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. 2015 May 22;290(21):13427-39.
doi: 10.1074/jbc.M114.626077. Epub 2015 Mar 30.

Nox2 mediates skeletal muscle insulin resistance induced by a high fat diet

Alvaro Souto Padron de Figueiredo  1 , Adam B Salmon  2 , Francesca Bruno  1 , Fabio Jimenez  1 , Herman G Martinez  1 , Ganesh V Halade  1 , Seema S Ahuja  3 , Robert A Clark  3 , Ralph A DeFronzo  3 , Hanna E Abboud  3 , Amina El Jamali  4
Affiliations

Nox2 mediates skeletal muscle insulin resistance induced by a high fat diet

Alvaro Souto Padron de Figueiredo et al. J Biol Chem. .

Abstract

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H2O2 decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H2O2 in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H2O2. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.

Keywords: Diabetes; Insulin Resistance; NADPH Oxidase; Reactive Oxygen Species (ROS); Skeletal Muscle; Superoxide Ion.

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Figures

FIGURE 1.
FIGURE 1.
Body weight composition and fat mass gain is similar in Nox2-KO mice and wild-type mice fed a high fat diet. A, body weight of wild-type and Nox2-KO mice fed either a standard or a high fat diet. B and C, lean mass and fat mass of wild-type and Nox2-KO mice fed either a standard or a high fat diet for 3 months (B) or 9 months (C). Lean mass (D) and fat mass (E) of mice fed for 9 months with either SD or HFD expressed in percent of total body weight. Data are the mean ± S.E. of results obtained from 6–10 mice/group. Statistical significance versus WT-SD mice is p <0.001 (***) versus WT-HFD p < 0.05 (#).
FIGURE 2.
FIGURE 2.
Nox2 deletion reduces insulin resistance induced by a high fat diet. In wild-type and Nox2-KO mice fed for 3 months with either a standard or high fat diet, fasting glucose (A), insulin (B), and HOMA-insulin resistance index (C) were determined. Intraperitoneally injected glucose tolerance test (GTT) and insulin tolerance test (ITT) (E) were performed in wild-type and Nox2-KO mice fed for 9 months with either a standard or a high fat diet. Overall insulin tolerance and glucose tolerance are expressed as the area under curve (F and G). Data are the mean ± S.E. of results obtained from 6–10 mice/group. Statistical significance versus WT-SD mice is p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) and versus WT-HFD is p < 0.05 (#) and p < 0.01 (##).
FIGURE 3.
FIGURE 3.
Adipocyte accumulation and increased lean mass in skeletal muscle of Nox2-KO mice fed a high fat diet. A, longitudinal and transverse paraffin sections of gastrocnemius muscle of wild-type and Nox2-KO mice fed either a standard or a high fat diet for 3 months were stained with H&E. B, hind limb fat and lean mass were measured by dual-energy x-ray absorptiometry before and after 3 months of diet. The gain in fat or lean mass is expressed as a percent of total fat or lean mass in the hind limb. C, gastrocnemius muscle fiber typing in wild-type and Nox2-KO mice. mATPase D, MHC I and GAPDH immunoblots were performed using muscle homogenates obtained from wild-type and Nox2 knockout mice fed a standard or a high fat diet. The levels of MHC I were calculated from their individual autoradiographic densities and normalized to the corresponding level of GAPDH. Data are the mean ± S.E. of results obtained from 5 mice/group. Statistical significance versus WT-SD mice is p < 0.05 (*) and p < 0.01 (**) and versus WT-HFD is p < 0.05 (#). A.U., arbitrary units.
FIGURE 4.
FIGURE 4.
Insulin resistance correlates with increased Nox2 expression. A, RNA extracted from frozen gastrocnemius muscle and from RAW cells (left panel) was reverse-transcribed (RTmRNA+) or not (RTmRNA−), then the products and control plasmid containing Nox4 cDNA were subjected to a PCR using primers for mouse p67phox, p47phox, p22phox, Nox2, and Nox4. In addition 50 μg of total protein extract were used to determine p67phox, p47phox, p22phox, Nox2, and Nox4 protein content by immunoblot analysis (right panel). B, wild-type and Nox2-KO mice genotyping (wild type = 240 bp, mutant = 195 bp). Raw cells were used as a positive control for the PCR. C, by quantitative RT-PCR, Nox2 and Nox4 mRNA expression levels were determined in skeletal muscle RNA extracts from mice fed the SD or the HFD for 3 and 9 months. The results were quantified by the ΔCt method using 18S for differences in RNA input and efficiency in cDNA synthesis and are expressed as arbitrary units compared with the average expression levels in WT mice. D, the protein expression levels of Nox2, p22phox, and Nox4 were determined by immunoblot in wild-type and Nox2-KO mice fed either a standard diet or a high fat diet. GAPDH was used as a loading control. Results are expressed as the ratio of the protein of interest and loading control immunoreactivity. All the data are the mean ± S.E. of results obtained from 5–10 mice/group. Statistical significance versus WT-SD mice is p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
FIGURE 5.
FIGURE 5.
Nox2 deletion reduces high fat diet-induced oxidative stress and improves insulin signaling in skeletal muscle tissue. A, confocal images of superoxide production in longitudinal sections of optimal cutting temperature compound (OCT)-embedded frozen gastrocnemius muscle isolated from wild-type mice fed for 3 months with a standard or a high fat diet were taken after 10 min of incubation with DHE. Where indicated, gastrocnemius muscle before imaging was preincubated with diphenylene iodonium or superoxide dismutase. Each pair of photomicrograph panels corresponds to superoxide production (red fluorescence) and the differential interference contrast image (gray picture). The scale bar indicates a length of 10 μm. A.U., arbitrary units. B, confocal images of superoxide production in longitudinal sections of optimal cutting temperature compound (OCT)-embedded frozen gastrocnemius muscle isolated from wild-type and Nox2-KO mice fed for 3 months with either a standard diet or a high fat diet. Fluorescence intensity levels were quantified using image J. Data are representative of results obtained from 6–10 mice/group. Statistical significance versus WT-SD mice is p < 0.001 (***). C, immunoblots were performed using either membrane fractions or homogenates of skeletal muscle tissue isolated from wild-type and Nox2-KO mice fed either a standard or a high fat diet for 9 months. Immunoblots with antibodies against Akt or Glut4 proteins were performed on the membrane fraction of skeletal muscle isolated from mice injected with insulin 15 min before sacrifice. The levels of phospho-Akt and membrane-associated p67phox and Glut4 were calculated from their individual autoradiographic densities and normalized to the corresponding level of Akt, protein disulfide isomerase (PDI), cadherin, or GAPDH. Data are representative of results obtained from 6–10 mice/group. Statistical significance versus WT-SD mice is p < 0.05 (*) and p < 0.01 (**). Statistical significance versus WT-HFD is p < 0.05 (#) and/or p < 0.001 (###).
FIGURE 6.
FIGURE 6.
Nox2 and Nox4 are expressed in C2C12 myoblasts and myotubes. A, digital images of C2C12 cells grown in either a proliferative (10% FBS) or a differentiating (1% FBS) medium were captured. B, differentiated (D) and undifferentiated (U) C2C12 cells were analyzed for myogenin expression by RT-PCR. C, expression of Nox2, Nox4, p22phox, p47phox, and p67phox in undifferentiated and differentiated C2C12 cells were determined by RT-PCR and immunoblot.
FIGURE 7.
FIGURE 7.
Chronic H2O2-dependent insulin resistance. Akt phosphorylation and glucose uptake were determined in myotubes cultured for 48 h with 5 mm (control) or 25 mm (HG) glucose (A and B), 200 μm palmitate (C and D), or with 100 μM H2O2 (E and F) and then stimulated for 20 min with 100 nm insulin. Where indicated, cells were pretreated with catalase (900 units/ml, 10 min) before HG, palmitate, or H2O2 exposure. Phosphorylation of Akt was calculated from individual autoradiographic densities and normalized to the corresponding total Akt levels (A, C, and E). These ratios were determined only in cells stimulated with insulin to accurately determine the effect of HG, palmitate, or H2O2 pretreatment. Glucose uptake was determined as the amount of deoxy-[14C]glucose in cells and expressed as -fold increase compared with values obtained in untreated cells (B, D, and F). Data are representative of results obtained from a total of 4–6 experiments. Statistical significance versus WT-SD mice is p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) and versus cells stimulated with insulin is p < 0.05 (#), p < 0.001 (###), and p < 0.01 (##).
FIGURE 8.
FIGURE 8.
Down-regulation of Nox2 counteracts the insulin resistance induced by high concentration of glucose but not by H2O2. A, RT-PCR was performed to determine the mRNA levels of myogenin and MHC expressed in control and Nox2 shRNA myoblasts (U) and myotubes (D). Akt phosphorylation (B, C, E, F, H, and I) and glucose uptake (D, G, and J) stimulated by insulin (Ins) were determined in control and Nox2 shRNA myotubes treated or not for 48 h with either 5 mm (control) or 25 mm (HG) of glucose (B–D), 200 μm palmitate (E–G), or with 100 μm H2O2 (H–J) and then stimulated for 20 min with 100 nm insulin. Immunoblots with Nox2 and Nox4 antibodies were performed to confirm the specific down-regulation of Nox2 by shRNA (B, E, and H). C, F, and I represent the quantitative analysis of the phosphorylation of Akt stimulated by insulin. Results were quantified and expressed as in Fig. 7. Data are the mean ± S.E. of 4–6 experiments. Statistical analysis is performed versus each untreated shRNA cell type (control (Ctrl) or Nox2 shRNA) p < 0.05 (*), p < 0.01 (**) and versus each shRNA cells type stimulated with insulin p < 0.05 (#), p < 0.001 (###), and p < 0.01 (##).
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