The Role of Ca in Insulin-stimulated Glucose Transport in 3T3-L1 Cells*

We have examined the requirement for Ca in the signaling and trafficking pathways involved in insulinstimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca, using 1,2-bis (o-aminophenoxy)ethane-N,N,N*,N*-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70–75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTAAM. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 °C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself. Insulin stimulates glucose uptake in skeletal muscle and adipose tissue by stimulating the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. In recent years considerable progress has been made in our understanding of the downstream signal transduction pathways that are activated by insulin to mediate the translocation of GLUT4 to the cell surface. Upon insulin binding, the activated insulin receptor (IR) tyrosine kinase phosphorylates a number of downstream substrates, most notably the insulin receptor substrate (IRS) family of proteins, including IRS-1 and IRS-2 (1). Tyrosyl-phosphorylated IRS-1 and IRS-2 serve as docking stations for SH2 domain-containing proteins such as the class Ia (p85/p110-type) PI 3-kinase (1). Activation of PI 3-kinase is essential for insulin-stimulated GLUT4 translocation and glucose uptake (2) with generation of phosphoinositide 3,4,5-trisphosphate at the plasma membrane (PM) (3) serving to recruit and activate pleckstrin homology domain-containing proteins. Recent evidence indicates that the pleckstrin homology domain-containing Ser/Thr kinase Akt (otherwise called protein kinase B) plays a fundamental role in mediating insulin-stimulated GLUT4 translocation (4–6). A direct link between the insulin-signaling cascade and the more distal events associated with GLUT4 trafficking is yet to be identified. The precise nature of the insulin-responsive GLUT4 storage vesicle (GSV) and a detailed molecular description of how insulin promotes translocation of the GSV to the PM remain to be defined. In contrast, the mechanism by which GSVs dock and fuse with the PM is better understood, in part because of the similarity with synaptic vesicle trafficking in neurons. Both of these events involve the pairing of protein complexes in the vesicle compartment (v-SNARES, for vesicle membrane SNAP receptors) with cognate receptor complexes at the target membrane (t-SNARES, for target membrane SNAP receptors). Interactions between v-SNARE and t-SNARE proteins, as well as additional accessory proteins, are responsible for formation of the core complex, which is required for membrane docking and fusion (7). In adipocytes the core complex is comprised of the v-SNARE, VAMP2, and the tSNAREs, syntaxin 4 and SNAP23 (8). In view of the similarity in molecular regulation between GLUT4 translocation in adipocytes and synaptic vesicle exocytosis in neurons, it has been suggested that GLUT4 translocation may represent a form of * This work was supported by the National Health and Medical Research Council of Australia and the Juvenile Diabetes Foundation International. The Institute for Molecular Bioscience is a Special Research Center of the Australian Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Wellcome Prize Traveling Fellow. To whom all correspondence should be addressed: Inst. for Molecular Bioscience, University of Queensland, Ritchie Research Bldg. , Research Rd., St. Lucia, QLD 4072, Australia. Tel. : 617-3365-4991; Fax: 617-3365-4388; E-mail: J.Whitehead@imb.uq. edu.au. § Recipient of a fellowship from Subprograma General de Perfeccionamiento de Doctores en el Extranjero, S.E.U.I. D, Ministerio de Educacion y Cultura, Spain. i Principal Research Fellow of the National Health and Medical Research Council of Australia. 1 The abbreviations used are: IR, insulin receptor; IRS, insulin receptor substrate; BAPTA-AM, 1,2-bis (o-aminophenoxy)ethane-N,N,N9,N9tetraacetic acid tetra(acetoxymethyl)ester; 2-DOG, 2-deoxyglucose; GSV, GLUT4 storage vesicle; SNAP, soluble NSF attachment protein; v-SNARE, vesicle membrane SNAP receptors; t-SNARE, target membrane SNAP receptors; PM, plasma membrane; BSA, bovine serum albumin; KRP, Krebs-Ringer phosphate; HA, hemagglutinin; CHO, Chinese hamster ovary; PI, phosphatidylinositol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 30, Issue of July 27, pp. 27816–27824, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.