Direct renin inhibition improves systemic insulin resistance and skeletal muscle glucose transport in a transgenic rodent model of tissue renin overexpression

Abstract

Renin is the rate-limiting enzyme in renin-angiotensin system (RAS) activation. We sought to determine the impact of renin inhibition on whole-body insulin sensitivity and skeletal muscle RAS, oxidative stress, insulin signaling, and glucose transport in the transgenic TG(mRen2)27 rat (Ren2), which manifests increased tissue RAS activity, elevated serum aldosterone, hypertension, and insulin resistance. Young (aged 6-9 wk) Ren2 and age-matched Sprague Dawley control rats were treated with aliskiren [50 mg/kg . d, ip] or placebo for 21 d and administered an ip glucose tolerance test. Insulin metabolic signaling and 2-deoxyglucose uptake in soleus muscle were examined in relation to tissue renin-angiotensin-aldosterone system [angiotensin (Ang) II, mineralocorticoid receptor (MR), and Ang type I receptor (AT(1)R)] and measures of oxidative stress as well as structural changes evaluated by light and transmission electron microscopy. Ren2 rats demonstrated systemic insulin resistance with decreased skeletal muscle insulin metabolic signaling and glucose uptake. This was associated with increased Ang II, MR, AT(1)R, oxidative stress, and reduced tyrosine insulin receptor substrate-1 phosphorylation, protein kinase B/(Akt) phosphorylation and glucose transporter-4 immunostaining. The Ren2 also demonstrated perivascular fibrosis and mitochondrial remodeling. Renin inhibition improved systemic insulin sensitivity, insulin metabolic signaling, and glucose transport along with normalization of Ang II, AT(1)R, and MR levels, oxidative stress markers, fibrosis, and mitochondrial structural abnormalities. Our data suggest that renin inhibition improves systemic insulin sensitivity, skeletal muscle insulin metabolic signaling, and glucose transport in Ren2 rats. This is associated with reductions in skeletal muscle tissue Ang II, AT(1)R, and MR expression; oxidative stress; fibrosis; and mitochondrial abnormalities.

Figure 1

Direct renin inhibition improves systemic insulin resistance and improves insulin-stimulated glucose uptake in the Ren2 rat. Insulin sensitivity was measured during an IPGTT performed after overnight fast on d 21. Samples for serum insulin (A) and glucose (B) were obtained and AUCs were calculated (C and D, respectively). The insulin resistance index (E) was calculated as the product of the AUC for glucose and insulin. Values presented as means ± se. *, P < 0.05 compared with SD-C; **, P < 0.05 when aliskiren-treated Ren2 (Ren2-A) are compared with Ren2 controls (Ren2-C). F, Insulin-stimulated 2-DOG uptake analyzed in ex vivo soleus muscle strips in the absence and presence of a maximally effective dose of insulin (100 nm). Values presented as means ± se. *, P < 0.05 compared with SD-C; #, P = 0.06 when compared with SD-C; **, P < 0.05 when Ren2-A are compared with Ren2-C.

Figure 2

Direct renin inhibition improves IRS-1, Akt, and GLUT-4 in soleus muscle of the Ren2 rat. A, Representative fluorescent images of total IRS-1 and quantification of converted signal intensities to the right. B, Representative fluorescent images of Tyr-phosphorylated (Tyr⁹⁴¹) IRS-1 and quantification of converted signal intensities. C, Representative fluorescent images of threonine (Thr³⁰)-phosphorylated Akt and quantification of converted signal intensities to the right. D, Representative fluorescent images of GLUT-4 and quantification of converted signal intensities to the right. Values presented as means ± se. *, P < 0.05 compared with SD-C; **, P < 0.05 when aliskiren-treated Ren2 (Ren2-A) are compared with Ren2 controls (Ren2-C). Scale bar, 50 μm.

Figure 3

Direct renin inhibition reduces NADPH oxidase activity and subunits in soleus muscle of the Ren2 rat. A, Total NADPH oxidase activity. B, Measures of intensities of immunohistochemical analysis of NADPH oxidase subunits as depicted by representative images in C. D, 3-Nitrotyrosine immunostaining and corresponding measures of intensity. Values presented as means ± se. *, P < 0.05 compared with SD-C; **, P < 0.05 when aliskiren-treated Ren2 (Ren2-A) are compared with Ren2 controls (Ren2-C). Scale bar, 50 μm.

Figure 4

Direct renin inhibition attenuates RAS activation in soleus muscle of the Ren2 rat. A, Representative images of immunohistochemistry analysis of Ang II in soleus and corresponding measures of intensities to the right. B, Representative images of immunohistochemistry analysis for AT₁R. C, Corresponding measures of intensities below. D, Western blot of AT₁R with corresponding densitometry analysis below. E, Representative images of immunohistochemistry analysis for MR and corresponding measures of intensities below (F). G, Western blot of MR with corresponding densitometry analysis below. Values presented as means ± se. *, P < 0.05 compared with Sprague Dawley controls (SD-C); **, P < 0.05 when aliskiren-treated Ren2 (Ren2-A) are compared with Ren2 controls (Ren2-C). Scale bar, 50 μm.

Figure 5

Direct renin inhibition attenuates fibrosis and ultrastructural mitochondrial abnormalities in the Ren2 rat. A, Representative images of Verhoeff-von Gieson (VVG) staining of soleus to demonstrate interstitial perivascular fibrosis and corresponding measures of area. B, Immunohistochemistry for number of mitochondria and measures of intensities below. Values presented as means ± se. *, P < 0.05 compared with SD-C; **, P < 0.05 when aliskiren-treated Ren2 (Ren2-A) are compared with Ren2 controls (Ren2-C). Scale bar, 50 μm. C, Representative TEM images of typical mitochondrial mounding (Mm) that is present in the vicinity of interstitial endomysial capillaries found in the soleus in the SD-C. Note the endothelial cell (EC) surrounding the capillary lumen (CL) and the pericyte foot process (Pc) traversing the endomysium (X). D, Marked increased in interdigitating mitochondria found within the Mm in the Ren2-C. Note the organized early collagen deposition (Co) adjacent to a capillary EC within the X and Pc. Insert (i), Exploded image of the early organized collagen fibrils indicative of early pericapillary fibrosis found in the Ren2. E, Abrogation of the pericapillary Mm found in the Ren2-A. Note the absence of the Mm in the vicinity of the interstitial endomysial capillary (CL) and the marked decrease in subsarcolemmal mitochondria (Mt). Also note the long connecting Pc traversing the interstitial X. F, Subsarcolemmal mitochondria (arrows) in the SD-C on either side of the interstitial X between two soleus skeletal muscle myocytes. Sarcomeres are bounded by Z line (double arrows). G, Marked increased in subsarcolemmal Mt (arrows) in the Ren2 untreated rat model. Note the marked increase in subsarcolemmal Mt compared with A and B. H, Representative image of the Ren2-A. Note the abrogation of the subsarcolemmal Mt (arrows). Magnification, ×2500. Scale bar, 0.5 μm.