Hyperosmolarity reduces GLUT4 endocytosis and increases its exocytosis from a VAMP2-independent pool in l6 muscle cells.
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.
Li
D
D
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1A8, Canada.
Randhawa
V K
VK
Patel
N
N
Hayashi
M
M
Klip
A
A
D000730:Androstadienes / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D005089:Exocytosis; D051275:Glucose Transporter Type 4; D008565:Membrane Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009132:Muscles / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050683:R-SNARE Proteins; D013744:Tetanus Toxin / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D051275:Glucose Transporter Type 4; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; D011505:Protein-Tyrosine Kinases; D011188:Potassium; D000077191:Wortmannin