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. 2013 Jun 14;288(24):17520-31.
doi: 10.1074/jbc.M113.467647. Epub 2013 May 2.

Rac-1 superactivation triggers insulin-independent glucose transporter 4 (GLUT4) translocation that bypasses signaling defects exerted by c-Jun N-terminal kinase (JNK)- and ceramide-induced insulin resistance

Tim Ting Chiu  1 , Yi Sun, Alexandra Koshkina, Amira Klip
Affiliations

Rac-1 superactivation triggers insulin-independent glucose transporter 4 (GLUT4) translocation that bypasses signaling defects exerted by c-Jun N-terminal kinase (JNK)- and ceramide-induced insulin resistance

Tim Ting Chiu et al. J Biol Chem. .

Abstract

Insulin activates a cascade of signaling molecules, including Rac-1, Akt, and AS160, to promote the net gain of glucose transporter 4 (GLUT4) at the plasma membrane of muscle cells. Interestingly, constitutively active Rac-1 expression results in a hormone-independent increase in surface GLUT4; however, the molecular mechanism and significance behind this effect remain unresolved. Using L6 myoblasts stably expressing myc-tagged GLUT4, we found that overexpression of constitutively active but not wild-type Rac-1 sufficed to drive GLUT4 translocation to the membrane of comparable magnitude with that elicited by insulin. Stimulation of endogenous Rac-1 by Tiam1 overexpression elicited a similar hormone-independent gain in surface GLUT4. This effect on GLUT4 traffic could also be reproduced by acutely activating a Rac-1 construct via rapamycin-mediated heterodimerization. Strategies triggering Rac-1 "superactivation" (i.e. to levels above those attained by insulin alone) produced a modest gain in plasma membrane phosphatidylinositol 3,4,5-trisphosphate, moderate Akt activation, and substantial AS160 phosphorylation, which translated into GLUT4 translocation and negated the requirement for IRS-1. This unique signaling capacity exerted by Rac-1 superactivation bypassed the defects imposed by JNK- and ceramide-induced insulin resistance and allowed full and partial restoration of the GLUT4 translocation response, respectively. We propose that potent elevation of Rac-1 activation alone suffices to drive insulin-independent GLUT4 translocation in muscle cells, and such a strategy might be exploited to bypass signaling defects during insulin resistance.

Keywords: AS160; Akt; Ceramide; Glut4; Insulin Resistance; Jun N-terminal Kinase (JNK); Rac1.

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Figures

FIGURE 1.
FIGURE 1.
Overexpression of CA-Rac-GFP or Tiam1-GFP stimulates GLUT4 translocation to the membrane of muscle cells. A and B, GLUT4myc myoblasts were transfected with GFP, WT-Rac-GFP, or CA-Rac-GFP followed by stimulation with/without insulin for 10 min to measure changes in surface GLUT4myc and quantified relative to GFP basal signal (mean ± S.E. (error bars); *, p < 0.05). C, myoblasts transfected with GFP, CA-Rac-GFP, or Tiam1-GFP were permeabilized and stained with rhodamine-phalloidin to visualize changes in F-actin. D and E, GLUT4myc myoblasts expressing GFP or Tiam1-GFP were stimulated with/without insulin for 10 min followed by staining for surface GLUT4myc and quantification relative to GFP basal signal (mean ± S.E. (error bars); *, p < 0.05). Representative images of three independent experiments are shown. Bar, 10 μm.
FIGURE 2.
FIGURE 2.
Acute Rac-1 activation via rapamycin-triggered heterodimerization induces GLUT4 translocation in muscle cells. YF, YF-CA-Rac, or YF-Tiam1 was co-transfected with/without Lyn-FRB in myoblasts. Cells were treated with/without rapamycin for 10 min followed by rounded-up myoblast assay and detecting the redistribution of the YFP signal (A), permeabilization and staining with rhodamine-phalloidin to measure changes in F-actin (B), and measurement of surface GLUT4myc (C and D). Changes were compared with those in YFP-expressing myoblasts stimulated with/without insulin and quantified relative to the YFP basal signal (mean ± S.E. (error bars); *, p < 0.05). Representative images of three independent experiments are shown. Bar, 10 μm.
FIGURE 3.
FIGURE 3.
Overexpression of CA-Rac-GFP results in a modest increase in plasma membrane PI(3,4,5)P3 and Akt phosphorylation without engaging IRS-1. A, HA-IRS-1 was co-transfected with GFP or CA-Rac-GFP before stimulating myoblasts with/without insulin for 10 min followed by immunoprecipitation (IP) using anti-HA antibody. Immunoprecipitates were stained with anti-Tyr(P) antibody to detect phosphorylation of IRS-1. B and C, PH-Akt-RFP was co-expressed along with GFP, WT-Rac-GFP, or CA-Rac-GFP. Rounded-up myoblasts were generated and stimulated for 10 min with/without insulin after which the redistribution of the PH-Akt-RFP to the membrane was determined. The plasma membrane to cytosolic (PM/Cyto) ratio of RFP in each condition was quantified and is expressed relative to the percentage of the parallel, maximal insulin response in units normalized to GFP-expressing control cells. D–F, myoblasts expressing HA-Akt + GFP, WT-Rac-GFP, or CA-Rac-GFP were stimulated with/without insulin for 10 min before immunoprecipitation with HA antibody. Immunoprecipitates were analyzed for P-Akt using anti-Thr(P)-308 or -Ser(P)-473 antibodies. Changes in P-Akt/total HA-Akt in each condition were quantified and are presented as the percentage of the parallel, maximal insulin response in units normalized to the GFP-expressing control. G, GLUT4myc myoblasts transfected with Lyn-FRB + YF-CA-Rac were pretreated with DMSO, 100 nm WM, 10 μm Akti1/2, 200 nm LB, or 10 μm compound C (CC) for 20 or 30 min (Akti1/2 and compound C) followed by 10-min rapamycin treatment in the presence of inhibitors. The changes in surface GLUT4myc were quantified and are illustrated as the percentage of the maximal rapamycin-induced response relative to DMSO-treated control cells (mean ± S.E. (error bars); *, p < 0.05 versus DMSO). Representative images of three to four independent experiments are shown. Bar, 10 μm. IB, immunoblot.
FIGURE 4.
FIGURE 4.
AS160 is phosphorylated upon CA-Rac-GFP overexpression and is required for the Rac-1-induced gain in surface GLUT4. A and B, FLAG-AS160 was co-expressed with GFP, WT-Rac-GFP, or CA-Rac-GFP before immunoprecipitation (IP) with anti-FLAG antibody. Immunoprecipitates were stained with phosphorylated Akt substrate (PAS) antibody to detect changes in AS160 phosphorylation. The intensity of phosphorylated Akt substrate/total FLAG-AS160 in each condition was quantified and is depicted as the percentage of the parallel, maximal insulin response in units normalized to the GFP-expressing control. C and D, FLAG-AS160–4A was co-transfected with GFP or CA-Rac-GFP followed by stimulation with/without insulin (INS) to measure changes in surface GLUT4. Quantification of each condition is presented as the percentage of the parallel, maximal insulin response normalized to GFP-expressing insulin-stimulated cells (mean ± S.E. (error bars); *, p < 0.05). Representative images of three independent experiments are shown. Bar, 10 μm. IB, immunoblot.
FIGURE 5.
FIGURE 5.
Signaling defects imposed by CA-JNK- and C2-ceramide-induced insulin resistance do not impede the Rac-1-mediated gain in surface GLUT4. A, HA-IRS-1 was co-transfected with FLAG or FLAG-CA-JNK followed by immunoprecipitation (IP) with HA antibody to detect changes in Ser-307 phosphorylation and degradation of IRS-1. B and C, GLUT4myc myoblasts co-expressing FLAG-CA-JNK + GFP or CA-Rac-GFP were stimulated with/without insulin followed by surface GLUT4 staining. Surface GLUT4myc in each condition was quantified and is presented as the percentage of the maximal, parallel insulin response normalized to GFP-expressing, insulin-stimulated cells. D, GLUT4myc myoblasts co-expressing Lyn-FRB + YF or YF-CA-Rac were pretreated with DMSO or 50 μm C2-ceramide for 2 h followed by treatment with/without insulin or rapamycin (Rap) before staining for surface GLUT4. Results in each condition were quantified and are represented as the percentage of the maximal, parallel insulin response relative to DMSO-treated, YF-expressing insulin-stimulated cells (mean ± S.E. (error bars); *, p < 0.05). Representative images of three independent experiments are shown. Bar, 10 μm.
FIGURE 6.
FIGURE 6.
Model of Rac-1 superactivation-induced GLUT4 translocation in muscle cells. Left, normal insulin response observed in cells expressing endogenous levels of Rac-1 (see Refs. and 79). Right, Rac-1 superactivation promotes membrane accumulation of PI(3,4,5)P3, which results in moderate phosphorylation of Akt and significant phosphorylation of AS160. These signaling components together with actin remodeling satisfied the molecular requirements for GLUT4 translocation without input of insulin.
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References

    1. Baron A. D., Brechtel G., Wallace P., Edelman S. V. (1988) Rates and tissue sites of non-insulin- and insulin-mediated glucose uptake in humans. Am. J. Physiol. Endocrinol. Metab. 255, E769–E774 - PubMed
    1. Foley K., Boguslavsky S., Klip A. (2011) Endocytosis, recycling, and regulated exocytosis of glucose transporter 4. Biochemistry 50, 3048–3061 - PubMed
    1. Huang C., Thirone A. C., Huang X., Klip A. (2005) Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes. J. Biol. Chem. 280, 19426–19435 - PubMed
    1. Patel N., Rudich A., Khayat Z. A., Garg R., Klip A. (2003) Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells. Mol. Cell. Biol. 23, 4611–4626 - PMC - PubMed
    1. Somwar R., Niu W., Kim D. Y., Sweeney G., Randhawa V. K., Huang C., Ramlal T., Klip A. (2001) Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport. J. Biol. Chem. 276, 46079–46087 - PubMed

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