Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Abstract

Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6-8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P < 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P < 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue.

Fig. 1.

Low-dose spironolactone does not reduce systolic blood pressure (SBP) in the transgenic TG(mRen2)27 rat (Ren2). SBP was measured before starting the experimental protocol and at days 19 and 20 before death (day 21). Sprague-Dawley control (SD-C, n = 6), Sprague-Dawley treated with spironolactone (SD-Sp, n = 4), Ren2 control (Ren2-C, n = 5), Ren2 treated with spironolactone (Ren2-Sp, n = 5). Values are means ± SE. *P < 0.05 compared with SD-C; #P > 0.05 compared with Ren2-C.

Fig. 2.

Low-dose spironolactone improves insulin resistance in Ren2. Insulin sensitivity measured during an intraperitoneal glucose tolerance test (IPGTT) performed after overnight fast on day 21. Samples for serum insulin (A) and glucose (B) were obtained at 0, 15, 30, 45, and 60 min after administering 50% dextrose, 1 g/kg ip. Areas under the curve (AUC) were calculated for insulin and glucose concentrations, and the insulin resistance index (C) was calculated as the product of the AUC for glucose and insulin. Sprague-Dawley control (SD-C, n = 4), Sprague-Dawley treated with spironolactone (SD-Sp, n = 4), Ren2 control (Ren2-C, n = 7), Ren2 treated with spironolactone (Ren2-SP, n = 4). Values are presented as means ± SE. **P < 0.05 compared with Ren2-C.

Fig. 3.

Low-dose spironolactone improves measures of oxidative stress in Ren2. A: NADPH oxidase activity. B and C: NADPH oxidase subunits. D: ROS formation by chemiluminescence. E: 4-hydroxy-2-nonenal (4-HNE) immunostaining was used to detect lipid peroxidation as a marker of reactive oxygen species (ROS). Sprague-Dawley control (SD-C; n = 6 for NADPH oxidase activity, NADPH oxidase subunits and ROS, n = 4 for 4-HNE), Sprague-Dawley treated with spironolactone (SD-Sp; n = 6 for NADPH oxidase activity, NADPH oxidase subunits and ROS, n = 4 for 4-HNE), Ren2 control (Ren2-C; n = 5 for NADPH oxidase, NADPH oxidase subunits and ROS, n = 4 for 4-HNE), and Ren2 treated with spironolactone (Ren2-Sp; n = 5 for NADPH oxidase, NADPH oxidase subunits and ROS, n = 4 for 4-HNE). Values are presented as means ± SE. *P < 0.01 compared with SD-C. **P < 0.05 compared with Ren2-C. Scale bar = 50 μm.

Fig. 4.

Low-dose spironolactone improves glucose transport in Ren2. 2-Deoxyglucose uptake analyzed in ex vivo soleus muscle strips in the absence and presence of a maximally effective dose of insulin. Sprague-Dawley control (SD-C, n = 6), Sprague-Dawley treated with spironolactone (SD-Sp, n = 4), Ren2 control (Ren2-C, n = 5), and Ren2 treated with spironolactone (Ren2-Sp, n = 5). Values are expressed as means ± SE. *P < 0.05 compared with SD-C. **P < 0.05 compared with Ren2-C.

Fig. 5.

Low-dose spironolactone improves insulin receptor substrate (IRS), GLUT4, and Akt in Ren2. A: representative fluorescent images of total IRS-1 and tyrosine (Tyr⁹⁴¹) phosphorylated (PO₄) IRS-1 and quantification of converted signal intensities in average gray scale intensities to the right. B: representative fluorescent images of total and serine (Ser⁴⁷³) phosphorylated (PO₄) Akt and quantification of converted signal intensities in average gray scale intensities to the right. C: representative fluorescent images of GLUT4 and quantification of converted signal intensities in average gray scale intensities to the right. Sprague-Dawley control (SD-C, n = 6), Sprague-Dawley treated with spironolactone (SD-Sp, n = 4), Ren2 control (Ren2-C, n = 5), and Ren2 treated with spironolactone (Ren2-Sp, n = 5). *P < 0.05 compared with SD-C. **P < 0.05 compared with Ren2-C. Scale bar = 50 μm.