Bridging the GAP Between Insulin Signaling and GLUT4 Translocation

Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006 Bridging the GAP between insulin signaling and GLUT4 translocation Robert T. Watson and Jeffrey E. Pessin Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA Upon binding and activating its cell-surface receptor, in both developing and developed countries, has galva- insulin triggers signaling cascades that regulate many nized the research efforts of scientists worldwide [6]. cellular processes. Regarding glucose homeostasis, At the cellular level, understanding glucose uptake insulin suppresses hepatic glucose production and involves two largely separate fields of inquiry: (i) insulin- increases glucose transport into muscle and adipose receptor signaling and (ii) GLUT4 membrane trafficking. tissues. At the cellular level, glucose uptake results from Not surprisingly, identifying the molecules that link insulin the insulin-stimulated translocation of the glucose signaling to GLUT4 translocation has been a major focus of transporter 4 (GLUT4) from intracellular storage sites research [7]. In addition, insulin-signaling pathways can to the plasma membrane. Although the signaling also be functionally separated into two distinct branches, molecules that function proximal to the activated insulin one defined by the Rho-family GTPase TC10 [8] and the receptor have been well characterized, it is not known other by the phosphatidylinositol 3-kinase (PI3-kinase) (see how the distal insulin-signaling cascade interfaces with Ref. [9] for abbreviations of the inositol phosphates and their and mobilizes GLUT4-containing compartments. kinases) (Figure 2). Although the polyphosphoinositide Recently, several candidate signaling molecules, (PPIn) 3-kinase pathway is well established, the TC10 including AS160, PIKfyve and synip, have been identified branch remains controversial [10,11]. Here, we will that might provide functional links between the insulin therefore focus on the PPIn 3-kinase pathway. In recent signaling cascade and GLUT4 compartments. Future years, several molecules, including AS160, PIKfyve, and work will focus on delineating the precise GLUT4 synip, have extended the reach of the PPIn 3-kinase arm of trafficking steps regulated by these molecules. the insulin-signaling cascade, bringing us closer to understanding the cellular mechanisms for bridging the gap between insulin signaling and GLUT4 membrane trafficking. Introduction Elevated levels of circulating sugars and amino acids, as occur following a meal, signal pancreatic b cells to release Insulin signaling: overview and update insulin into the bloodstream. Insulin stimulates periph- When activated, most receptor tyrosine kinases directly eral tissues, primarily muscle and adipose, to absorb recruit downstream effector molecules to their phosphory- glucose from the vascular system. At the cellular level, lated cytoplasmic-tail domains [12]. By contrast, several glucose crosses the plasma membrane through aqueous cytosolic scaffold proteins serve as substrates for the pores formed by facilitative transporters of the GLUT activated insulin receptor, thereby greatly expanding the (glucose transporter) family [1,2]. One GLUT isoform in repertoire of potential downstream signaling opportu- particular, GLUT4, translocates from intracellular nities (Figure 2). With respect to glucose uptake, the four storage sites to the plasma membrane in response to members of the insulin-receptor-substrate family (IRS1, insulin (Figure 1). At the cell surface, GLUT4 facilitates IRS2, IRS3 and IRS4), in addition to Cbl and APS, the passive transport of glucose into muscle and fat cells. undergo tyrosine phosphorylation in response to insulin By contrast, during periods between meals, the liver stimulation [13]. Identified at the molecular level in 1991 normally provides sufficient glucose output to maintain [14], the IRS family has enjoyed considerable attention. circulating levels in the range of 4–7 mM in humans. Both IRS1 and IRS2 have key roles in insulin-stimulated Deviations from this range can have major health glucose uptake in fat and muscle, and the independent consequences. Low blood glucose levels, for example, can genetic ablation of either isoform leads to peripheral lead to seizures, coma and death. Far more common, insulin resistance [15]. However, only Irs2K/K mice show a however, are prolonged elevated glucose levels, as occurs diabetic phenotype, in part, because IRS2 also has in the diabetic state [3–5]. Frank diabetes (when fasting important roles in b-cell function [16]. Mechanistically, blood glucose levels are 126 mg/dl or higher) has many the insulin-dependent tyrosine phosphorylation of IRS associated health risks, including blindness, renal failure, proteins generates docking sites (Tyr–P-Xaa-Xaa-Met) for neuropathy and cardiovascular disease. Indeed, the many Src homology 2 (SH2)-domain-containing down- seemingly inexorable increase in cases of type-2 diabetes, stream effectors, notably the type-1A PPIn 3-kinase. The Corresponding author: Pessin, J.E. ([email protected]). PPIn 3-kinase has been implicated in numerous biological responses, including mitogenesis, anti-apoptosis, protein www.sciencedirect.com 0968-0004/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2006.02.007 216 Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006 insulin-stimulated recruitment of GLUT4 to the cell (a) Myc surface [13]. Conversely, expression of a constitutively active p110 subunit, or introduction of a PtdIns(3,4,5)P3 analog into cells, induces GLUT4 translocation [20–24]. Lumen/extracellular The PPIn 3-kinase-catalyzed increase in PtdIns(3,4,5)P3 in the membrane is thought to form lipid-based platforms Cytoplasm for recruiting and concentrating downstream signaling NH2 molecules containing pleckstrin-homology (PH) domains EGFP [25]. PH domains are 100–120 amino acid modules that bind to PtdIns(3,4,5)P3 and other membrane phosphoino- sitides [26]. PtdIns(3,4,5)P3 promotes the cell-surface (b) EGFP Myc Merge localization of two PH-domain-containing enzymes (i) (ii) (iii) involved in GLUT4 translocation, the 3-phosphoino- sitide-dependent protein kinase-1 (PDK1) and Akt. The Basal atypical protein kinase C l and z isoforms (aPKCl/z) might also be regulated by PtdIns(3,4,5)P3, although not through PH-domain interactions [27]. In addition, sub- (iv) (v) (vi) stantial evidence supports a role for aPKCl/z in GLUT4 translocation, and the interested reader is referred to Refs Insulin [27,28] for recent authoritative reviews. Activation of Akt requires that it undergoes phosphorylation at two sites [29]. PDK1 phosphorylates Akt at Thr308, a residue located in its kinase-domain activation loop. In addition, Ti BS Ser473 in the C-terminal hydrophobic motif of Akt has Figure 1. The glucose transporter 4 (GLUT4) redistributes to the plasma membrane long been known to undergo phosphorylation but the in response to insulin. (a) Model of the predicted membrane topology of GLUT4. identity of the kinase responsible has been controversial. The N and C termini are cytoplasmic, and the first extracellular loop can accommodate engineered epitope tags, including Myc (shown) and hemagglutinin. Recently, however, an enzyme complex consisting of In non-permeabilized cells transfected with this type of GLUT4 reporter, fully fused mTOR (mammalian target of rapamycin) and RICTOR transporters can be detected by incubating cells with the appropriate antibody. In (rapamycin insensitive companion of mTOR) has been addition, the internalization and recycling of GLUT4 can also be investigated by incubating live cells with the appropriate antibody at 48C, then washing out the shown to phosphorylate Akt at Ser473 in response to antibody for various times at 378C and following its endocytosis with a fluorescent- insulin [30,31] (Figure 3). By contrast, the rapamycin- secondary antibody. The C terminus of GLUT4 can also be modified, for example, sensitive mTOR/RAPTOR (regulatory associated protein with enhanced green fluorescent protein (EGFP), to facilitate live-cell imaging and other applications requiring intrinsic fluorescence. (b) 3T3L1 adipocytes were of mTOR) complex is a downstream target of Akt [32], transiently transfected with myc–GLUT4–EGFP and treated with insulin. Cells were placing mTOR both upstream and downstream of Akt then fixed (without detergent) and labeled with an anti-myc antibody followed by function depending on its associated interacting partner. Texas Red secondary. In the basal state, GLUT4 is present in the perinuclear region and small vesicular structures dispersed in the cytoplasm, as revealed by the In addition to positive regulators of GLUT4 transloca- intrinsic EGFP fluorescence (i). Little myc labeling, other than background tion, negative regulators have also been studied intensely fluorescence, is detectable in the basal state (ii). The merged images are shown because they might provide opportunities for pharmaco- in (iii). In response to insulin, GLUT4 translocates and fuses with the cell surface, thereby exposing the myc-epitope to the exterior of the cell. A prominent ring of logical interventions. For example, the endogenous EGFP fluorescence (iv) and myc labeling (v) is readily visible at the plasma attenuation of PPIn 3-kinase signaling occurs through membrane. Merged images are shown in (vi). the dephosphorylation of PtdIns(3,4,5)P3, and two phos- synthesis and glycogen synthesis [17]. In addition, apart phoinositide phosphateses,

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