Nox2 contributes to hyperinsulinemia-induced redox imbalance and impaired vascular function

Figures

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  Fig. 1. Impaired FID in isolated skeletal muscle arterioles after incubation with insulin. FID was measured during intraluminal flow corresponding to pressure gradients of 10–100 cmH2O in the absence or presence of insulin (10 nmol/L). (A) The effect of insulin on arteriolar FID at different time points (10, 30, and 60 min) (n = 6). (B) The effect of insulin on FID following a preincubation with the Nox2 inhibitor, VAS2870 or NF-κB inhibitory peptide (n = 6). (C) & (D) The effect of L-NAME and PEG-catalase on FID, respectively in the presence and absence of insulin (n = 6). All values are plotted as means ± SE. *(P < 0.05) for comparison with baseline. † and # (P < 0.05) for insulin+ VAS2870 and insulin+ NF-κB vs insulin alone, respectively.

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  Fig. 2. ROS and NO production in isolated skeletal muscle arterioles in response to insulin. (A) Representative images of ROS and NO fluorescence in arterioles at baseline and after incubation with insulin (10 nmol/L) for 10–60 min or insulin (60 min) following a preincubation with VAS2870. BSO and Ach were used as positive controls for ROS and NO production, respectively. (B) & (C) Fluorescence intensities of ROS and NO were measured in arbitrary units using NIH Image J software. All values are plotted as means± SE. *(P < 0.05) for comparison with baseline. † (P < 0.05) for comparison with insulin. BSO, buthionine sulfoximine; Ach, acetylcholine.

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  Fig. 3. Insulin induces Nox2 expression and P47phoxphosphorylation in HAMECs. (A) & (B) Western blot analysis and quantification of the normalized signal intensity of Nox2 and Nox4 protein in response to increasing doses of insulin (0.01–100 nmol/L) for 4 h and insulin (1 nmol/L) at different time points (30 min-4 h). (C) mRNA expression of Nox2 in response to insulin (1 nmol/L for 4 h). (D) & (E) Western blot analysis and quantification of the ratio between p. P47phox and total P47phox proteins in response to different doses of insulin (0.01–100 nmol/L for 4 h) and incubation periods (30 min-4 h) of 1 nmol/L insulin. (F) HAMECs treated with insulin (0.01–100 nmol/L for 4 h) were lysed, immunoprecipitated (IP) with Nox2 antibody or rabbit IgG (R-IgG) as a control for the non-specific IgG (NS-IgG) and immunoblotted for p. P47phox. Untreated cells were used as a negative control and total cell lysate before IP was used to demonstrate that protein bands are corresponding to the expected molecular weight. Signal intensity of the IP Western bands were normalized to the untreated control (Cont). Images were then quantified using Image Studio ver. 4.0 (LI-COR). Results represent the means ± SE…

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  Fig. 4. Dose- and time-dependent effects of insulin on ROS production in HAMECs. (A) Visualization of ROS using H2DCFDA fluorescent staining in HAMECs treated with insulin (0.01–100 nmol/L) for 30 min (B) Quantification of ROS fluorescent signal at different time points (0–60 min) using a multi-mode plate reader; *, #, † p < 0.05 for comparing signals at different time points with the baseline (0 min) at insulin doses 1, 10, and 100 nmol/L, respectively. (C) Quantification of ROS signal in HAMECs treated with insulin with and without antioxidants. All results represent the means ± SE for three different experiments. *p < 0.05 for comparison with control and † (P < 0.05) for comparison with insulin.

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  Fig. 5. Response of SOD2 and SOD3 proteins to insulin treatment in HAMECs. Western blot analysis and quantification of the relative signal intensity of SOD2 and SOD3 proteins in response to different incubation periods (30 min to 4 h) of 1 nmol/L insulin (A) and in response to insulin incubation (1 nmol/L) for 4 h along with the antioxidants, VAS2870, apocynin, NSC23766 trihydrochloride and Tempol (B). (C) Western blot analysis and quantification of the relative signal intensity of Nox2 and Nox4 proteins in cells transfected with Nox2 siRNA, cells transfected with a scrambled sequence of siRNA (scam) and non-transfected cells (cont). (D) Western blot analysis and quantification of the relative signal intensity of SOD2 and SOD3 proteins in response to insulin (1 nmol/L for 4hrs) in cells transfected with Nox2 siRNA, cells transfected with a scrambled sequence of siRNA (scam) and non-transfected cells (insulin). Untreated, non-transfected cells were used as a baseline control (cont). Results represent the means ± SE for three different experiments. * and † (P < 0.05) for comparison with control and ‡ and # (P < 0.05) for comparison with insulin for SOD2 and SOD3, respectively.

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  Fig. 6. The effect of insulin on eNOS phosphorylation and uncoupling. Western blot analysis and quantification of the relative signal intensity of phosphorylated eNOS (ser 1177) protein to total eNOS in response to incubation with insulin (1nmol/L) for different time periods (30 min to 4 h) (A) and in response to insulin incubation for 4 h along with the antioxidants, Tempol, VAS2870, and BH4 (B). (C) Western blot analysis and quantification of the relative signal intensity of phosphorylated eNOS in response to insulin (1 nmol/L for 4hrs) in cells transfected with Nox2 siRNA, cells transfected with a scrambled sequence of siRNA (scam) and non-transfected cells (insulin). Untreated, non-transfected cells were used as a baseline control (cont). (D) Western blot analysis using low-temperature SDS-PAGE (LT-PAGE) to assess eNOS monomer to dimer ratio in HAMECs treated with insulin (1 nmol/L) for 30 min to 4 h and with insulin plus Tempol, VAS2870, or BH4 for 4 h. Results represent the means ± SE for three different experiments. *(P < 0.05) for comparison with control. † (P < 0.05) for comparison with insulin.

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  Fig. 7. Transient induction of NO levels in HAMECs in response to insulin. (A) Visualization of NO and ROS in HAMECs using NO detector and H2DCFDA fluorescent staining, respectively at baseline (1st column) or in response to insulin (1nmol/L) for 20 min (2nd column), 30 min (3rd column), and 60 min (4th column). (B) Visualization of NO and ROS in HAMECs at baseline (1st column) or in response to insulin (1nmol/L) for 60 min in scram siRNA-transfected cells (2nd column) and Nox2 siRNA-transfected cells (3rd column). (C) Quantification of NO to ROS fluorescence intensity ratio in HAMECs in response to insulin (1 nmol/L) at incubation periods that ranged from 0 to 60 min with and without VAS2870, Tempol, and NSC23766 trihydrochloride. (D) Quantification of NO to ROS fluorescence intensity ratio in HAMECs in response to insulin (1 nmol/L) at incubation periods that ranged from 0 to 60 min in scram siRNA and Nox2 siRNA-transfected cells. (* p < 0.05) for comparison with insulin in (C) and for comparison with scram-siRNA in (D).

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  Fig. 8. Persistent increases in nitrosylated proteins after insulin incubation. Western blot analysis and quantification of the relative signal intensity of nitrotyrosine in HAMECs treated with insulin (1 nmol/L) for 1–24 h (A) or insulin plus L-NAME, VAS2870, NSC23766 trihydrochloride, or apocynin for 4 h (B). Results represent the means ± SE for three different experiments. *(P < 0.05) for comparison with control. † (P < 0.05) for comparison with insulin.