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

Abstract

Insulin 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 µm), 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.

Keywords: Adipocyte; GLUT4; Insulin; Intracellular trafficking; Rab14.

Fig. 1.

Rab14 gives rise to enlarged vesicular structures containing Rab and GLUT4. (A) 3T3-L1 adipocytes were serum starved for 3 hours, fixed, permeabilised and stained with antibodies against Rab14 and GLUT4. White arrowheads indicate puncta positive for Rab14 and GLUT4. (B) 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP and mCherry-Rab14WT. After 24 hours, cells were serum starved for 3 hours and fixed. (C) 3T3-L1 adipocytes were electroporated with a plasmid encoding GFP-Rab14WT. After 24 hours, cells were serum starved for 3 hours, fixed, permeabilised, and stained with an antibody against GLUT4. (D) 3T3-L1 adipocytes were electroporated with plasmids encoding mCherry-Rab14WT and IRAP-GFP. After 24 hours, cells were serum starved for 3 hours and fixed. All cells were imaged by confocal microscopy. White arrowheads indicate enlarged vesicular structures positive for Rab14 and either GLUT4 or IRAP. Scale bars: 10 µm.

Fig. 2.

Rab14Q70L and Rab14WT, but not Rab14S25N, give rise to enlarged vesicular structures containing Rab14 and GLUT4. (A) 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP and either mCherry, mCherry-Rab8AQ67A, mCherry-Rab10Q68A, mCherry-Rab11Q70L, mCherry-Rab14Q70L or mCherry-Rab31Q64L. After 24 hours, cells were serum starved for 3 hours, stimulated for 30 minutes with 87 nM insulin as required, fixed and imaged by confocal microscopy. Shown are the mean number of enlarged vesicular structures positive for Rab and GLUT4 (or in the case of mCherry alone, positive for mCherry and GLUT4) per cell ± s.e.m. from three independent experiments, with 20 cells analysed per experiment. (B) 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP and either mCherry, mCherry-Rab14Q70L, mCherry-Rab14WT or mCherry-Rab14S25N. After 24 hours, cells were serum starved for 3 hours, fixed and imaged by confocal microscopy. Shown are the mean number of enlarged vesicular structures positive for Rab14 and GLUT4 (or in the case of mCherry alone, positive for mCherry and GLUT4) per cell ± s.e.m. from three independent experiments, with 20 cells analysed per experiment. *P<0.05 versus Rab14Q70L.

Fig. 3.

Rab14Q70L enlarged vesicular structures contain predominantly early endosomal markers. (A) 3T3-L1 adipocytes were electroporated with plasmids encoding mCherry-Rab14Q70L and GFP-2×FYVE/Hrs. After 24 hours, cells were serum starved for 3 hours and fixed. (B) 3T3-L1 adipocytes were electroporated with plasmids encoding GFP-Rab14Q70L and the human transferrin receptor. After 24 hours, cells were serum starved for 3 hours, incubated for 30 minutes with 30 µg/ml transferrin Alexa Fluor 633 and fixed. (C) 3T3-L1 adipocytes were electroporated with plasmids encoding mCherry-Rab14Q70L and GFP-CI-MPR. After 24 hours, cells were serum starved for 3 hours and fixed. (D) 3T3-L1 adipocytes were electroporated with a plasmid encoding mCherry-Rab14Q70L. After 24 hours, cells were serum starved for 3 hours, fixed, permeabilised and stained with an antibody against TGN38. All cells were imaged by confocal microscopy. White arrowheads indicate enlarged vesicular structures positive for Rab14 and the compartment marker. Scale bars: 10 µm.

Fig. 4.

Rab14Q70L enlarged vesicular structures contain predominantly early endosomal associated Rabs. (A–D) 3T3-L1 adipocytes were electroporated with plasmids encoding mCherry-Rab14Q70L and GFP-Rabs 4WT, 5WT, 7WT and 11WT. After 24 hours, cells were serum starved for 3 hours, fixed and imaged by confocal microscopy. White arrowheads indicate enlarged vesicular structures positive for Rab14 and the respective Rab. Scale bars: 10 µm.

Fig. 5.

Rab14Q70L enlarged vesicular structures contain no intra-lumenal vesicles. 3T3-L1 adipocytes were electroporated with a plasmid encoding GFP-Rab14Q70L. After 24 hours, cells were serum starved for 3 hours, fixed, embedded in gelatin and copper finder grids prepared, as described in Materials and Methods. (A) Rab14 enlarged vesicular structures were identified by widefield fluorescence microscopy. The white arrowhead indicates an enlarged vesicular structure positive for Rab14. Scale bar: 10 µm. (B) Grids were labelled with an antibody against GFP and 15 nm gold, and imaged by transmission electron microscopy. Black dots indicate 15 nm gold.

Fig. 6.

Rab14Q70L enlarged vesicular structures are accessible to internalised GLUT4. 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4 and mCherry-Rab14Q70L. After 24 hours, cells were serum starved for 3 hours, stimulated for 30 minutes with 87 nM insulin, incubated with an antibody against the HA epitope for 30 minutes at 15°C and incubated at 37°C for 10 minutes prior to fixation. Fixed cells were permeabilised and HA visualised using a fluorescently labelled secondary antibody. White arrowheads indicate enlarged vesicular structures positive for Rab14 and internalised GLUT4. Scale bar: 10 µm.

Fig. 7.

Rab14Q70L reduces cell surface GLUT4 in the basal and insulin-stimulated states. 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP and either mCherry or mCherry-Rab14Q70L. After 24 hours, cells were serum starved for 3 hours, stimulated for 30 minutes with 87 nM insulin, as required, and fixed. Fixed, non-permeabilised cells were stained with an antibody against the HA epitope to detect cell surface GLUT4. Cells were imaged and GLUT4 translocation measured as described in Materials and Methods. Shown are the mean amounts of cell surface GLUT4 ± s.e.m. from three independent experiments, with 20 cells analysed per experiment. The values in each experiment have been normalised to a value of 1 for the control with insulin. *P<0.05 versus control basal, ∧P<0.05 versus control insulin.

Fig. 8.

GLUT4 collects in early endocytic compartments in the absence of Rab14. (A) Lysates of either Rab14 knockdown or scrambled 3T3-L1 adipocytes were immunoblotted for Rab14 or α tubulin (loading control). (B) Rab14 knockdown or scrambled 3T3-L1 adipocytes expressing HA-GLUT4-GFP were serum starved for 3 hours and stimulated for 20 minutes with 87 nM insulin. Cells were incubated with anti-HA antibody, rabbit anti-mouse bridging antibody and 5 nm Protein A gold for 20 minutes at 15°C. Cells were warmed to 37°C for 60 minutes before fixation, processing for transmission electron microscopy and quantification, as described in Materials and Methods. The graph represents the amount of gold in each compartment as a percentage of total gold (mean ± s.e.m.; *P<0.05 or **P<0.01 by Kruskal–Wallis ANOVA). Data are derived from two independent experiments with 11 and 9 scrambled and Rab14 knockdown cells analysed, respectively.