Department of Joint Surgery, The 3rd Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang, Hebei, China 050000 (e-mail: email@example.com).
ABSTRACT: Diabetes Mellitus (DM) is an important risk factor of erectile dysfunction (ED). We sought to investigate the nature and mechanism of the vasoreactivity in Akita (Ins2 [WT/C96Y]) mice, a model of genetic nonobese Type 1 diabetes that recapitulates human Type 1 diabetes. Eight wild-type (WT) mice (group 1) and 8 Akita mice (group 2) were used for this study. Corporal tissues were harvested and studied for endothelium-dependent and endothelium-independent vasoreactivities by isometric tension study; levels of vascular endothelial growth factor (VEGF) and cyclic guanosine monophosphate (cGMP) were studied with the use of enzyme-linked immunosorbent assay (ELISA). Endothelium-dependent and endothelium-independent vasoreactivities, cGMP, and VEGF were significantly decreased in the corporal tissues of Akita mice. Corporal tissues from Akita mice demonstrate many of the major functional and biochemical changes found in humans with ED. This model could serve as a valuable tool for advancing our understanding of the role DM plays in the pathogenesis of ED.
Diabetes mellitus (DM) is one of the major risk factors for erectile dysfunction. It has been estimated that 50%–75% of diabetic men have some degree of erectile dysfunction and the incidence of erectile dysfunction is higher in diabetic men than in age-matched nondiabetic men (Lue, 2000; Siu et al, 2001). A diabetic mouse model provides excellent opportunities to explore potential mechanisms and therapeutic approaches for human erectile dysfunction (ED) (Nangle et al, 2006a,b; Xie et al, 2007a; Carneiro et al, 2008; Chitaley and Luttrell, 2008; Kolavennu et al, 2008; Luttrell et al, 2008; Jin et al, 2009). The Akita (Ins2 [WT/C96Y]) mouse has significant spontaneous diabetes and has been used in multiple studies (Hong et al, 2007; Liu et al, 2007; Sullivan et al, 2007). An autosomal dominant mutation in Akita mice causes chronic hypoinsulinemia and hyperglycemia. In effect, they suffer from chronic insulin-dependent diabetes (Chang et al, 2005; Choeiri et al, 2005). It is unknown if there are functional or molecular changes in the corporal tissues of Akita mice. Therefore, the purpose of our study is to examine, ex vivo, endothelium-dependent and endothelium-independent vasoreactivity in the corporal tissues of Akita mice, as well as in wild-type mice, and determine temporal changes in biochemical measures linked to vasoreactivity.
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in controlling survival and apoptosis (Kuhn et al, 2010; Moberg et al, 2010). One of the downstream effects of VEGF includes VEGF receptor (VEGFR) dimerization and autophosphorylation, the phosphorylation and activation of Akt and endothelial nitric oxide synthase (eNOS), which has been shown to mediate VEGF-induced penile erection by further mediating activation of guanosine 3′,5′-cyclic monophosphate (cGMP) (Musicki et al, 2004; Olsson et al, 2006). We predicted that vasoreactivity would be reduced in the corporal tissue of the Akita mouse compared with wild-type mouse. We sought to investigate this possibility and its mechanism.
Materials and Methods
Eight WT mice (group 1) and 8 Akita mice (group 2) fed with standard chow were used in this study. All mice were kept in a temperature-controlled, air-conditioned animal house with a 12-hour light-dark cycle and were given free access to food and water. Mice were 20–22 weeks old when their corporal tissues were harvested for study, as previously described (Xie et al, 2007). All protocols were approved by the Animal Care and Use Committee of Hebei Medical University.
Tissue Procurement; Protein Isolation
At study termination, all mice were deeply anesthetized with ketamine and xylazine and penectomy was performed with careful dissection of the corpora cavernosa from the tunica albuginea before sacrifice, as previously described. Protein lysates were prepared, and concentrations were determined by Bradford assay, as previously described (Xie et al, 2007, 2008).
After equilibration at 0.2 g for 1 hour, a dose-response curve for norepinephrine was obtained by cumulative addition of norepinephrine (10−9–10−4 M) in logarithmic increments. Strips were then submaximally precontracted with 10−5 M norepinephrine, and after a contractile plateau was reached, 10−8–10−3 M acetylcholine or 10−8–10−4 M sodium nitroprusside was added cumulatively in logarithmic increments. Endothelium-dependent relaxation was assessed with acetylcholine (Ach), whereas smooth muscle relaxation was assessed by sodium nitroprusside (SNP) on corporal strips. Data were analyzed and expressed as a percentage of the active tension generated by norepinephrine. These values were plotted against the negative logarithm of the agonist dose to produce relaxation dose-response curves. Polyview software (Grass-Telefactor, West Warwick, Rhode Island) was used for calculation of concentrations (EC50s) causing half-maximal and maximal relaxation. Groups were compared at the EC50 and maximal relaxation of Ach and SNP, as previously described (Xie et al, 2007, 2008).
Measurement of VEGF Protein
VEGF was determined with the use of a solid-state ELISA system with a Quantikine VEGF ELISA Kit (R&D Systems China Co Ltd, Beijing), as previously described (Xie et al, 2008). At the final step, the optical density of samples and standards were measured. The amount of VEGF protein in each sample was calculated on the basis of a standard curve.
Quantitative Determination of cGMP Nucleotides
Determination of the cyclic nucleotide concentration was done by using a Parameter cyclic GMP Assay commercial kit (R&D Systems China Co Ltd). This method is based on the ELISA, a competitive immunoassay for the quantitative determination of the relevant nucleotide in samples. At the final step, the optical densities of samples and standards were measured. The amount of nucleotide in each sample was calculated on the basis of a standard curve.
Assessments of Akt, p-Akt, eNOS, p-eNOS, nNOS, and iNOS
Western analysis for Akt, phosphorylated Akt (p-Akt), eNOS, phosphorylated eNOS (p-eNOS), neuronal NOS (nNOS), and inducible NOS(iNOS) expression was performed according to methods previously described (Xie et al, 2007, 2008). Alphatubulin was used as a protein loading control. The intensity for each band was quantified with the use of National Institutes of Health (Bethesda, Maryland) image software on the basis of pixel values. The p-Akt or p-eNOS density was calculated relative to total Akt or eNOS, giving the phosphorylated/total fraction. The primary antibodies were purchased from Beijing GBI Biotechnology Co Ltd (Beijing, China); all secondary antibodies were purchased from Beijing Chief-East Tech Co Ltd (Beijing, China). The working concentration for all the first antibodies was 1:1000, and for all the secondary antibodies, the concentration was 1:5000.
Results are expressed as x̄ ± standard deviation. The mean values for the measured parameters were compared by analysis of variance (ANOVA). For vasoreactivity data, if the overall F test for the ANOVA was at least of borderline significance (P ≤ .05) then post hoc pairwise comparisons of the group means were performed; P < .05 was considered statistically significant.
Of the 8 Akita mice, all had blood glucose higher than 525 mg/dL, with significant differences compared with the wild-type mice. The body weight of Akida mice is significantly less than that of wild-type mice. Blood glucose level and body weight of the mice are shown in the Table.
Table Table. . Results of body weight, blood glucose, VEGF, and cGMP (group 2 vs group 1)
Diabetes Mellitus Was Associated With Decreased Vasoreactivities in Corporal Tissue—
For endothelium-dependent vasoreactivity, the overall difference between the 2 groups by F test was P = .013. As shown in Figure 1, endothelium-dependent vasoreactivity was significantly decreased when measured by the EC50 for Ach (group 1 = 5.10 ± 0.28, group 2 = 3.17 ± 0.30; P < .01 for group 2 vs group 1) or percent maximal relaxation to Ach (group 1 = 73.00 ± 5.43%, group 2 = 52.80 ± 4.97%; P < .01 for group 2 vs group 1) in Akita compared with wild-type mice. Similar changes were seen in endothelium-independent vasoreactivity. The overall difference between the 2 groups by F test was P = .047. Between the 2 groups, significant differences were seen in EC50 for SNP (group 1 = 5.72 ± 0.33, group 2 = 4.79 ± 0.27; P < .01 for group 2 vs group 1) or percent maximal relaxation to SNP (group 1 = 97.60 ± 3.36%, group 2 = 82.30 ± 3.49%; P < .01 for group 2 vs group 1).
Diabetes Mellitus Was Associated With Decrease of VEGF Protein in Corporal Tissue—
The levels of VEGF protein were significantly different between the wild-type and Akita mouse groups (see the Table; Figure 2).
Diabetes Mellitus Is Associated With Abnormalities in cGMP Expression—
The levels of cGMP were significantly different between the wild-type and Akita mouse groups (see the Table; Figure 3).
Diabetes Mellitus Was Associated With a Reduction in VEGF Downstream Protein Expression—
Total Akt and total eNOS protein expression were not different between the 2 groups. However, the level of Akt phosphorylation at Ser 473 or eNOS phosphorylation at Ser 1177 was significantly decreased in the corporal tissue of Akita mouse. With respect to p-Akt or p-eNOS expression, a 1.58-fold or 1.99-fold decrease was observed (group 2 vs group 1; P < .05, and P < .01, respectively). The decrease in nNOS protein expression was 1.43-fold, significantly different (P < .05) in Akita mice. With respect to iNOS expression, no significant difference was observed between the 2 groups (see Figure 4).
Diabetes mellitus is a major cause of ED. The purpose of this study was to determine whether diabetes mellitus in Akita mice would result in any abnormalities in vasoreactivity of corporal tissues relevant to ED. We were able to demonstrate changes in vasoreactivities and levels of VEGF and cGMP. Many of these changes are known to be part of the pathophysiology of ED in humans.
The first major finding of our study was that diabetes mellitus resulted, sequentially, in abnormalities in endothelium-dependent and endothelium-independent vasoreactivity. Abnormalities in vasoreactivity in corporal tissue are the result of dysfunctional physiological interactions between endothelium and vascular smooth muscle, which could lead to ED.
VEGF binds to 1 of 3 VEGF receptors that lead to receptor dimerization and autophosphorylation. This activates the enzyme phoshitidylinosityl 3 kinase (PI3K). PI3K in turn converts 4,5-inositylphophate (PI) to 3,4,5-PI, and this phosphorylates and thereby activates the protein kinase Akt. One of the downstream effects of Akt activation is the phosphorylation and activation of eNOS (Olsson et al, 2006), which is an important source of nitric oxide (NO) and plays an important role in modulating intracorporal blood flow to maintain tumescence (Lue, 2000; Musicki et al, 2004). nNOS, which has been found to be important in initiating erection (Burnett, 2004), is also an important source of NO. The NO-dependent signal transduction system contains several molecular targets available for pharmacologic manipulation to treat ED. The most prominent target identified thus far is phosphodiesterase 5 (PDE5), which enzymatically converts the intracellular second messenger molecule, cGMP, to its inactive form (Burnett, 2005). In this study, the decrease in cGMP in Akita mice could be a result of decreased expression of VEGF and its downstream markers. It also could be the result of decreased expression of nNOS, which could signify a neuropathy in Akita mice.
Even though we have seen a significant decrease in vasoreactivity in Akita mice, the results need to be supplemented in future research by investigating EFS-induced relaxation and responses to different agonists. It would be useful to check the possible involvement of metabolic and hormonal factors. Finally, it would be very useful to confirm that the Akita mouse can be used as a model of diabetic ED by measuring intracavernosal pressure/mean arterial pressure and electrical field stimulation.
To our knowledge, this is the first report about the use of Akita mice in investigating ED. This model could serve as a valuable tool for advancing our understanding of the role DM plays in the pathogenesis of ED.
This study was supported by the Grant of Health Bureau of Hebei Province of China (Grant No.20090136) and the National Natural Science Foundation of China (grant 30772082).