Liraglutide Ameliorates Erectile Dysfunction via Regulating Oxidative Stress, the RhoA/ROCK Pathway and Autophagy in Diabetes Mellitus

Abstract

Background: Erectile dysfunction (ED) occurs more frequently and causes a worse response to the first-line therapies in diabetics compared with nondiabetic men. Corpus cavernosum vascular dysfunction plays a pivotal role in the occurrence of diabetes mellitus ED (DMED). The aim of this study was to investigate the protective effects of glucagon-like peptide-1 (GLP-1) analog liraglutide on ED and explore the underlying mechanisms in vivo and in vitro.

Methods: Type 1 diabetes was induced in rats by streptozotocin, and the apomorphine test was for screening the DMED model in diabetic rats. Then they were randomly treated with subcutaneous injections of liraglutide (0.3 mg/kg/12 h) for 4 weeks. Erectile function was assessed by cavernous nerve electrostimulation. The corpus cavernosum was used for further study. In vitro, effects of liraglutide were evaluated by primary corpus cavernosum smooth muscle cells (CCSMCs) exposed to low or high glucose (HG)-containing medium with or without liraglutide and GLP-1 receptor (GLP-1R) inhibitor. Western blotting, fluorescent probe, immunohistochemistry, and relevant assay kits were performed to measure the levels of target proteins.

Results: Administration of liraglutide did not significantly affect plasma glucose and body weights in diabetic rats, but improved erectile function, reduced levels of NADPH oxidases and ROS production, downregulated expression of Ras homolog gene family (RhoA) and Rho-associated protein kinase (ROCK) 2 in the DMED group dramatically. The liraglutide treatment promoted autophagy further and restored expression of GLP-1R in the DMED group. Besides, cultured CCSMCs with liraglutide exhibited a lower level of oxidative stress accompanied by inhibition of the RhoA/ROCK pathway and a higher level of autophagy compared with HG treatment. These beneficial effects of liraglutide effectively reversed by GLP-1R inhibitor.

Conclusion: Liraglutide exerts protective effects on ED associated with the regulation of smooth muscle dysfunction, oxidative stress and autophagy, independently of a glucose- lowering effect. It provides new insight into the extrapancreatic actions of liraglutide and preclinical evidence for a potential treatment for DMED.

Keywords: autophagy; diabetes mellitus; erectile dysfunction; liraglutide; oxidative stress.

Figure 1

Metabolic parameters in rats. (A) Time course of fasting blood glucose (mmol/L) measurements. (B) Time course of body weight (g) measurements. Before means the initial body weight and plasma glucose level measured after 7-day adaptive feeding. Liraglutide was treated from the 10th to the 14th week. Data are expressed as means ± SD. *P < 0.05 vs. the control group. DMED, diabetes mellitus-induced erectile dysfunction; Lir, liraglutide.

Figure 2

Assessment of erectile function in rats. (A) Representative traces of ICP and MAP (stimulation parameters: 15-Hz frequency; 1.2-ms width; 5.0 V). (B) Max ICP/MAP among the groups. Data are expressed as means ± SD. *P < 0.05 vs. the control group; #P < 0.05 vs. the DMED group. DMED, diabetes mellitus-induced erectile dysfunction; Lir, liraglutide; ICP, intracavernous pressure; MAP, mean atrial blood pressure.

Figure 3

Effects of liraglutide on reducing oxidative stress in vivo and in vitro. Representative immunofluorescence (×200) (A) and quantification (B) of ROS among the groups in the corpus cavernosum. Representative immunofluorescence (×200) (C) and quantification (D) of ROS among the groups in CCSMCs. Representative immunoblot (E) and quantification (F) of NOX1, NOX2 and NOX4 among the groups in the corpus cavernosum. Levels of MDA (G) and SOD (H) among the groups in the corpus cavernosum. The quantitative data of β-actin in 3F: 1, 1.16, and 1.25, respectively. Data are expressed as means ± SD. *P < 0.05 vs. the control group; #P < 0.05 vs. the DMED group or the HG group; & P < 0.05 vs. the HG + Lir group. CCSMCs, corpus cavernosum smooth muscle cells; DMED, diabetes mellitus-induced erectile dysfunction; Lir, liraglutide; HG, high glucose; Exe, Exendin (9-39); ROS, reactive oxygen species; DHE, Dihydroethidium; DAPI, 4’,6-diamidino-2-phenylindole; NOX, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; MDA, malondialdehyde; SOD, superoxide dismutase; IOD, Integrated option density.

Figure 4

Effects of liraglutide on regulating RhoA/ROCK pathway in vivo and in vitro. Representative immunoblot (A) and quantification (B) of ROCK1, ROCK2 and RhoA among the groups in the corpus cavernosum. Representative immunoblot (C) and quantification (D) of ROCK1, ROCK2 and RhoA among the groups in CCSMCs. The quantitative data of β-actin: 1, 1.15, and 0.91, respectively in 4B and 1, 1.04, 1.19, and 1.28, respectively in 4D. Data are expressed as means ± SD. *P < 0.05 vs. the control group; ^#P < 0.05 vs. the DMED group or the HG group; ^&P < 0.05 vs. the HG + Lir group. CCSMCs, corpus cavernosum smooth muscle cells; DMED, diabetes mellitus-induced erectile dysfunction; Lir, liraglutide; HG, high glucose; Exe, Exendin (9-39); RhoA, Ras homolog gene family; ROCK, Rho-associated protein kinase.

Figure 5

Effects of liraglutide on regulating autophagy in vivo and in vitro. Representative immunoblot (A) and quantification (B, C) of Beclin1 and LC3II/LC3I among the groups in the corpus cavernosum. Representative immunofluorescence (×200) (D) and quantification (E) of LC3 among the groups in the corpus cavernosum. Representative immunofluorescence (×200) (F) and quantification (G) of LC3 among the groups in CCSMCs. The quantitative data of β-actin in 5B: 1, 1.18 and 1.32, respectively. Data are expressed as means ± SD. *P < 0.05 vs. the control group; #P < 0.05 vs. the DMED group or the HG group. CCSMCs, corpus cavernosum smooth muscle cells; DMED, diabetes mellitus-induced erectile dysfunction; Lir, liraglutide; HG, high glucose; Exe, Exendin (9-39); LC3, microtubule-associated protein 1 light chain; DAPI, 4’,6-diamidino-2-phenylindole; IOD, Integrated option density.

Figure 6

Effects of liraglutide on expression of GLP-1R in the corpus cavernosum. Representative immunohistochemistry (A) and quantification (B) of GLP-1R among the groups. Data are expressed as means ± SD. *P < 0.05 vs. the control group; #P < 0.05 vs. the DMED group. DMED, diabetes mellitus-induced erectile dysfunction; Lir, liraglutide; GLP-1R, glucagon-like peptide-1 receptor; IOD, Integrated option density.