Disruption of microtubules ablates the specificity of insulin signaling to GLUT4 translocation in 3T3-L1 adipocytes.
Although the cytoskeletal network is important for insulin-induced glucose uptake, several studies have assessed the effects of microtubule disruption on glucose transport with divergent results. Here, we investigated the effects of microtubule-depolymerizing reagent, nocodazole and colchicine, on GLUT4 translocation in 3T3-L1 adipocytes. After nocodazole treatment to disrupt microtubules, GLUT4 vesicles were dispersed from the perinuclear region in the basal state, and insulin-induced GLUT4 translocation was partially inhibited by 20-30%, consistent with other reports. We found that platelet-derived growth factor (PDGF), which did not stimulate GLUT4 translocation in intact cells, was surprisingly able to enhance GLUT4 translocation to approximately 50% of the maximal insulin response, in nocodazole-treated cells with disrupted microtubules. This effect of PDGF was blocked by pretreatment with wortmannin and attenuated in cells pretreated with cytochalasin D. Using confocal microscopy, we found an increased co-localization of GLUT4 and F-actin in nocodazole-treated cells upon PDGF stimulation compared with control cells. Furthermore, microinjection of small interfering RNA targeting the actin-based motor Myo1c, but not the microtubule-based motor KIF3, significantly inhibited both insulin- and PDGF-stimulated GLUT4 translocation after nocodazole treatment. In summary, our data suggest that 1) proper perinuclear localization of GLUT4 vesicles is a requirement for insulin-specific stimulation of GLUT4 translocation, and 2) nocodazole treatment disperses GLUT4 vesicles from the perinuclear region allowing them to engage insulin and PDGF-sensitive actin filaments, which can participate in GLUT4 translocation in a phosphatidylinositol 3-kinase-dependent manner.
Huang
Jie
J
Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, USA.
Imamura
Takeshi
T
Babendure
Jennie L
JL
Lu
Juu-Chin
JC
Olefsky
Jerrold M
JM
D041721:3T3-L1 Cells; D000199:Actins / Q000378:metabolism; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction
D000199:Actins; D051275:Glucose Transporter Type 4; D007328:Insulin; D010982:Platelet-Derived Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt