Progress in the research of insulin-stimulated GLUT4 translocation has revealed a model in which AS160 and Rab10 play important roles in the regulation of GLUT4 translocation in adipocytes [3
]. AS160 has a functional GAP domain, which remains active in the basal state [3
]. Insulin-activated Akt phosphorylation of AS160 inhibits its GAP activity, thus shifting more Rab10 to the active GTP-bound form. GTP-bound Rab10 could recruit a variety of Rab effectors to facilitate the transport of GLUT4 storage vesicles towards the PM [8
Apparent colocalization of GLUT4 and Rab10 revealed in the present study is consistent with the report by Sano et al. [8
], where Rab10 was found at GSVs purified from 3T3-L1 adipocytes with anti-GLUT4 antibody. In combination with previous findings that the GAP domain of AS160 shows activity against Rab10 [5
], that overexpression of Rab10(Q67L), which favours the GTP-binding form, increases GLUT4 on the cell surface in the absence of insulin stimulation [9
], and that Rab10 knock-down inhibits insulin-stimulated GLUT4 translocation [8
], the model centring AS160 and Rab10 in GLUT4 translocation in adipocytes is firmly favoured, although potential roles of other Rab proteins should also not be neglected [5
Aggregation of GLUT4, Rab10 and both GDIs at the TGN reveals that the TGN might be the donor membrane where Rab10 initiates GSV formation. This hypothesis has previously been proposed in some of the models depicting GLUT4 translocation [2
]. Supportive evidence includes that a large fraction of GLUT4 is distributed at the TGN [35
], that internalized GLUT4 rapidly traverses the endosomal system en route to Syntaxin 6 and 16-positive TGN [36
], that newly synthesized GLUT4 enters GSVs at the TGN in a GGA [Golgi-associated γ-adaptin ear homology domain Arf (ADP-ribosylation factor)-interacting protein]-dependant manner [37
], and that disturbance of the function of Syntaxin 6, a TGN-localized protein, affects GLUT4 translocation [39
]. Although previous studies using BFA to interfere with TGN function gave ambiguous results about the role of the TGN in GLUT4 translocation [23
], in the present study it was found that BFA treatment partially inhibits insulin-stimulated GLUT4 translocation (). Although the inhibitory effect is minor, it is noteworthy that a model assigning GSV formation at the TGN does not imply that the TGN-localized GLUT4 would be directly translocated to the cell surface in response to insulin stimulation. GSVs represent the standing insulin-sensitive pool of GLUT4 and have already been formed in the resting state. They are capable of redistributing to the cell surface in a short time when stimulated with insulin. In this way, it should not be expected that the disturbance of TGN function with BFA could produce a severe inhibition of insulin-stimulated GLUT4 translocation.
Although both GDI-1 and GDI-2 could bind to a wide range of Rab proteins [22
], either GDI shows distinct functional roles in the context of living cells [21
]. Specifically, the expression level of the two GDIs is different, with a higher expression level of GDI-1 in 3T3-L1 adipocytes [22
], and their subcellular distribution is also distinct [21
]. The demonstration that GDI-1 binds to Rab10 with higher affinity, whereas GDI-2 favours Rab4A, in the present study, reveals a certain selectivity of GDIs towards different Rab proteins and probably implies a general mechanism underlying the interaction between GDIs and Rab proteins in the cell.
Overexpression of GDI inhibits insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These results confirm the participation of GDIs in insulin-stimulated GLUT4 translocation. The defect probably lies in that overexpressed GDI could compete with downstream Rab effectors, e.g. GDFs (GDI-displacement factors) [6
], in binding Rab10, and then sequester Rab10 in its GDP-bound form in the cytosol. Thus inefficient delivery of Rab10 back to the donor membrane could result in reduced GLUT4 translocation. Moreover, it is noteworthy that overexpression of GDI-2 inhibited insulin-stimulated GLUT4 translocation to a similar extent to GDI-1, although its affinity to Rab10 is lower. This may imply that some other Rab protein(s) (e.g. Rab4A) favoured by GDI-2 is also involved in GLUT4 translocation, and the inhibition of which by overexpressed GDI-2 also causes reduction in insulin-stimulated GLUT4 translocation.
Taken together, our results propose that the TGN could be the donor membrane of insulin-stimulated GLUT4 translocation, and GDI-1 is preferably involved in insulin-stimulated GLUT4 translocation through interaction with Rab10.