GLUT4 Overexpression or Deficiency in Adipocytes of Transgenic Mice Alters the Composition of GLUT4 Vesicles and the Subcellular Localization of GLUT4 and Insulin-responsive Aminopeptidase

Carvalho, Eugénia; Schellhorn, Sarah E.; Zabolotny, Janice M.; Martin, Sally; Tozzo, Effie; Peroni, Odile D.; Houseknecht, Karen L.; Mundt, Adrian P.; James, David E.; Kahn, Barbara B.

doi:10.1074/jbc.m312269200

Cited by 56 publications

(43 citation statements)

References 52 publications

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“…striated muscle and adipose tissue (Keller et al, 2002). In addition, the genetic ablation of GLUT4 altered the subcellular distribution and expression levels of the IRAP protein (Abel et al, 2004;Carvalho et al, 2004). These data suggest a functional relationship between IRAP and GLUT4; however, a molecular mechanism has not been elucidated.…”

Section: Introductionmentioning

confidence: 74%

Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

Watson

Hou

Pessin

2008

Journal of Cell Science

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Insulin recruits two transmembrane proteins, GLUT4 and IRAP, to the plasma membrane of muscle cells and adipocytes. The subcellular trafficking and localization of GLUT4, and to a lesser extent IRAP, have been intensely studied, yet the molecular mechanisms responsible for their insulin-responsive compartmentalization remain unknown. Herein we have investigated the endocytosis and recycling of IRAP from the cell surface back to the insulin-responsive compartment (IRC). Our results show that a key dileucine motif at position 76,77 (LL76,77), although required for the initial biosynthetic entry of IRAP into the IRC, is dispensable for entry into the IRC via the endosomal system. Indeed, we found that an AA76,77 mutant of IRAP is fully capable of undergoing endocytosis and is correctly routed back to the IRC. To verify that the AA76,77 mutant enters the bona fide IRC, we show that the internalized IRAP-AA76,77 construct is sequestered in an IRC that is insensitive to brefeldin A yet sensitive to a dominant-interfering mutant of AS160 (AS160-4P). In addition, we show that the GGA clathrin adaptors are not required for the re-entry of IRAP from the cell surface back into the IRC, whereas the Q-SNARE syntaxin 6 is required for this process.

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Section: Introductionmentioning

confidence: 74%

Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

Watson

Hou

Pessin

2008

Journal of Cell Science

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“…Although many mutant proteins inhibit insulin-stimulated Glut4 exocytosis in adipocytes, S10A synapsin is one of only a small number of proteins that specifically inhibits basal Glut4 retention. Others include activated signaling molecules (PI3 kinase and Akt) that presumably modify the activity of the retention machinery proteins (Eyster et al, 2005;Frevert et al, 1998) and overexpression of 'cargo' (Glut4 or IRAP), presumably through saturation of the retention mechanism (Carvalho et al, 2004;Waters et al, 1997). Glut4 retention is also inhibited by siRNA-induced knockdown of two proteins, AS160 and Golgin-160 (Eguez et al, 2005;Larance et al, 2005;Williams et al, 2006).…”

Section: Discussionmentioning

confidence: 96%

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

Muretta

Romenskaia

Cassiday

et al. 2007

Journal of Cell Science

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Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a `reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.

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“…A number of studies have been reported where the GLUT4 gene was manipulated specifically within muscle tissue, and it was shown that GLUT4 expression was critical for whole body glucose homeostasis (3,6,37). In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression was shown to be decreased in adipose cells but not in muscles (1).…”

Section: Discussionmentioning

confidence: 99%

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Lizunov

Stenkula

Lisinski

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a musclespecific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.hemagglutinin; glucose transporter 4; green fluorescent protein; insulin; fusion MUSCLE, ESPECIALLY SKELETAL MUSCLE, is a major direct contributor to mammalian systemic glucose homeostasis (1, 10). It is now well established that insulin stimulates glucose transport in adipose and muscle cells through the translocation of glucose transporter 4 (GLUT4) from intracellular sites to the plasma membrane (5, 13, 15). However, whereas the molecular mechanism of GLUT4 translocation has been extensively studied in primary adipose cells and cultured adipocytes during the past years, relatively few studies have focused on GLUT4 trafficking in primary skeletal muscle cells (18,20). In part, this is due to the presence of abundant microfibrillar protein and large amounts of nuclei such that most fractionation protocols suffer from poor resolution for the analysis of the subcellular distribution of GLUT4 in skeletal muscle. Likewise, because of technical limitations, morphological analyses of GLUT4 in skeletal muscle by photolabeling techniques, immunofluorescence, and electron microscopy have not provided sufficient information about the kinetics and dynamics of GLUT4 recycling through the multiple intracellular compartments.Recent alternative approaches involving ectopic expression of tagged glucose transporters in culture cells offer novel opportunities for the molecular analysis of GLUT4 translocation (9,11,35). In these studies, recombinant GLUT4 reporters carrying extracellular ep...

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GLUT4 Overexpression or Deficiency in Adipocytes of Transgenic Mice Alters the Composition of GLUT4 Vesicles and the Subcellular Localization of GLUT4 and Insulin-responsive Aminopeptidase

Cited by 56 publications

References 52 publications

Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

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