Aerobic Exercise Reduces NOX2 in Skeletal Muscle of Obese Insulin‐resistant Adults Via Interfering with RAGE/p‐IkB‐α Axis
Authors: Mahmoud Abeer M , Solomon Thomas P.J. , Phillips Shane A , Kirwan John P , Haus Jacob M 
Published: November 2023

Obesity and diabetes lead to enhanced generation of advanced glycation end products (AGEs) and activation of their receptor, RAGE, which promotes persistent activation of pro‐inflammatory signaling. Previously, we found that levels of RAGE protein and inflammatory cytokines in skeletal muscle of obese insulin‐resistant individuals (OB‐IR) were significantly higher than lean healthy controls (LHCs) and that they were markedly reduced after aerobic exercise (AE) training. Therefore, we hypothesized that the upregulation of RAGE signaling in skeletal muscle of OB‐IR subjects leads to induction of NADPH oxidase (NOX2), an enzyme that is responsible for the production of reactive oxygen species and ultimately results in exacerbation of inflammation and oxidative stress. NOX2 is transcriptionally regulated by nuclear factor kappa B (NF‐kB) and the phosphorylation state of the inhibitor of kappa B‐α (IkB‐α). Thus, we sought to examine the phosphorylation of IkB‐α (p‐IkB‐α) as a surrogate marker of NF‐kB that links elevated RAGE signaling with NOX2 upregulation in OB‐IR. Finally, we assessed the effects of AE training on normalizing p‐IkB‐α/total IkB‐α ratio and NOX2 protein expression in skeletal muscle of OB‐IR individuals. Skeletal muscle biopsies were obtained from OB‐IR subjects (age 66±1, BMI 34±1, n=22) and LHCs (age 31±2, BMI 23±1, n=6) at baseline and following AE training (85% HRmax, 60 min/d, 5d/wk, 12 wks) in OB‐IR. Protein levels of NOX2, p‐IkB‐α and total IkB‐α were measured via immunoblotting (AU). At baseline, OB‐IR subjects showed increased NOX2 protein levels (LHC: 0.3±0.1, OB‐IR: 3.7±0.8, p<0.001) and p‐IkB‐α/total IkB‐α ratio (LHC: 0.5±0.2, OB‐IR: 6.6±1.8, p=0.002) relative to LHCs. Following AE in OB‐IR, NOX2 and p‐IkB‐α protein levels were reduced by 38.5±7.4% (p=0.003) and 28.9±6.5% (p=0.0008), respectively. Total IkB‐α protein expression was elevated by 1.8 folds in OB‐IR subjects after AE training and accordingly the p‐IkB‐α to total IkB‐α ratio was markedly reduced (pre: 11.6±2.1, post: 3.2±0.6, p<0.0001). Interestingly, baseline levels of p‐IkB‐α were correlated with body mass index (r=0.55, p=0.002), fasting plasma glucose (r=0.44, p=0.04), and hyperinsulinemia (40 mU/m2/min)–euglycemic (5mM) clamp‐derived insulin sensitivity (M: mg/kg/min) (r=−0.50, p=0.008) in OB‐IR subjects and LHCs. AE‐induced changes in p‐IkB‐α/total IkB‐α ratio were correlated with changes in RAGE (r=0.66, p=0.001) and NOX2 (r= 0.47, p=0.03) in OB‐IR participants. These data suggest that the IkB‐α and NOX2 pathways are initiated by upregulation of RAGE in skeletal muscle of OB‐IR individuals and that regular AE is effective in attenuating this pro‐inflammatory signaling mechanism.Support or Funding InformationCTSA grants UL1RR024989 and UL1RR029879 (JMH), Central Society for Clinical and Translational Research – Junior Faculty Award (JMH), American Diabetes Association Grant 1‐14‐JF‐32 (JMH), NIH R01s HL095701, HL095701‐01A2S (SAP), DK089547 (JPK), and American Heart Association Grant 15POST24480172 (AMM).