Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Nov 16;107(46):19909-14.
doi: 10.1073/pnas.1009523107. Epub 2010 Nov 1.

Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells

Yi Sun  1 , Philip J Bilan, Zhi Liu, Amira Klip
Affiliations

Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells

Yi Sun et al. Proc Natl Acad Sci U S A. .

Abstract

Skeletal muscle is the primary site of dietary glucose disposal, a function that depends on insulin-mediated exocytosis of GLUT4 vesicles to its cell surface. In skeletal muscle and adipocytes, this response involves Akt signaling to the Rab-GAP (GTPase-activating protein) AS160/TBC1D4. Intriguingly, the AS160-targeted Rabs appear to differ, with Rab8A participating in GLUT4 exocytosis in muscle cells and Rab10 in adipocytes, and their activation by insulin is unknown. Rabs 8A, 10, and 13 belong to the same subfamily of Rab-GTPases. Here we show that insulin promotes GTP loading of Rab13 and Rab8A but not Rab10 in rat L6 muscle cells, Rab8A activation preceding that of Rab13. siRNA-mediated Rab13 knockdown blocked the insulin-induced increase of GLUT4 at the muscle cell surface that was rescued by a Rab13 ortholog but not by Rab8A. Constitutively active AS160 lowered basal and insulin-stimulated levels of surface GLUT4, effects that were reversed by overexpressing Rab8A or Rab13, suggesting that both Rabs are targets of AS160-GAP activity in the context of GLUT4 traffic. Rab13 had a broader intracellular distribution compared with the perinuclear restriction of Rab8A, and insulin promoted Rab13 colocalization with GLUT4 at the cell periphery. We conclude that Rab13 and Rab8A are Rab-GTPases activated by insulin, and that downstream of AS160 they regulate traffic of GLUT4 vesicles, possibly acting at distinct steps and sites. These findings close in on the series of events regulating muscle GLUT4 traffic in response to insulin, crucial for whole-body glucose homeostasis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Rab13 mRNA is expressed in muscle and fat. RT-PCR detection of Rab13, Rab8A, and Rab10 in total RNA from rat L6GLUT4myc (L6) and mouse C2C12 muscle cells; rat (R) and mouse (M) skeletal muscle (SKM); mouse 3T3-L1 adipocytes (3T3-L1); rat and mouse perigonadal white adipose tissue (WAT); and analysis by agarose gel electrophoresis. Rab13 detection was confirmed by sequencing the PCR products from L6.
Fig. 2.
Fig. 2.
Rab8A and Rab13, but not Rab10 are GTP-loaded in response to insulin. L6-GLUT4myc-IR myoblasts were transiently transfected with GFP-Rab8A, GFP-Rab13, or GFP-Rab10 for 48 h. Serum-deprived cells (3 h) were permeabilized to introduce the biotinylated GTP-photoprobe before incubation with insulin (100 nM) for the indicated times, followed by UV illumination. Cells were immediately processed for pull-down with streptavidin resin, SDS/PAGE, and immunoblotting with anti-GFP antibody to detect GFP-Rab as detailed in Methods. Representative gels and the fold change of GTP-loaded GFP-Rab relative to basal are illustrated for (A) GFP-Rab8A, (B) GFP-Rab13, and (C) GFP-Rab10. *P < 0.05 and ***P < 0.001 versus basal. Other statistical comparisons are shown by brackets.
Fig. 3.
Fig. 3.
Rab13 knockdown prevents insulin-induced GLUT4myc translocation to the plasma membrane without affecting basal levels of surface GLUT4myc. L6-GLUT4myc (A and B) or L6-GLUT4myc-AS160 (C) myoblasts were transfected with the indicated siRNA to rat Rab13 (siRab13) or nonrelated (nontargeting) siRNA (siNR) for 72 h. Cells were serum-deprived for 3 h before treatment without (basal, open bars) or with insulin (100 nM, 20 min, filled bars) and lysed to detect (A) Rab13 and loading control actinin1; (B) Rab8, phospho-Akt S473 (p-Akt), and actinin1; and (C) phospho-T642-AS160 (p-AS160), p-Akt, and actinin1. (D) L6-GLUT4myc myoblasts were transfected with siRNA for 24 h and then retransfected with GFP-Rab8A (canine), GFP-Rab13 (human), or GFP cDNA for 24 h and processed for cell-surface GLUT4myc levels as described in Methods. Shown are mean ± SEM fold changes from four experiments. ***P < 0.001 versus basal in siNR+GFP-transfected myoblasts. Other statistical comparisons are shown by brackets.
Fig. 4.
Fig. 4.
Rab8A and Rab13 relieve the repression of basal and insulin-stimulated cell-surface GLUT4myc levels caused by AS160-4A. L6-GLUT4myc myoblasts were cotransfected with Flag-tagged AS160-4A plus GFP-Rab8A, GFP-Rab13, or GFP for 24 h, and then incubated without (open bars) or with insulin (filled bars) and processed for cell-surface GLUT4myc levels as described in Methods. Results are the mean ± SEM fold change over basal in cells expressing AS160-4A and indicated GFP-Rab from four experiments. ***P < 0.001 versus basal in GFP-transfected control myoblasts. Other statistical comparisons are shown by brackets.
Fig. 5.
Fig. 5.
Rab13 colocalizes with GLUT4myc in perinuclear regions in basal conditions and at the cell periphery upon insulin stimulation. L6-GLUT4myc myoblasts were transfected with (A) GFP-Rab8A or (B) GFP-Rab13 for 24 h. Basal or insulin-stimulated (100 nM, 5 and 15 min) cells were permeabilized and processed for confocal fluorescence microscopy as described in Methods: GFP-Rab (green) or GLUT4myc (red). Shown are representative images of merged signals at the middle and dorsal optical planes from basal and insulin-treated cells from three experiments. Insets show magnifications of the boxed areas of interest. (Scale bars, 10 μm.) The pixel coincidence analysis of GLUT4myc with each GFP-Rab at the dorsal plane in insulin-stimulated cells is shown in grayscale.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zaid H, Antonescu CN, Randhawa VK, Klip A. Insulin action on glucose transporters through molecular switches, tracks and tethers. Biochem J. 2008;413:201–215. - PubMed
    1. Karylowski O, Zeigerer A, Cohen A, McGraw TE. GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes. Mol Biol Cell. 2004;15:870–882. - PMC - PubMed
    1. Govers R, Coster AC, James DE. Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway. Mol Cell Biol. 2004;24:6456–6466. - PMC - PubMed
    1. Seabra MC, Wasmeier C. Controlling the location and activation of Rab GTPases. Curr Opin Cell Biol. 2004;16:451–457. - PubMed
    1. Stenmark H, Olkkonen VM. The Rab GTPase family. Genome Biol. 2001;2 REVIEWS3007. - PMC - PubMed

Publication types

MeSH terms