A role for Rab14 in the endocytic trafficking of GLUT4 in 3T3-L1 adipocytes

Abstract

SummaryInsulin enhances the uptake of glucose into adipocytes and muscle cells by promoting the redistribution of the glucose transporter isoform 4 (GLUT4) from intracellular compartments to the cell surface. Rab GTPases regulate the trafficking itinerary of GLUT4 and several have been found on immunopurified GLUT4 vesicles. Specifically, Rab14 has previously been implicated in GLUT4 trafficking in muscle although its role, if any, in adipocytes is poorly understood. Analysis of 3T3-L1 adipocytes using confocal microscopy demonstrated that endogenous GLUT4 and endogenous Rab14 exhibited a partial colocalisation. However, when wild-type Rab14 or a constitutively-active Rab14Q70L mutant were overexpressed in these cells, the colocalisation with both GLUT4 and IRAP became extensive. Interestingly, this colocalisation was restricted to enlarged 'ring-like' vesicular structures (mean diameter 1.3 mm), which were observed in the presence of overexpressed wild-type Rab14 and Rab14Q70L, but not an inactive Rab14S25N mutant. These enlarged vesicles contained markers of early endosomes and were rapidly filled by GLUT4 and transferrin undergoing endocytosis from the plasma membrane. The Rab14Q70L mutant reduced basal and insulin-stimulated cell surface GLUT4 levels, probably by retaining GLUT4 in an insulin-insensitive early endosomal compartment. Furthermore, shRNA-mediated depletion of Rab14 inhibited the transit of GLUT4 through early endosomal compartments towards vesicles and tubules in the perinuclear region. Given the previously reported role of Rab14 in trafficking between endosomes and the Golgi complex, we propose that the primary role of Rab14 in GLUT4 trafficking is to control the transit of internalised GLUT4 from early endosomes into the Golgi complex, rather than direct GLUT4 translocation to the plasma membrane.

Insulin-regulated Glut4 Translocation

“…The fact that LRP1 also cycles through this slow pathway indicates that it may be a recycling intermediate on the late endosome/lysosome degradative pathway. This is consistent with electron microscopy data (7,48). The fact that there are both fast and slow exocytic pathways means that a possible mechanism to increase cell surface Glut4 (or LRP1) would be to redirect Glut4 from the slow constitutive recycling pathway through the ERC into the more rapidly recycling Tf receptor pathway.…”

“…Consistent with this, knockdown of the AS160 substrates Rab10 and Rab14 affect sequestration of Glut4 and decrease cell surface Glut4 in adipocytes. 7 Modeling and simulations of this data indicate that Rab10 is required for release of Glut4 from sequestration in GSVs, whereas Rab14 is required in packaging/sorting of proteins from endosomes into GSVs, consistent with recent microscopy studies (40,48). Interestingly, although Glut4 traffics through specialized GSVs in muscle cells, kinetics studies show that this compartment is not as tightly regulated as in adipocytes (49,50).…”

Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation

“…Therefore, there may be multiple functional interactions between Rab10 and effectors that take place during the rate-limiting vesicle priming/fusion step in Glut4 exocytosis. Consistent with the known function of these Rab10 effector proteins, Rab10 knockdown decreased the accumulation of Glut4 in and near the plasma membrane after insulin stimulation (22)(23)(24). However, it did not inhibit the insulin-stimulated increase in Glut4 insertion efficiency, indicating that fusion itself is regulated through an additional mechanism downstream of the GTP-loading/activation of Rab10, perhaps through direct regulation of the Rab10 effectors (11,15,16) or through other elements of the downstream fusion machinery (50).…”

“…Loss of Rab14 also attenuates insulin-stimulated Glut4 translocation in adipocytes, whereas expression of an AS160-resistant mutant acts as a dominant negative inhibitor (22)(23)(24). Likewise, depletion of Rab8 and -14 inhibits Glut4 translocation in muscle cells (25)(26)(27).…”

Rab Proteins and the Compartmentalization of the Endosomal System

“…The interconnection of Rab GTPases in cascades may incorporate entire circuits involved in specific physiological responses, connecting exocytosis to cargo return via endocytosis. This is particularly evident in specialized pathways such as the coordinate function of exocytic and endocytic Rab GTPases in transport to the primary cilium, insulin-stimulated GLUT4 vesicle secretion, and recycling, as well as responses to metabolic stress that trigger autophagy or lipid droplet formation (Sano et al 2008;Longatti and Tooze 2009;Murphy et al 2009;Ward et al 2011;Westlake et al 2011;Chen et al 2012;Longatti et al 2012;Reed et al 2013). …”