Intracellular Delivery of Phosphatidylinositol (3,4,5)-Trisphosphate Causes Incorporation of Glucose Transporter 4 into the Plasma Membrane of Muscle and Fat Cells without Increasing Glucose Uptake

Gary Sweeney,Rami R Garg,Rolando B Ceddia,Dailin Li,Manabu Ishiki,Romel Somwar,Leonard J Foster,Paul O Neilsen,Glenn D Prestwich,Assaf Rudich,Amira Klip

doi:10.1074/jbc.m402897200

Abstract

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P(3)) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P(3) intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P(3) caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phospho-Akt and GLUT4. Intracellular delivery of PI(3,4,5)P(3) using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P(3) was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P(3) elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P(3) did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P(3) to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.

Highlights

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B
PI[3,4,5]P3 suffices to elicit translocation and insertion of GLUT4 at the plasma membrane. Because such action is insufficient to mimic the insulin-dependent gain in glucose uptake, we propose that input(s) additional to PI[3,4,5]P3 are required for the culmination of increased transport of glucose into muscle and fat cells through the translocated transporters
PI[3,4,5]P3-AM Elicits GLUT4 Translocation in 3T3-L1 Adipocytes—PI[3,4,5]P3-AM is a derivative of PI[3,4,5]P3 in which all of the negatively charged phosphates are masked by AM esters to allow the compound to cross cell membranes readily [31]

Summary

EXPERIMENTAL PROCEDURES

Materials—Di-C12-PI[3,4,5]P3-AM was synthesized as described previously [31]. All phosphoinositides, as well as the nitrobenzoxadiazoyl (NBD)-labeled or BodipyTMR (Bodipy)-conjugated versions of PI[3,4,5]P3, carrier (neomycin), and anti-PI[3,4,5]P3 mouse IgG were from Echelon Biosciences (Salt Lake City, UT). Akt Phosphorylation in L6 Myoblasts upon Carrier-mediated Delivery of PI[3,4,5]P3—L6 myoblasts grown on coverslips were treated with insulin or PI[3,4,5]P3 plus carrier as described above, washed three times with cold PBS supplemented by 100 nM okadaic acid and 1 mM Na3VO4, fixed in 3% paraformaldehyde in PBS supplemented with the phosphatase inhibitors for 30 min, after which excess paraformaldehyde was quenched with 50 mM NH4Cl. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked with 1% bovine serum albumin in PBS. A p value of Ͻ0.05 was considered statistically significant

RESULTS

DISCUSSION

Amira Klip