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Signal Transduction

Ceramide- and Oxidant-Induced Insulin Resistance Involve Loss of Insulin-Dependent Rac-Activation and Actin Remodeling in Muscle Cells

  1. Lellean JeBailey1,2,
  2. Oshrit Wanono1,
  3. Wenyan Niu1,
  4. Jessica Roessler1,
  5. Assaf Rudich1 and
  6. Amira Klip1,2
  1. 1Programme in Cell Biology, the Hospital for Sick Children, Toronto, Ontario, Canada
  2. 2Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
  1. Address correspondence and reprint requests to Dr. Amira Klip, Programme in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8. E-mail: amira{at}sickkids.ca
Diabetes 2007 Feb; 56(2): 394-403. https://doi.org/10.2337/db06-0823
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  • FIG. 1.
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    FIG. 1.

    Glucose oxidase (GO) and ceramide (C2) reduce GLUT4 translocation and glucose uptake in L6 myotubes. A: Glucose oxidase caused a dose-dependent increase in H2O2 in the medium. B and C: Glucose oxidase dose-dependent inhibition of insulin-induced gain in surface GLUT4myc (B) or in 2-deoxyglucose uptake (C). D: NAC prevents the glucose oxidase–induced decrease in insulin-induced GLUT4 translocation. E and F: C2-ceramide dose-dependent inhibition of insulin-induced gain in surface GLUT4myc (E) or in 2-deoxyglucose uptake (F). Results in B–F are expressed as the percent of maximal insulin response observed in the absence of glucose oxidase or C2-ceramide. #P < 0.001, *P < 0.05, *P < 0.01 (n = 4–13), relative to the insulin response in absence of glucose oxidase or C2-ceramide. Basal surface GLUT4myc levels were not reduced by glucose oxidase or C2-ceramide (see supplemental Tables 1 and 2).

  • FIG. 2.
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    FIG. 2.

    Glucose oxidase treatment prevents insulin-induced actin remodeling. Myotubes pretreated without or with 6.25–100 mU/ml glucose oxidase (GO) were stimulated for 10 min with 100 nmol/l insulin where indicated. Cells were then fixed and actin filaments stained with rhodamine-phalloidin, and then images were collected by confocal fluorescence microscopy. A: Organized actin stress fibers in unstimulated (Basal) cells. B: Insulin-induced actin remodeling. Glucose oxidase doses progressively inhibited insulin-induced actin remodeling (F, H, J, and L) without affecting basal-state stress fibers (E, G, I, and K). Less than half of the fields examined showed any of the small actin bundles illustrated in F. See also supplemental Figs. 2 and 3.

  • FIG. 3.
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    FIG. 3.

    C2-ceramide (C2) treatment prevents insulin-induced actin remodeling. Myotubes pretreated without or with 6.25–100 μmol/l C2-ceramide were stimulated for 10 min with 100 nmol/l insulin where indicated, and otherwise they were processed as in Fig. 2. A: Organized actin stress fibers in unstimulated (Basal) cells. B: Insulin-induced actin remodeling. C2-ceramide doses progressively inhibited insulin-induced actin remodeling (F, H, J, and L) without affecting basal-state stress fibers (E, G, I, and K). See also supplemental Fig. 3.

  • FIG. 4.
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    FIG. 4.

    Dose-dependent inhibition of insulin-induced Rac activation by glucose oxidase (GO) (A) or C2-ceramide (C2) (B). Myotubes were pretreated with glucose oxidase or C2-ceramide and exposed to insulin for 10 min, and then GTP-Rac bound to glutathione-S-transferase–CRIB (Cdc 42/Rac interactive binding) as well as total Rac in lysates were determined by immunoblotting. Representative gels with doses up to 50 mU/ml glucose oxidase or 50 μmol/l C2-ceramide are illustrated. Additional gels tested the effect of higher concentrations. These experiments were repeated 3–12 times, and the averaged results are expressed as a percent of the maximal insulin response in the absence of pretreatments. #P < 0.001, **P < 0.01 relative to the stimulation in the absence of glucose oxidase or C2-ceramide. Basal levels were not altered by glucose oxidase or C2-ceramide (see supplemental Tables 1 and 2).

  • FIG. 5.
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    FIG. 5.

    Glucose oxidase (GO) does not alter whole-cell levels of insulin-induced IRS-1 tyrosine phosphorylation or its association with p85 or PI 3-kinase activity. A: Myotubes pretreated with glucose oxidase or C2-ceramide as in Fig. 1 were incubated without or with insulin for 10 min, followed by lysis, immunoprecipitation with polyclonal anti–IRS-1 or IgG, and immunoblotting for phosphotyrosine or p85. Supernatants were immunoblotted with anti–IRS-1 to ascertain immunoprecipitation efficiency. B: PI 3-kinase activity associated with anti–IRS-1 immunoprecipitates was determined by in vitro incorporation of radioactive ATP into PI. Shown are representative thin-layer chromatography plates and averaged results of seven experiments quantifying the fold insulin response normalized to basal activity in the absence of treatments. ns, not statistically significant.

  • FIG. 6.
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    FIG. 6.

    Glucose oxidase (GO) and C2-ceramide (C2) inhibit insulin-induced Akt phosphorylation on Thr308 and Ser473. Myotubes retreated with glucose oxidase or C2-ceramide were incubated without or with insulin for 10 min, followed by lysis and immunoblotting for phosphorylated Akt (pAkt) on Thr308 and Ser473. A and D: Representative gels illustrate the response to 0–100 mU/ml glucose oxidase or 0–100 μmol/l C2-ceramide. Parallel experiments were carried out with 100 μmol/l C2-ceramide and C2-dihydroceramide. The results from 4–12 experiments analyzing Thr308 phosphorylated Akt (B and E) or Ser473 phosphorylated Akt (C and F) in cells pretreated with glucose oxidase (B and C) or C2-ceramide (E and F) were quantified and expressed as a percent of the maximal insulin response observed in the absence of treatments. **P < 0.01,*P < 0.05, #P < 0.001 (n = 4–12) relative to the stimulation in absence of glucose oxidase or C2-ceramide.

  • FIG. 7.
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    FIG. 7.

    Rac1 expression silencing via siRNA inhibits insulin-induced actin remodeling and GLUT4 translocation, but not Akt phosphorylation. Myotubes were exposed to 400 nmol/l of unrelated siRNA (siUR) or Rac1 siRNA (siRac1) for up to 72 h, incubated without or with insulin for 10 min, and used in the following assays. A: Rac expression assessed by immunoblotting of cell lysates. B: Filamentous actin visualization in fixed and permeabilized myotubes. C: Surface GLUT4myc in 15 experiments, expressed as a percent of the maximal insulin response in unrelated siRNA cells, and corresponding fractional expression of Rac. ▪, surface GLUT4myc; □, fractional expression of Rac. #P < 0.001 relative to unrelated siRNA. D: Phosphorylation of Rac-target PAK on Thr423, phosphorylation of Akt on Ser423 and Thr308, and immunoblotting of actin levels to assess protein loading. NT, no treatment; p-Akt, phosphorylated Akt.

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Ceramide- and Oxidant-Induced Insulin Resistance Involve Loss of Insulin-Dependent Rac-Activation and Actin Remodeling in Muscle Cells
Lellean JeBailey, Oshrit Wanono, Wenyan Niu, Jessica Roessler, Assaf Rudich, Amira Klip
Diabetes Feb 2007, 56 (2) 394-403; DOI: 10.2337/db06-0823

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Ceramide- and Oxidant-Induced Insulin Resistance Involve Loss of Insulin-Dependent Rac-Activation and Actin Remodeling in Muscle Cells
Lellean JeBailey, Oshrit Wanono, Wenyan Niu, Jessica Roessler, Assaf Rudich, Amira Klip
Diabetes Feb 2007, 56 (2) 394-403; DOI: 10.2337/db06-0823
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Keywords

GLUT4myc, c-myc epitope–tagged GLUT4
IRS, insulin receptor substrate
NAC, N-acetyl-l-cysteine
PAK, p21-activated kinase
PI, phosphatidylinositol
siRNA, small interfering RNA

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