Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast Microscopy

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Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actinrelated protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-⌬WA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-⌬WA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.Insulin stimulates glucose uptake by skeletal muscle and adipose tissues primarily through regulation of the subcellular distribution of the glucose transporter 4 (GLUT4) 1 (1, 2). In response to insulin, a fraction of GLUT4 present in intracellular membranes is redistributed to the plasma membrane, resulting in an increase of GLUT4 on the cell surface and enhanced glucose uptake by these cells. This effect of insulin is important in maintaining glucose homeostasis in humans, and impaired insulin action can contribute to the pathogenesis of type 2 diabetes (3). The precise mechanism by which insulin directs exocytosis of GLUT4-containing membrane vesicles remains obscure. However, it is established that activation of the insulin receptor tyrosine kinase catalyzes tyrosine phosphorylation of insulin receptor substrate proteins that bind to Srchomology 2 domain-containing molecules, including the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (4, 5). This results in activation of the p110 catalytic subunit of the kinase, which then phosphorylates cellular polyphosphoinositides at the D-3 position, forming signaling molecules such as phosphatidylinositol 3,4,5-trisphosphate. Multiple studies using various pharmacologic inhibitors, overexpression of constitutively active or dominant negative mutants, and microinjection of blocking antibodies have suggested a necessary role of the p85/p110-type PI 3-kinase in insulin-stimulated GLUT4 translocation and glucose transport (1, 2, 6 -8).On the other hand, several lines of evidence suggest the requirement of PI 3-kinase-independent pathway(s) in G...

Section: Fig 3 Effects Of N-wasp Mutants On Insulin-induced Corticasupporting

confidence: 93%

“…Interestingly, previous findings (18,24) indicated the inhibition of insulin-stimulated GLUT4 recycling by actin depolymerization reagents such as latrunculin B is about 50%, similar to our present findings with dominant negative N-WASP (Fig. 5).…”

Section: Fig 3 Effects Of N-wasp Mutants On Insulin-induced Corticasupporting

confidence: 93%

A Phosphatidylinositol 3-Kinase-independent Insulin Signaling Pathway to N-WASP/Arp2/3/F-actin Required for GLUT4 Glucose Transporter Recycling

Jiang

Chawla

Bose

et al. 2002

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151

“…On the other hand, while there is a controversy on the mechanism of action of nocodazole especially in 3T3-L1 adipocytes (14,15,16,19,20), our results show that nocodazole has no direct inhibitory effect on the glucose transporter activity (Fig. 10).…”

Section: Discussionmentioning

confidence: 53%

“…While the role of the actin filaments in insulin-induced GLUT4 translocation has been reported with relatively consistent results (10 -13), the relevance of the microtubules to the insulin action has been controversial. Thus, depolymerization of the microtubules caused a complete cessation of the basal linear tracking movements of green fluorescent protein (GFP)-tagged GLUT4, and inhibited insulin-induced GLUT4 translocation and glucose uptake by 40 -70% in 3T3-L1 adipocytes (14,15). Likewise, Olson et al (16) also showed that nocodazole treatment of 3T3-L1 adipocytes resulted in about 80% inhibition of insulin-stimulated translocation of GLUT4.…”

mentioning

confidence: 95%

Insulin Recruits GLUT4 from Distinct Compartments via Distinct Traffic Pathways with Differential Microtubule Dependence in Rat Adipocytes

Liu

Omata

Kojima

et al. 2003

In the present study, we investigated the physiological significance of the microtubules in the subcellular localization and trafficking of GLUT4 in rat primary adipocytes. Morphological and biochemical analyses revealed a dose-and time-dependent disruption of the microtubules by treatment with nocodazole. With nearly complete disruption of the microtubules, the insulinstimulated glucose transport activity was inhibited by 55%. This inhibition was concomitant with a comparable inhibition of GLUT4 translocation measured by the subcellular fractionation and the cell-surface GLUT4 labeling by trypsin cleavage. In addition, the time-course of insulin stimulation of the glucose transport activity was significantly delayed by microtubule disruption (t ½ ½ were 7 and 2.3 min in nocodazole-treated and control cells, respectively), while the rate of GLUT4 endocytosis was little affected. The impaired insulin-stimulated glucose transport activity was not fully restored to the level in control cells by blocking GLUT4 endocytosis, suggesting that the inhibition was due to the existence of a microtubule-dependent subpopulation in the insulin-responsive GLUT4 pool. On the other hand, nocodazole partially inhibited insulin-induced translocation of the insulin-regulated aminopeptidase and the vesicle-associated membrane protein (VAMP)-2 without affecting GLUT1 and VAMP-3. In electrically permeabilized adipocytes, the insulin-stimulated glucose transport was inhibited by 40% by disruption of the microtubules whereas that stimulated with GTP␥S was not affected. Intriguingly, the two reagents stimulated glucose transport to the comparable level by disruption of the microtubules. These data suggest that insulin recruits GLUT4 to the plasma membrane from at least two distinct intracellular compartments via distinct traffic routes with differential microtubule dependence in rat primary adipocytes.Insulin stimulates glucose uptake mainly by promoting subcellular redistribution of a facilitative glucose transporter isoform GLUT4 from intracellular compartments to the plasma membrane in adipocyte and skeletal/cardiac muscles (1-3). While the subcellular trafficking pathways and the molecular mechanisms by which insulin recruits GLUT4 to the plasma membrane still remain obscure, several lines of evidence have suggested that GLUT4 is associated with more than one intracellular compartment. Morphologically, studies with immunoelectron microscopy have shown that most of GLUT4 localizes intracellularly to tubulovesicular structures clustered near the stacks of Golgi and the endosomes, or scattered throughout the cytoplasm in unstimulated adipocytes and that insulin decreases GLUT4 from all the intracellular compartments (4, 5). On the other hand, many biochemical studies have indicated that insulin-responsive GLUT4 is associated with the endosomal recycling pathway and with a more specialized postendosomal compartment (for review, see Ref. 1). Additionally, more recent works have suggested that insulin recruits GLUT4 to the plasma membrane f...

“…The cytoskeleton plays a significant role in facilitating GLUT4 exocytosis mediated by both microtubules and actin filaments (5)(6)(7)(8)(9). Microtubules direct the transport of GLUT4 vesicles to the cell cortex via the kinesin motor protein KIF5B (10).…”

mentioning

confidence: 99%

Identification of P-Rex1 as a Novel Rac1-Guanine Nucleotide Exchange Factor (GEF) That Promotes Actin Remodeling and GLUT4 Protein Trafficking in Adipocytes

Balamatsias

Kong

Waters

et al. 2011

Background: PREX1 maps to a Type 2 diabetes susceptibility locus; however, its role in insulin-stimulated GLUT4 trafficking is unknown. Results: P-Rex1 activates Rac1 in adipocytes and thereby actin rearrangement and GLUT4 trafficking, facilitating glucose uptake. Conclusion: P-Rex1 may contribute to insulin-stimulated glucose homeostasis. Significance: These studies identify a novel regulator of GLUT4 trafficking in adipocytes.