Varinder K. Randhawa

Insulin action on glucose transporters through molecular switches, tracks and tethers

Glucose entry into muscle cells is precisely regulated by insulin, through recruitment of GLUT4 (glucose transporter-4) to the membrane of muscle and fat cells. Work done over more than two decades has contributed to mapping the insulin signalling and GLUT4 vesicle trafficking events underpinning this response. In spite of this intensive scientific research, there are outstanding questions that continue to challenge us today. The present review summarizes the knowledge in the field, with emphasis on the latest breakthroughs in insulin signalling at the level of AS160 (Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) and their target Rab proteins; in vesicle trafficking at the level of vesicle mobilization, tethering, docking and fusion with the membrane; and in the participation of the cytoskeleton to achieve optimal temporal and spatial location of insulin-derived signals and GLUT4 vesicles.

GLUT4 Vesicle Recruitment and Fusion Are Differentially Regulated by Rac, AS160, and Rab8A in Muscle Cells

Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.

Insulin but not PDGF relies on actin remodeling and on VAMP2 for GLUT4 translocation in myoblasts.

Insulin promotes the translocation of glucose transporter 4 (GLUT4) from intracellular pools to the surface of muscle and fat cells via a mechanism dependent on phosphatidylinositol (PtdIns) 3-kinase, actin cytoskeletal remodeling and the v-SNARE VAMP2. The growth factor PDGF-BB also robustly activates PtdIns 3-kinase and induces actin remodeling, raising the question of whether it uses similar mechanisms to insulin in mobilizing GLUT4. In L6 myoblasts stably expressing Myc-tagged GLUT4, neither stimulus affected the rate of GLUT4 endocytosis, confirming that they act primarily by enhancing exocytosis to increase GLUT4 at the cell surface. Although surface GLUT4myc in response to insulin peaked at 10 minutes and remained steady for 30 minutes, PDGF action was transient, peaking at 5 minutes and disappearing by 20 minutes. These GLUT4myc translocation time courses mirrored that of phosphorylation of Akt by the two stimuli. Interestingly, insulin and PDGF caused distinct manifestations of actin remodeling. Insulin induced discrete, long (>5 μm) dorsal actin structures at the cell periphery, whereas PDGF induced multiple short (<5 μm) dorsal structures throughout the cell, including above the nucleus. Latrunculin B, cytochalasin D and jasplakinolide, which disrupt actin dynamics, prevented insulin- and PDGF-induced actin remodeling but significantly inhibited GLUT4myc translocation only in response to insulin (75-85%, P<0.05), not to PDGF (20-30% inhibition). Moreover, transfection of tetanus toxin light chain, which cleaves the v-SNAREs VAMP2 and VAMP3, reduced insulin-induced GLUT4myc translocation by >70% but did not affect the PDGF response. These results suggest that insulin and PDGF rely differently on the actin cytoskeleton and on tetanus-toxin-sensitive VAMPs for mobilizing GLUT4.

α-Actinin-4 Is Selectively Required for Insulin-induced GLUT4 Translocation

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64–75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of α-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton.

The Pleckstrin Homology (PH) Domain-Interacting Protein Couples the Insulin Receptor Substrate 1 PH Domain to Insulin Signaling Pathways Leading to Mitogenesis and GLUT4 Translocation

ABSTRACT Receptor-mediated tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) is required for the propagation of many of insulins biological effects. The amino-terminal pleckstrin homology (PH) domain of IRS-1 plays a pivotal role in promoting insulin receptor (IR)-IRS-1 protein interactions. We have recently reported the isolation of a PH domain-interacting protein, PHIP, which selectively binds to the IRS-1 PH domain and is stably associated with IRS-1 in mammalian cells. Here we demonstrate that overexpression of PHIP in fibroblasts enhances insulin-induced transcriptional responses in a mitogen-activated protein kinase-dependent manner. In contrast, a dominant-negative mutant of PHIP (DN-PHIP) was shown to specifically block transcriptional and mitogenic signals elicited by insulin and not serum. In order to examine whether PHIP/IRS-1 complexes participate in the signal transduction pathway linking the IR to GLUT4 traffic in muscle cells, L6 myoblasts stably expressing a myc-tagged GLUT4 construct (L6GLUT4myc) were transfected with either wild-type or dominant-interfering forms of PHIP. Whereas insulin-dependent GLUT4myc membrane translocation was not affected by overexpression of PHIP, DN-PHIP caused a nearly complete inhibition of GLUT4 translocation, in a manner identical to that observed with a dominant-negative mutant of the p85 subunit of phosphatidylinositol 3-kinase (Δp85). Furthermore, DN-PHIP markedly inhibited insulin-stimulated actin cytoskeletal reorganization, a process required for the productive incorporation of GLUT4 vesicles at the cell surface in L6 cells. Our results are consistent with the hypothesis that PHIP represents a physiological protein ligand of the IRS-1 PH domain, which plays an important role in insulin receptor-mediated mitogenic and metabolic signal transduction.

Dissecting GLUT4 traffic components in L6 myocytes by fluorescence-based, single-cell assays.

Postprandial blood glucose homeostasis is regulated by an insulin-stimulated increase in glucose transport into muscle and fat tissues via glucose transporter isoform 4 (GLUT4). In the basal state, this constitutively recycling membrane protein predominantly resides intracellularly. In order to achieve the insulin-stimulated increase in glucose flux, GLUT4 increases its cell surface abundance at the expense of preformed intracellular depots. By confocal microscopy of cultured L6 muscle cells stably expressing myc-tagged GLUT4 (L6-GLUT4myc), we can visualize the two arms of GLUT4 traffic: exocytosis (movement to the cell surface) and endocytosis (internalization from the cell surface).