Glucose transporter GLUT4 allows glucose uptake into muscle and adipose cells. Insulin promotes recruitment and plasma membrane insertion of GLUT4 vesicles that can recycle constitutively. Obesity and type 2 diabetes mellitus are associated with defects in insulin-induced GLUT4 recruitment. Knowledge of alternative modes and steps of GLUT4 traffic in L6-GLUT4myc muscle cells may reveal possible targets for therapeutic intervention in insulin-resistant patients. Hypertonicity and Platelet Derived Growth Factor also increase surface GLUT4 levels but it was unknown if they tap on the same intracellular GLUT4 depots as insulin.
We explored whether GLUT4 vesicles recycle using different compartments and mechanisms for the surface gain elicited by each stimulus. We hypothesized that all vesicle fusion steps require NSF but depend on individual v-SNAREs. Specifically, we tested effects of ATPase-deficient NSF or VAMP7 siRNA transfections, and endosomal ablation on GLUT4 traffic. We show that VAMP7 was required for basal and hypertonic recycling, while VAMP2 is exclusively used in response to insulin.
As insulin action bifurcates downstream of phosphatidylinositol 3-kinase, we also hypothesized that the Rac-to-actin and Akt-to-AS160 branches regulate distinct GLUT4 traffic steps. For this we determined GLUT4myc localization in rounded myoblasts relative to a surface marker. Interfering with Rac, actin dynamics or actin-binding α-actinin4 maintained GLUT4 in a perinuclear region even under insulin-stimulation. Interfering with AS160 allowed significant GLUT4 accumulation beneath the membrane, but not fusion. We propose that actin dynamics and α-actinin4 are required for cortical GLUT4 tethering mechanisms, and AS160 contributes to GLUT4 docking/fusion. We confirmed that VAMP2 facilitates GLUT4 fusion, as tetanus toxin-based cleavage did not inhibit peripheral GLUT4 recruitment. Finally, AS160 targets Rab8A and Rab14 in muscle respectively affected GLUT4 availability for membrane fusion, and basal GLUT4 retention.
This work will lead to future testing of strategies to selectively enhance vesicle availability, tethering, or surface fusion, for bypassing insulin resistance.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/16719 |
| Date | 19 January 2009 |
| Creators | Randhawa, Varinder |
| Contributors | Klip, Amira |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 2785524 bytes, application/pdf |
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