NADPH Oxidase Contributes to Oxidative Stress and Reduced eNOS Phosphorylation during Hyperinsulinemia in Human Skeletal Muscle Arterioles and Microvascular Endothelial Cells
Authors: Mahmoud Abeer M , Ali Mohamed M , Miranda Edwin , Mey Jacob , Blackburn Brian , Robinson Austin , Haus Jacob M , Phillips Shane A 
Published: November 2023

Insulin resistance promotes vascular endothelial dysfunction and subsequent development of cardiovascular disease. Previously we found that skeletal muscle arteriolar flow induced dilation (FID) was reduced following 2‐hours of a 40 mU/m2/min hyperinsulinemic clamp in healthy adults. Therefore, we hypothesized that hyperinsulinemia contributes to microvascular endothelial cell inflammation and oxidative stress generation through NADPH oxidase (NOX). We examined 1) flow induced vasodilation of isolated human skeletal muscle arterioles exposed to high insulin levels and 2) the effect of a broad range of insulin doses (0.1–10 nmol/L) on human adipose microvascular endothelial cells (HAMECs) expression of nitric oxide synthase and pathways that regulate oxidative stress. Determination of superoxide production in HAMECs was made using flow cytometry (MitoSOXTM Red). In four lean healthy participants (mean age 23.4±0.8 y, BMI 21.6±0.5), arteriolar FID (% of dilation at Δ60 pressure gradient) measured by videomicroscopy was reduced following insulin incubation (10 nmol/L; 1 hour) (baseline FID: 83.3±3.3; post insulin: 73.6±2.3, p=0.01). This impairment in FID was reversed after co‐incubation with either the NOX2 inhibitor, VAS (2μmol/L) (85.8±3.3, p=0.008) or nuclear factor kappa B (NF‐κB) SN50 cell permeable inhibitory peptide; AAVALLPAVLLALLAPVQRKRQKLMP (15 μg/ml; 85.6±3.7, p=0.008). Informed by these human data, we sought to further investigate the mechanism of insulin action using HAMECs, a microvascular endothelial model. We found that insulin induced dose‐dependent increases in mRNA and protein levels of NADPH oxidase (NOX2 and NOX4) isoforms that was accompanied by a dose‐dependent elevation of O2− production. In addition, mRNA and protein levels of superoxide dismutase (SOD1, SOD2 and SOD3) isoforms were increased. In a separate group of experiments, we found that hydrogen peroxide (H2O2; 2×10−4 mol/L) also induced SOD 1, SOD 2, and SOD 3. The mRNA and protein levels of endothelial nitric oxide synthases (eNOS) were increased in response to insulin. We measured eNOS phosphorylation (ser1177) up to 4 hours in 30 min increments following insulin exposure and found that phosphorylated eNOS peaks at 60 min (8 fold increase) followed by a time‐dependent reduction. The insulin‐induction of eNOS phosphorylation was abrogated by the H2O2 scavenger (PEG‐catalase; 500 U/ml). These data suggest that the effect of insulin on eNOS phosphorylation in microvascular endothelial cells may be related to a biphasic effect of H2O2 on eNOS phosphorylation. In conclusion, high insulin levels may reduce flow induced vasodilation via inducing inflammatory signaling and NADPH sources of oxidative stress in microvascular endothelial cells.Support or Funding InformationNIH RO1s HL095701, HL095701‐01A2S (SAP), American Diabetes Association Grant 1‐14‐JF‐32 (JMH), American Heart Association Grant 15POST24480172 (AMM)