Abstract
AS160 and Tbc1d1 are key Rab GTPase-activating proteins (RabGAPs) that mediate release of static GLUT4 in response to insulin or exercise-mimetic stimuli, respectively, but their cooperative regulation and its underlying mechanisms remain unclear. By employing GLUT4 nanometry with cell-based reconstitution models, we herein analyzed the functional cooperative activities of the RabGAPs. When both RabGAPs are present, Tbc1d1 functionally dominates AS160, and stimuli-inducible GLUT4 release relies on Tbc1d1-evoking proximal stimuli, such as AICAR and intracellular Ca2+ Detailed functional assessments with varying expression ratios revealed that AS160 modulates sensitivity to external stimuli in Tbc1d1-mediated GLUT4 release. For example, Tbc1d1-governed GLUT4 release triggered by Ca2+ plus insulin occurred more efficiently than that in cells with little or no AS160. Series of mutational analyses revealed that these synergizing actions rely on the phosphotyrosine-binding 1 (PTB1) and calmodulin-binding domains of Tbc1d1 as well as key phosphorylation sites of both AS160 (Thr642) and Tbc1d1 (Ser237 and Thr596). Thus, the emerging cooperative governance relying on the multiple regulatory nodes of both Tbc1d1 and AS160, functioning together, plays a key role in properly deciphering biochemical signals into a physical GLUT4 release process in response to insulin, exercise, and the two in combination.
Highlights
Akt substrate of 160 kDa (AS160) and Tbc1d1 are key Rab GTPase–activating proteins (RabGAPs) that mediate release of static GLUT4 in response to insulin or exercise-mimetic stimuli, respectively, but their cooperative regulation and its underlying mechanisms remain unclear
We found that cells with an increased intensity ratio of Tbc1d1 relative to AS160 showed dampening of insulin-responsive increases in the speed of GLUT4 movement; in other words, by plotting insulin-induced percentage increases in the mean speed of GLUT4 movement, which resulted from GLUT4 liberation, versus the relative ratio of Tbc1d1 to AS160 obtained from each cell, we identified a negative correlation between these two values (Fig. 1, D and E)
Our present study provides compelling evidence that AS160 and Tbc1d1 cooperatively regulate stimuli-responsive GLUT4releasing activities, based on single-molecule analysis of GLUT4 behavior in cell-based reconstitution models (Fig. 6) and in 3T3-L1 adipocytes
Summary
To analyze GLUT4 behavior in the co-presence of AS160 and Tbc1d1 in detail, we took advantage of the cell-based reconstitution model employing 3T3-L1 fibroblasts exogenously expressing Myc-GLUT4, HA-sortilin, and AS160 and Tbc1d1, the first two alone and the other two in combination (Fig. 1A). Insulin resulted in the liberation of static GLUT4 in the Tbc1d1-expressing cells acquiring regulatory mode shift status by prior exposure to AICAR, and statistically significant enhancement of the speed of movement was observed at 30 min, but not at 5 min, of insulin stimulation (Fig. 3B, left). In differentiated adipocytes expressing exogenous Tbc1d1 (by transfection) with marginal AS160 (by siRNAmediated knockdown) due to prior AICAR treatment, insulin liberated static GLUT4 at 30 min (Fig. S4A, left), indicating that the regulatory mode shift can be recapitulated in 3T3-L1 adipocytes under this experimental condition. We detected significant liberation of static GLUT4 after 10 min of exposure to elevated [Ca2ϩ]i in cells expressing Tbc1d1 accompanied by insulin (Fig. 4B, left), implying that [Ca2ϩ]i changes induced the regulatory mode shift [9]. Dynamic GLUT4 repackaging is not always necessary for rendering the regulatory mode shift phenomenon
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
