Biogenesis and regulation of insulin-responsive vesicles containing GLUT4

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Insulin regulates the trafficking of GLUT4 glucose transporters in fat and muscle cells. In unstimulated cells, GLUT4 is sequestered intracellularly in small, insulin-responsive vesicles. Insulin stimulates the translocation of these vesicles to the cell surface, inserting the transporters into the plasma membrane to enhance glucose uptake. Formation of the insulin-responsive vesicles requires multiple interactions among GLUT4, IRAP, LRP1, and sortilin, as well as recruitment of GGA and ACAP1 adaptors and clathrin. Once formed, the vesicles are retained within unstimulated cells by the action of TUG, Ubc9, and other proteins. In addition to acting at other steps in vesicle recycling, insulin releases this retention mechanism to promote the translocation and fusion of the vesicles at the cell surface.

Introduction

Several examples of regulated, non-secretory exocytosis of membrane vesicles have been described [1]. In vertebrates, the exocytic translocation of various membrane proteins occurs in response to hormones and other extracellular stimuli, providing a mechanism for the regulation of systemic physiology. One of the best-studied examples of this process is the insulin-stimulated translocation of GLUT4 glucose transporters [2, 3, 4]. A 12-transmembrane protein, GLUT4 is the predominant glucose transporter present in fat and muscle. Insulin regulates glucose uptake by controlling the number of transporters at the cell surface, and acts within minutes to mobilize GLUT4 from intracellular stores. Because this action is compromised during the development of type 2 diabetes, much work has been carried out to understand both the insulin signaling and GLUT4 trafficking pathways that mediate this process.

Multiple insulin signaling pathways have been implicated in GLUT4 regulation, and may intersect with GLUT4 trafficking pathways at distinct steps. Greatest attention has been given to signaling through Akt2 to AS160/TBC1D4 and TBC1D1, Rab GTPase-activating proteins that direct GLUT4 targeting in fat and muscle, respectively [5]. Other signaling pathways that have been implicated involve atypical protein kinase C isoforms and the Rho-family GTPase, TC10α [6, 7•]. These pathways are reviewed elsewhere [3, 4, 5].

Here, we focus on protein trafficking mechanisms that regulate GLUT4. In particular, we summarize recent work that has led to improved understanding of how GLUT4 and other proteins assemble in insulin-responsive vesicles (IRVs), how these vesicles are retained intracellularly in unstimulated cells, and how they may be mobilized by insulin.

Section snippets

Protein sorting into IRVs

Immunoelectron microscopy of adipose and skeletal muscle cells as well as biochemical data demonstrate that under basal conditions, up to 75% of total intracellular GLUT4 is localized in small (50–80 nm in diameter, ca. 80S) vesicles and short tubules [2, 3]. The rest of the transporter is present in large, rapidly sedimenting intracellular membranes that probably represent endosomes and trans-Golgi network (TGN) structures. Small GLUT4-containing vesicles do not represent a homogeneous

Biogenesis of IRVs

The formation of membrane vesicles is driven by protein coats that are recruited to specific sites on donor membranes by adaptor proteins. The latter associate with donor membranes through multiple interactions with the cytoplasmic domains of cargo proteins, Arf-GTP and phosphatidylinositol phosphates [22]. Formation of the IRVs on intracellular donor membranes requires clathrin coats [23••, 24] and GGA adaptors [25•, 26]. The only protein component of the IRVs that is known to interact with

Targeting of the newly synthesized IRV proteins

Newly synthesized GLUT4 and IRAP are not targeted to the plasma membrane like most membrane proteins, but arrive in the insulin-responsive compartment within 6–9 h [26, 33, 34]. Presumably, these proteins traffic directly from the secretory pathway to IRVs. Reaching the insulin-responsive compartment requires specific targeting signals in the cytoplasmic regions of the proteins. For GLUT4, these signals reside in the N-terminus and large central loop [35]; for IRAP, the dileucine motif at

Trapping and intracellular retention of IRVs

Once formed, IRVs are retained very efficiently within cells not exposed to insulin. Exactly how these vesicles are retained intracellularly is not known, but they probably participate in an intracellular cycle involving endosomes and/or the TGN [2, 17••, 37•]. Most recent data suggest that IRVs do not coalesce with transferrin receptor-containing endosomes [37•, 38]. Moreover, efficient intracellular retention of endocytosed GLUT4 requires SNARE components that drive membrane fusion at the TGN

IRV mobilization by insulin

Insulin stimulates dissociation of GLUT4 from TUG, both in 3T3-L1 adipocytes and in muscle [40••, 42]. Dissociation occurs rapidly, and precedes GLUT4 translocation in 3T3-L1 adipocytes [40••, 43]. More important, the number of TUG-GLUT4 complexes that dissociate controls the number of GLUT4 proteins that are translocated rapidly upon insulin addition, and that appear as an initial burst at the cell surface. This combination of kinetic and biochemical data initially suggested that TUG controls

Conclusions

This summary highlights selected aspects of insulin-regulated GLUT4 trafficking. Much important work on insulin signaling, modulation of Rab activity, and vesicle fusion at the plasma membrane is not covered. It will be interesting to learn which aspects of GLUT4 trafficking are compromised in the setting of insulin resistance, and whether these processes may contribute to the pathogenesis of type 2 diabetes. Additionally, the mechanisms that are involved in IRV cargo recruitment, biogenesis,

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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