Insulin regulates the membrane arrival, fusion, and C-terminal unmasking of glucose transporter-4 via distinct phosphoinositides.
Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) to intact cells significantly increased lawn-associated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P(3) causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.
Ishiki
Manabu
M
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8.
Randhawa
Varinder K
VK
Poon
Vincent
V
Jebailey
Lellean
L
Klip
Amira
A
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016259:Genes, myc; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D008561:Membrane Fusion; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology; D010716:Phosphatidylinositols / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009025:Morpholines; D009124:Muscle Proteins; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C495694:Slc2a4 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000077191:Wortmannin