu SC, Asara JM, Lane WS, Garner CC, Lienhard GE 2002 A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J Biol Chem 277:2211522118[Abstract/Free Full Text]
- Zerial M, McBride H 2001 Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107117[CrossRef][Medline]
- Shibata H, Omata W, Kojima I 1997 Insulin stimulates guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adipocytes. J Biol Chem 272:1454214546[Abstract/Free Full Text]
- Kessler A, Tomas E, Immler D, Meyer HE, Zorzano A, Eckel J 2000 Rab11 is associated with GLUT4-containing vesicles and redistributes in response to insulin. Diabetologia 43:15181527[CrossRef][Medline]
- Cormont M, Gautier N, Ilc K, le Marchand-Brustel Y 2001 Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B. Biochem J 356:143149[CrossRef][Medline]
- Gerst JE 2003 SNARE regulators: matchmakers and matchbreakers. Biochim Biophys Acta 1641:99110[CrossRef][Medline]
- Randhawa VK, Thong FS, Lim DY, Li D, Garg RR, Rudge R, Galli T, Rudich A, Klip A 2004 Insulin and hypertonicity recruit GLUT4 to the plasma membrane of muscle cells using NSF-dependent SNARE mechanisms but different v-SNAREs: role of TI-VAMP. Mol Biol Cell 15:55655573[Abstract/Free Full Text]
- Chen D, Elmendorf JS, Olson AL, Li X, Earp HS, Pessin JE 1997 Osmotic shock stimulates GLUT4 translocation in 3T3L1 adipocytes by a novel tyrosine kinase pathway. J Biol Chem 272:2740127410[Abstract/Free Full Text]
- Li D, Randhawa VK, Patel N, Hayashi M, Klip A 2001 Hyperosmolarity reduces GLUT4 endocytosis and increases its exocytosis from a VAMP2-independent pool in l6 muscle cells. J Biol Chem 276:2288322891[Abstract/Free Full Text]
- Lawrence JT, Birnbaum MJ 2001 Adp-ribosylation factor 6 delineates separate pathways used by endothelin 1 and insulin for stimulating glucose uptake in 3T3L1 adipocytes. Mol Cell Biol 21:52765285[Abstract/Free Full Text]
- Millar CA, Shewan A, Hickson GR, James DE, Gould GW 1999 Differential regulation of secretory compartments containing the insulin-responsive glucose transporter 4 in 3T3L1 adipocytes. Mol Biol Cell 10:36753688[Abstract/Free Full Text]
- James DE, Strube M, Mueckler M 1989 Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 338:8387[CrossRef][Medline]
- Shewan AM, Marsh BJ, Melvin DR, Martin S, Gould GW, James DE 2000 The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal. Biochem J 350:99107[CrossRef][Medline]
- van Dam EM, Stoorvogel W 2002 Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles. Mol Biol Cell 13:169182[Abstract/Free Full Text]