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* This work was supported by Canadian Institutes for Health Research Grant MOP-7307 (to A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Funded by a Canadian Institutes for Health doctoral studentship. ¶ Funded by a post-doctoral fellowship from the Banting and Best Diabetes Center, University of Toronto. ** Funded by a studentship from the Banting and Best Diabetes Center, University of Toronto.
Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GLUT4), but it is unknown whether insulin regulates GLUT4 traffic through endosomal compartments. In L6 myoblasts expressing Myc-tagged GLUT4, insulin markedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribution in space and time using confocal immunofluorescence microscopy. Surface-labeled GLUT4myc internalized rapidly (t 3 min), reaching the early endosome by 2 min and the transferrin receptor-rich, perinuclear recycling endosome by 20 min. Upon re-addition of insulin, the t of GLUT4 disappearance from the plasma membrane was unchanged (3 min), but strikingly, GLUT4myc reached the recycling endosome by 10 and left by 20 min. This effect of insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002 or by transiently transfected dominant-negative phosphatidylinositol 3-kinase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate inter-endosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulin-dependent GLUT4 recycling.
protein kinase B
early endosome antigen 1
N-ethylmaleimide-sensitive factor attachment protein receptor protein
Transmembrane proteins are constitutively removed from the cell surface and directed to the early endosome. From here, proteins destined for recycling enter the recycling endosome (
). This pathway is thought to work in a constitutive fashion, but little is known about possible regulation of the individual steps, i.e. direction and speed of sorting out of the early endosome, fusion with the recycling endosome, and exit from this compartment.
The mammalian glucose transporter GLUT4 is an integral membrane protein specific to muscle and fat cells that undergoes both constitutive recycling (
). Despite extensive work, the intracellular donor compartments remain poorly defined as do the effects of insulin on the route or the velocity of inter-compartmental GLUT4 traffic. This paucity in knowledge is largely because of shortcomings of biochemical approaches to isolate and characterize the diverse intracellular compartments populated by GLUT4 and by the limited intracellular space available for detailed immunolocalization in primary fat and muscle tissue and in cultured adipocytes.
Here we used L6 myoblasts stably expressing GLUT4 bearing an extracellular Myc epitope (GLUT4myc) to examine the time course and route of GLUT4 intracellular transit. The subcellular distribution of GLUT4myc in L6 muscle cells was previously characterized (
). Insulin-dependent translocation of GLUT4myc differed from basal state recycling of GLUT4myc in its sensitivity to tetanus toxin, suggesting that basal and insulin-stimulated GLUT4myc arrive at the plasma membrane on distinct vesicles (
When partially detached from the substratum, L6 myoblasts round up and thus offer the opportunity to discern with clarity the intracellular compartments populated by GLUT4myc using fluorescence microscopy. Here we take advantage of the exofacial exposure of the Myc epitope to follow the journey of GLUT4myc during its recycling back to the cell surface. After labeling with an anti-Myc antibody the transporter summoned to the cell surface in response to insulin, we analyze its temporal and spatial intracellular coordinates during its internalization in the absence and presence of insulin. Strikingly, we observed that insulin expedites GLUT4 transit into and out of the recycling endosome. Dominant-negative mutants of PI 3-kinase or PKB blocked the insulin-dependent acceleration of GLUT4myc traffic. These results provide evidence for a previously unknown action of insulin, i.e. to speed up inter-endosomal GLUT4 traffic. This increase may be the basis for the acceleration of GLUT4 recycling caused by the hormone.
Biochemical and morphological approaches have been used to date in an effort to characterize the endomembranes populated by GLUT4. Gradient centrifugation of adipose cells has revealed at least two if not three distinct intracellular compartments containing GLUT4 (
). Moreover, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs) vesicle-associated membrane protein-2, syntaxin 4, and synaptosome-associated protein of 23 kDa are required for about 50% of the insulin-induced GLUT4 translocation in muscle and fat cells (see Ref.
), suggesting that insulin draws GLUT4 from two pools distinguishable by their complement of fusogens. Consistent with this scenario, oxidative ablation of transferrin-containing compartments by transferrin-coupled horseradish peroxidase obliterates 30–50% of the intracellular GLUT4 of 3T3-L1 adipocytes (
), indicating that the recycling endosome is one of the compartments populated by GLUT4. Until now there had been no information on whether insulin regulates the traffic through intracellular compartments in either muscle or fat cells. Answering this question is key to our understanding of GLUT4 availability for translocation.
For this study we selected L6 myoblasts stably expressing GLUT4 tagged at its first exofacial loop with an Myc epitope. We have previously shown that GLUT4myc is effectively sequestered intracellularly in L6 myoblasts and myotubes such that at steady state only 10% of the transporter is found at the cell surface (
). In the present study we further characterize the recycling rate of this molecule in L6 myoblasts and confirm that insulin increases its recycling rate to the cell surface (Fig. 1). Moreover, we show by confocal microscopy that GLUT4myc is concentrated in a perinuclear compartment and that it markedly increases at the cell surface upon insulin stimulation in rounded-up myoblasts (Fig. 4). This behavior is highly reminiscent of the sequestration and movement of GLUT4 in rat and mouse adipose cells and suggests that rounded-up L6 myoblasts expressing GLUT4myc constitute a useful and reliable cell system to study spatial/temporal coordinates of GLUT4 traffic in response to insulin and other stimuli.
In the present study we measured the rates of GLUT4myc recycling and disappearance of GLUT4myc from the cell surface and followed the localization of the transporter once internalized in L6 myoblasts. Insulin markedly increased the rate of GLUT4myc recycling (Fig. 1) without altering the rate of GLUT4myc removal from the cell surface within 10 min of initiation of endocytosis (Fig. 2). Given that insulin markedly increases GLUT4myc recycling back to the cell surface, this observation suggests that the primary effect of insulin in muscle cells is to promote the externalization of GLUT4myc. Studies with rat primary adipocytes have yielded conflicting results, some failing and others detecting a small effect of insulin on the rate of removal of GLUT4 from the cell surface (
). There is evidence for a small reduction in the rate of GLUT4 internalization caused by insulin, detected by either photolabeling of surface transporters in 3T3-L1 adipocytes followed by immunoprecipitation (
). In the present study GLUT4myc internalization was measured by a simpler procedure not requiring either immunoprecipitation or subcellular fractionation, techniques that have complications of incomplete recoveries. It is not clear whether the use of different techniques or a cell-type specificity are responsible for the lack of evidence for an insulin-dependent reduction in GLUT4myc removal from the cell surface in L6 myoblasts. Consistent with the present results, insulin did not alter the rate of removal from the cell surface of IRAP (
The stimulation of the externalization of GLUT4myc could conceivably involve events of intracellular sorting/transit as well as events of mobilization of vesicles directly to the plasma membrane. To begin to explore these possible phenomena, we resorted to a spatial and temporal analysis of the localization of surface-labeled GLUT4myc. For these studies, insulin was given to cells for 10 min to summon GLUT4myc to the cell surface, where it was labeled with antibody at 4 °C, and then its intracellular transit was examined in space and time under conditions where insulin signals had waned or with re-added insulin. Once antibody-labeled GLUT4myc was internalized in the absence of insulin, the transporter was chased and found to be present in a compartment marked by the early endosomal marker EEA1. The presence of insulin did not appear to alter the time course of appearance of labeled-GLUT4myc in this organelle.
Within 10 min of initiation of its internalization, some GLUT4myc reached the TfR-containing compartment, but a more significant accumulation was noted at 20 min. Strikingly, insulin cut by half the time required for labeled-GLUT4myc to accumulate in the TfR-containing compartment. Moreover, by 20 min, GLUT4myc internalized in the presence of insulin had exited from the recycling endosome. We interpret these results to reveal a novel, insulin-induced acceleration of GLUT4myc through the endosomal system. In contrast, internalized rhodamine-transferrin was detected in this compartment 20 min after initiation of loading with this ligand, whether in the absence or presence of insulin. Importantly, the PI 3-kinase inhibitor LY294002 and a dominant negative form of the p85α subunit of PI 3-kinase prevented the early arrival and departure of GLUT4myc to and from the recycling endosome stimulated by insulin. Quantitation revealed that treatment of cells with LY294002 alone had a slightly retarding effect on the movement of GLUT4myc through the endosomal system. This is consistent with previous reports that wortmannin slows inter-endosomal movement of TfR (
Although finding surface-labeled GLUT4myc in the recycling endosome was not surprising, the observed effect of insulin on the inter-endosomal traffic of GLUT4 was unexpected. At steady state, intracellular GLUT4myc was found both in the recycling endosomes and in punctate structures within the cytosol (Fig. 4). Immunoadsorption of GLUT4-containing bodies results in segregation of over half of GLUT4myc away from GLUT1 (
). These results suggest that, as in rodent adipocytes, a portion of GLUT4 segregates from the recycling compartment. However, the site of such segregation and the origin of the specialized segregated compartment were unknown. The findings reported here lead us to propose a revised model of intracellular GLUT4 traffic and its regulation by insulin as presented in Fig.10. In this model, internalized GLUT4 travels through the early endosome defined by the presence of EEA1 and progresses to the recycling endosome defined by TfR. Insulin accelerates GLUT4 arrival at the recycling endosome. Our results also suggest that insulin accelerates the exit of GLUT4 from this compartment since the residence time of labeled GLUT4myc in the TfR-positive endosome was >10 min in the absence of insulin but <10 min in the presence of the hormone. The above results suggest that insulin input is required for at least two distinct functions, movement of GLUT4 into the recycling endosome (Fig. 10) and budding out of the recycling endosome. We propose that, from the recycling endosome GLUT4myc would travel to the cell surface, possibly via generation of specialized exocytic vesicles. Our model also proposes that sorting of GLUT4 occurs in the recycling endosome but does not rule out that a portion of the exocytic vesicle pool may form directly from the early endosome, since there was always a fraction of the internalized GLUT4myc that did not colocalize with TfR at either 10 or 20 min after internalization. That the TGN does not appear to be involved in insulin-dependent GLUT4 traffic is borne out by previous studies showing that brefeldin A does not prevent insulin-dependent GLUT4 exocytosis (
). Our study suggests that the TGN is not a stage in GLUT4 endocytosis and sorting in L6 myoblasts either in the absence or presence of insulin, given that chased, surface-labeled GLUT4 did not appear in the furin-containing compartment (TGN).
) a lag time was noted between the disappearance from the plasma membrane of 3T3-L1 adipocytes of the IRAP, whose traffic closely parallels that of GLUT4, and its appearance in a low density microsome fraction previously shown to contain TfR. It is conceivable that the early endosome defined by EEA1 segregates from the low density microsomal fraction. In this way, the time differential between the disappearance of IRAP from the surface and its appearance in low density microsomes could be because of the transit of IRAP through the early endosome, as demonstrated here for GLUT4myc.
There is general agreement that increased GLUT4 insertion into the plasma membrane in response to insulin requires the action of PI 3-kinase (
). However, the precise point(s) of action of PI 3-kinase and PKB were unknown, since previous studies only used glucose uptake and/or a gain in GLUT4 in the plasma membrane as end point measurements to study the effects of perturbing PI 3-kinase or PKB mutants. The present study raises the hypothesis that insulin input is required at two distinct loci (entrance to and departure from the recycling endosome) and is consistent with a model whereby PI 3-kinase and PKB mediate both inputs. The overall effect of insulin on inter-endosomal GLUT4 traffic would be to expedite movement of GLUT4 through the endosomal system, presumably culminating in the genesis of the exocytic GLUT4 vesicles, which would then be mobilized to the plasma membrane where they would dock and fuse. Additional regulation of the movement of the vesicle and of its interaction with the plasma membrane is not ruled out by this study. Accelerated inter-endosomal transit would provide the cell with a means to maintain levels of plasma membrane GLUT4 in the presence of a continued insulin challenge by regulating the production of plasma membrane-destined GLUT4 vesicles.
An alternative possibility would be that, in the absence of insulin, GLUT4 is sorted directly from the early endosomes to the sequestration compartment. Upon insulin stimulation this compartment could be envisaged to undergo fusion with the recycling compartment, from which the vesicles that finally arrive at the plasma membrane would emanate. Further rounds of GLUT4 traffic could conceivably bypass the sequestration compartment altogether, thereby enhancing the rate of GLUT4 recycling. Although this is also a very attractive model, it is not borne out by the observation that tetanus toxin prevents insulin-dependent incorporation of GLUT4 at the plasma membrane but does not alter the incorporation of GLUT4 either at the basal state (i.e. during continuous recycling) or under conditions of hyperosmolarity stimulation of GLUT4 translocation (
). One would have to consider that, if the recycling endosome is the ultimate stop for GLUT4 before departing to the plasma membrane, different vesicles must bud out from this compartment in the presence or absence of insulin, which are differentiated by their dependence on tetanus toxin-sensitive vesicle SNARES. Further studies should focus on discerning among these different possibilities.
We thank Dr. Timothy McGraw for helpful discussion and Drs. Heidi McBride, Marino Zerial, Mike Moran, Jim Woodgett, Julian Downward, Juan Bonifacino, Sergio Grinstein, Bill Trimble, Phil Bilan, and Zhi Liu for generous supplies of constructs, reagents, and advice.