Hyperosmolarity Reduces GLUT4 Endocytosis and Increases Its Exocytosis from a VAMP2-independent Pool in L6 Muscle Cells

Dailin Li,Varinder K Randhawa,Nish Patel,Michiko Hayashi,Amira Klip

doi:10.1074/jbc.m010143200

Dailin Li, Varinder K Randhawa + Show 3 more

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https://doi.org/10.1074/jbc.m010143200

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Abstract

The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t(12) = 4 min) gain, whereas hyperosmolarity (0.45 m sucrose) caused a slow (t(12) = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t(12) = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool.

Highlights

The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored
The total myc epitope present in L6 myoblasts was determined by permeabilization with 0.1% Triton X-100 before immunolabeling with anti-myc antibody, followed by Horseradish peroxidase (HRP)-conjugated secondary antibody coupled to an o-phenylenediamine dihydrochloride (OPD) optical densitometric assay
We have previously reported that expression of GLUT4myc in L6 myoblasts leads to the segregation of the protein to a GLUT4-specific pool, conferring insulin sensitivity to glucose uptake [24]

Summary

Introduction

The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. Inhibition of clathrin-coated vesicle formation via Kϩ depletion [7], interference with dynamin-amphiphysin pairing [8], or expression of GTPase-deficient dynamin [9, 10], all prevent GLUT4 internalization in adipocytes. Insulin shifts the subcellular distribution of GLUT4 resulting in a new steady state where a large fraction of GLUT4 resides at the plasma membrane of skeletal muscle [11,12,13], primary adipose cells [14, 15], L6 muscle cells in culture [16], and 3T3-L1 adipocytes [1]. The contribution of exocytic and endocytic pathways to insulin action in muscle cells has not been explored

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