Nox2 Mediates Skeletal Muscle Insulin Resistance Induced by a High Fat Diet J. Biol. Chem., 22 May, /10/26 M1 眞野 僚 【背景と目的】 近年、インスリン抵抗性が発症する原因として ROS が関わることが示唆されている。これは、フ リーラジカルのスカベンジャーの添加や抗酸化酵素の過剰発現によって、インスリン抵抗性が改 善したことが理由として挙げられる。骨格筋における ROS の発生源は、ミトコンドリア、小胞体に 加えて、 Nox2(NADPH oxidase) 由来の ROS がある。しかし、 Nox2 由来の ROS がインスリン抵抗性の発 症に与える影響は未だ解明されておらず、本研究では高脂肪食によるインスリン抵抗性に Nox2 由 来の ROS がどのように関わるのか検討を行った。 【略語】 ROS; reactive oxygen species; DHE, dihydroethidine; HFD, high fat diet; HG, high concentration of glucose; ITT, insulin tolerance test; GTT, glucose tolerance test; GTTHOMA, homeostasis model of assessment of insulin resistance Nox, NADPH oxidase; RT mRNA: Reverse-transcribed mRNA ; SD, standard diet; WT-HFD: wild-type mice fed a high fat diet; WT-SD, wild-type fed a standard diet. 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 < (***) versus WT-HFD p < 0.05 (#). 高脂肪食による体重の増加は見られたが、 食事、 Nox2 の有無による体組成の変化は見られなかっ た。

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 < (***) and versus WT-HFD is p < 0.05 (#) and p < 0.01 (##). Nox2 のノックアウトにより、高脂肪食によるインスリン抵抗性が改善 された。 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. Nox2 のノックアウトは、骨格筋の構成には影響を与えな かった。

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 p67 phox, p47 phox, p22 phox, Nox2, and Nox4. In addition 50 μg of total protein extract were used to determine p67 phox, p47 phox, p22 phox, 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, p22 phox, 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 < (***). 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 < (***). 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 p67 phox 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 < (###). 高脂肪食により、 Nox2,Nox4(mRNA のみ ),p22 phox の発現が上昇し た。 高脂肪食により産生する ROS は Nox2 由来である。 また、 GLUT4 の膜移行と発現量は Nox2 に依存してい る。

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, p22 phox, p47 phox, and p67 phox in undifferentiated and differentiated C2C12 cells were determined by RT-PCR and immunoblot. Figure 7. Chronic H 2 O 2 -dependent insulin resistance. Akt phosphorylation and glucose uptake were determined in myotubes cultured for 48 h with 5 m M (control) or 25 m M (HG) glucose (A and B), 200 μ M palmitate (C and D), or with 100 μM H 2 O 2 (E and F) and then stimulated for 20 min with 100 n M insulin. Where indicated, cells were pretreated with catalase (900 units/ml, 10 min) before HG, palmitate, or H 2 O 2 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 H 2 O 2 pretreatment. Glucose uptake was determined as the amount of deoxy-[ 14 C]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 < (***) and versus cells stimulated with insulin is p < 0.05 (#), p < (###), and p < 0.01 (##). C2C12 には、 Nox2 複合体、 Nox4 が発現している。 また、分化後の方が発現量が 多い。 高濃度グルコース、高濃度パルミチン酸の添加によって、糖取り込みが減 少した。 これらは、 H 2 O 2 を介して誘導される。

Figure 8. Down-regulation of Nox2 counteracts the insulin resistance induced by high concentration of glucose but not by H 2 O 2. 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 m M (control) or 25 m M (HG) of glucose (B–D), 200 μ M palmitate (E–G), or with 100 μ M H 2 O 2 (H–J) and then stimulated for 20 min with 100 n M 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 < (###), and p < 0.01 (##). Fig. 7 高濃度グルコース、高濃度パルミチン酸の添加によって、糖取り込みが減 少した。 これらは、 H 2 O 2 を介して誘導される。 まとめ ・今回の実験で、 Nox2 が高脂肪食によるインスリン抵抗性の発症に重要 である。 ・ Nox2 をノックダウン・ノックアウトによりインスリン感受性が回復し た。 ⇒Ⅱ型糖尿病治療の新しいターゲットとして有用であるかも しれない。