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Taurine Supplementation Improves Erectile Function in Rats with Streptozotocin-induced Type 1 Diabetes via Amelioration of Penile Fibrosis and Endothelial Dysfunction
Taurine Supplementation Improves Erectile Function in Rats with Streptozotocin-induced Type 1 Diabetes via Amelioration of Penile Fibrosis and Endothelial Dysfunction Yajun Ruan, MD,1,2 Mingchao Li, MD, PhD,1,2 Tao Wang, MD, PhD,1,2 Jun Yang, MD, PhD,1,2 Ke Rao, MD, PhD,1,2 Shaogang Wang, MD, PhD,1,2 Weiming Yang, MD, PhD,1,2 Jihong Liu, MD, PhD,1,2 and Zhangqun Ye, MD, PhD1,2
ABSTRACT
Introduction: For patients with diabetes, erectile dysfunction (ED) is common and greatly affects quality of life. However, these patients often exhibit a poor response to first-line oral phosphodiesterase type 5 inhibitors. Aim: To investigate whether taurine, a sulfur-containing amino acid, affects diabetic ED (DED). Methods: Type 1 diabetes mellitus was induced in male rats by using streptozotocin. After 12 weeks, an apomorphine test was conducted to confirm DED. Only rats with DED were administered taurine or vehicle for 4 weeks. Age-matched nondiabetic rats were administered saline intraperitoneally for 4 weeks. Main Outcome Measures: Erectile function was evaluated by electrical stimulation of the cavernous nerve. Histologic and molecular alterations of the corpus cavernosum also were analyzed. Results: Erectile function was significantly reduced in the diabetic rats compared with in the nondiabetic rats, and was improved in the diabetic rats treated with taurine. The corpus cavernosum of the rats with DED exhibited severe fibrosis and decreased smooth muscle content. Deposition of extracellular matrix proteins was increased in the diabetic rats, while expression of endothelial nitric oxide synthase/cyclic guanosine monophosphate/nitric oxide pathwayerelated proteins was reduced. Taurine supplementation ameliorated erectile response as well as histologic and molecular alterations. Conclusion: Taurine supplementation improves erectile function in rats with DED probably by potential antifibrotic activity. This finding provides evidence for a potential new therapy for DED. J Sex Med 2016;-:1e8. Copyright 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. Key Words: Taurine; Diabetes; Erectile Dysfunction; Fibrosis; Extracellular Matrix
INTRODUCTION Erectile dysfunction (ED), defined as the inability to attain or maintain a penile erection sufficient for successful vaginal intercourse, is becoming more common worldwide.1 Approximately 15% to 20% of the general male population experiences ED.2,3 Patients with diabetes are nearly 3 times more likely to suffer from ED compared to patients without diabetes. In addition, ED appears to arise 10 years earlier in patients with diabetes and tends to be more severe, significantly decreasing health-related quality of life.4 Furthermore, the prevalence of ED is 95% in patients with diabetes above the age of 60.3 Received September 25, 2015. Accepted February 17, 2016. 1
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China;
2
Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China
Copyright ª 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsxm.2016.02.164
J Sex Med 2016;-:1e8
Introduction of phosphodiesterase type 5 (PDE5) inhibitors revolutionized the treatment of ED. PDE5 inhibitors enhance nitric oxide (NO)-mediated relaxation of the corpus cavernosum by preventing cyclic guanosine monophosphate (cGMP) degradation. Currently, PDE5 inhibitors are the first-line therapy for ED.5e6 However, because ED is caused by multiple factors, a certain proportion of patients, especially those with diabetes, exhibit a poor response to PDE5 inhibitors.7 Moreover, the failure rate for all PDE5 inhibitors is higher in men with diabetes than in men without.8 In addition, diabetes could induce a series of pathophysiologic changes that could contribute to the decreased response to PDE5 inhibitors. Therefore, it is urgent to develop novel treatment modalities for men with diabetic ED (DED). Taurine (2-aminoethanesulfonic acid), a sulfur-containing amino acid, is one of the most abundant free amino acids in the human body. It plays a role in several physiologic functions, including neuromodulation or neurotransmission, modulation of calcium flow, osmoregulation, stabilization of membranes, and bile formation in the liver.9 Data from existing studies have 1
2
indicated that taurine might decrease cholesterol level, control high blood pressure, and have protective potential in the cardiovascular system.10 Previous studies also have shown a beneficial effect of taurine on diabetes-associated complications. Importantly, it has been demonstrated that taurine supplementation can enhance sexual response and mating ability in aged rats.11 However, whether taurine could mitigate DED has not been investigated. In the present study, we evaluated the effect of taurine in rats with DED in vivo. Our results show that taurine improves erectile function in diabetic rats and could serve as an alternative therapy for ED.
METHODS Treatment of Animals This study was approved by the Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China. Forty-five 8-week-old male Sprague-Dawley rats were used in the present study. After overnight fasting, the rats were injected with freshly prepared streptozotocin (60 mg/kg; Sigma-Aldrich, St. Louis, MO, USA; n ¼ 37) or vehicle (0.1 mol/L citratephosphate buffer; pH, 4.2; n ¼ 8; nondiabetic control group) intraperitoneally. Blood glucose levels were measured 72 hours after streptozotocin injection using a blood glucose meter (ACCU-CHEK Performa; Roche Diagnostics, Shanghai, China). Only rats with a fasting glucose concentration 16.7 mmol/L were considered as diabetic rats (n ¼ 35). At 12 weeks, 30 diabetic rats survived. An apomorphine (APO) test was conducted to confirm DED. Eighteen APOnegative rats were used for further experiments.12,13 Rats with DED were administered taurine (400 mg/kg; Sigma-Aldrich; DED þ taurine group) or vehicle (saline, DED group) intraperitoneally daily for another 4 weeks (n ¼ 9 per group). This dose of taurine is within the range used in previous studies.14e16
In Vivo Erection Studies At 12 weeks after diabetes was induced, erectile function was evaluated by observing APO-induced erection in all surviving rats. APO was injected into the loose skin of the cervical vertebra subcutaneously (80 mg/kg). Then a camera was set in the bottom of the cage for 30 minutes, and the number of erections was recorded. The criteria for penile erection were based on previous studies as follows: stretching of the penis with a congested glans, repeated pelvic thrusts immediately followed by an upright position, and complete emergence of an engorged glans penis and distal penile shaft; otherwise, the result was negative.13,17 At week 16, following a 2-day washout of taurine, intracavernosal pressure (ICP) was measured in all groups as described previously.18 Under proper anesthesia (pentobarbital sodium, 40 mg/kg, i.p.), the rats were fixed on the table, and the left carotid artery was exposed. A PE-50 tube filled with heparinized saline
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(100 IU/mL) was cannulated into the artery and connected to a pressure transducer to monitor mean arterial pressure (MAP) continuously. A 25-G needle was inserted into the left penile crura to monitor ICP. Pressure data were recorded by a data acquisition system (PowerLab/4SP; AD Instruments, Bella Vista, Australia). Erectile response was elicited by electrical stimulation using a bipolar electrode hooked to the cavernous nerve. Stimulation parameters were as follows: 7.5 v, 12 Hz, and 1.2 ms; this was judged to cause a maximal reaction.18e20 The ratio between maximum ICP and MAP (ICP max/MAP) was calculated to normalize for variations in systemic blood pressure. Total ICP was determined by the area under the curve (AUC, mmHg $ s) during stimulation. After finishing these in vivo studies, the rats were sacrificed by intraperitoneal overdose of pentobarbital. Penile midshafts were maintained in 4% paraformaldehyde overnight and embedded in paraffin for subsequent histologic studies. Remaining penile tissues were snap frozen in liquid nitrogen and stored at 80 C for subsequent molecular detection. Blood samples were obtained from rats via the vena cava. The total testosterone levels were measured in the central laboratory of our institution.
Western Blot Analysis Equal amounts (40 mg/lane) of protein extracts were electrophoresed on 10% sodium dodecylsulfateepolyacrylamide gels, transferred to polyvinylidene fluoride membranes (Immobilon-P Transfer Membrane; Millipore Corporation, Billerica, MA, USA), and probed with antibodies against endothelial nitric oxide synthase (eNOS; 1:500; BD Biosciences, San Jose, CA, USA), plasminogen activator inhibitor type 1 (PAI-1; 1:2500; BD Biosciences), collagen I (1:500; Boster, Wuhan, China), collagen IV (1:500; Proteintech, Wuhan, China), transforming growth factor b1 (TGF-b1; 1:500; Abcam, Cambridge, UK), aesmooth muscle actin (a-SMA; 1:1000; Abcam), or b-actin (1:500; Boster). Primary antibodies were incubated overnight at 4 C followed by incubation with horseradish peroxidase-labeled secondary antibody (1:10,000; Jackson ImmunoResearch, West Grove, PA, USA), then detected with Bio-Rad Clarity Western ECL Substrate (Bio-Rad Laboratories, CA, USA). The resulting image was analyzed using ImageJ software (http://rsb.info.nih.gov/ij) to determine the integrated density value of each protein band.
cGMP Measurement cGMP was measured in extracts from penile samples using a commercial cGMP enzyme immunoassay kit (Cayman Chemicals Inc., Ann Arbor, MI, USA) following the manufacturer’s instructions. Protein content in each sample was measured using the BCA assay and expressed as range of pmol cGMP/mg protein. All samples were analyzed in triplicate.20
Histologic Assessment Penile tissue sections (5 mm thickness) were processed for immunohistochemistry as described previously.21 The primary antibody was mouse antiea-SMA (1:150; Boster). Masson’s J Sex Med 2016;-:1e8
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Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis
trichrome staining was performed according to the standard protocol. Semiquantitative analysis was performed to evaluate intensity using Image-Pro plus software (Media Cybernetics, Silver Spring, MD, USA).
Statistical Analysis Results were analyzed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard error of the mean. Statistical analyses were performed using 1-way analysis of variance followed by the Tukey-Kramer test for post hoc comparisons. Differences among groups were considered significant at P < .05.
RESULTS Metabolic Variables and ICP/MAP Assessment There were no significant differences in initial body weight or serum glucose concentration among the 3 groups. At 16 weeks after diabetes was induced, fasting glucose concentration was significantly higher, and body weight along with total testosterone levels were significantly lower in the diabetic rats than in the age-matched normal controls. No significant differences in body weight or glucose concentration were found between the DED and DED þ taurine groups (Table 1). Erectile function was evaluated by measuring maximum ICP, ICP max/MAP, and total ICP. All erectile function variables were significantly lower in the diabetic rats than in the control rats (Figure 1). Partial but significant recovery of erectile function was seen in the DED þ taurine group. MAP did not differ significantly among the groups (Table 1, Figure 1).
EXTRACELLULAR MATRIX PROTEIN PRODUCTION We performed Western blot analysis to evaluate production of extracellular matrix (ECM) proteins, including PAI-1, collagen I, and collagen IV. Cavernous ECM protein production was
significantly higher in the DED group than in the normal controls (P < .05); however, taurine reversed this change (Figure 2). We also performed Western blot analysis to evaluate expression of a-SMA and TGF-b1 in each group. The diabetic rats showed lower a-SMA expression than the normal controls. Expression of a-SMA was higher in the DED þ taurine group than in the DED group (all P < .05; Figures 4B and 4E). Meanwhile, expression of TGF-b1 was higher in the DED group than in the normal controls; however, taurine partially reversed its upregulation.
eNOS Expression We performed eNOS Western blot analysis to quantify endothelial function in all groups. Penile tissue in the rats with DED showed significantly lower eNOS immunoexpression compared with the normal controls (P < .05). This decline was attenuated in part by taurine (Figures 3A and 3B).
cGMP Concentration Cavernous tissue cGMP concentration in the DED group was significantly lower than that in the normal controls (P < .05). The DED þ taurine group had a higher concentration of cGMP than the DED group (P < .05; Figure 3C).
Histologic Changes As displayed in Figure 4, corporal tissue in the diabetic rats showed a significantly lower smooth muscle/collagen ratio compared with tissue in the normal controls (0.13 and 0.30, respectively; P < .05). Taurine increased the ratio in the DED þ taurine group compared with that in the DED group. Immunohistochemical staining showed that taurine-treated DED group had a significant increase of a-SMA in corpus cavernosum compared with DED group (13.53% and 7.73%; P < .05).
DISCUSSION In this study, we demonstrated that administration of taurine, not as a food supplement, in rats with streptozotocin-induced
Table 1. Metabolic and Physiological Variables Variable
Controla (n ¼ 8)
Initial weight (g) Final weight (g) Initial fasting glucose (mmol/L) Final fasting glucose (mmol/L) Testosterone (ng/mL) MAP (mm Hg) ICP (mm Hg) AUC (mm Hg $ s)
240.90 522.90 6.25 6.23 2.55 112.50 97.58 5125
± ± ± ± ± ± ± ±
11.47 60.31 0.41 0.47 0.61 9.73 9.04 723.6
DEDa (n ¼ 9) 233.10 243.00 6.12 30.54 0.73 114.10 43.28 1504
MAP ¼ mean arterial pressure; ICP ¼ intracavernous pressure; AUC ¼ area under the curve. a Data are presented as mean value ± standard deviation. * P< .05 compared with the control group. † P< .05 compared with the DED þ taurine group. J Sex Med 2016;-:1e8
± ± ± ± ± ± ± ±
11.09 42.66* 0.44 2.48* 0.05* 10.30 12.10*,† 505.2*,†
DED þ taurinea (n ¼ 9) 233.00 257.50 6.11 30.86 1.60 115.80 71.99 3687
± ± ± ± ± ± ± ±
12.06 33.35* 0.48 2.11* 0.25*,† 10.56 16.08* 1308*
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Figure 1. Increase of erectile function in taurine-treated diabetic rats. (A) Representative tracing of intracavernosal pressure (ICP) and mean arterial pressure (MAP) for age-matched normal control, diabetic erectile dysfunction (DED) and taurine-treated DED groups. (B, C) Erectile function presented as maximum ICP/MAP and total ICP (AUC) in each group. *P < .05 compared with the control group. †P < .05 compared with the DED þ Taurine group. diabetes significantly decreased penile fibrosis; significantly decreased production of ECM proteins, including PAI-1, collagen I, and collagen IV; and upregulated eNOS expression; and that these changes were associated with physiologically relevant changes in erectile function. Taurine is known to play a role in a variety of cellular processes, including calcium regulation, inflammation, osmoregulation, oxidative stress, and cysteine detoxification, all of which may
contribute to cardiovascular homeostasis. Taurine has become a potential therapeutic target for cardiovascular disease, preventing stroke, atherosclerosis, and hypertension.22 Maia et al reported that taurine supplementation was beneficial to control high blood pressure and vascular remodeling induced via postweaning protein restriction.16 Besides, the protective effect of taurine on endothelial function in diabetic rats already has been proved.14 As a benchmark for cardiovascular disease,23 erectile function also has been
Figure 2. Taurine reduces cavernous extracellular matrix protein deposition. (A) Representative Western blot for plasminogen activator inhibitor type1 (PAI-1, 47 kDa), transforming growth factor beta 1 (TGF-b1, 37 kDa), collagen I (139 kDa), and collagen IV (161 kDa) in the control group, DED group and DED rats treated with taurine group. (B) Data are presented as the relative density of each protein compared with that of b-actin. *P < .05 compared with the control group. †P < .05 compared with the DED þ Taurine group. J Sex Med 2016;-:1e8
Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis
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Figure 3. Taurine treatment restores cavernous endothelial nitric oxide synthase (eNOS) expression. (A) Representative Western blot for eNOS (140 kDa) in each group. (B) Data are presented as the relative density values of eNOS to the b-actin loading control. The relative ratio measures in the control group is arbitrarily presented as 1. (C) ELISA demonstrating quantity of cGMP. *P < .05 compared with the control group. †P < .05 compared with the DED þ Taurine group. investigated. A previous ex vivo study indicated that chronic treatment with taurine could prevent development of corpus cavernosum dysfunction after induction of diabetes.15 Dalaklioglu et al found that the protective effect of taurine might be associated with suppression of nicotinamide adenine dinucleotide phosphate oxidase and Rho-kinase activity. However, the NO/cGMP pathway, which is the most classic pathway in the erectile process, has not been discussed. Our results suggest that taurine may partially restore endothelial function via upregulation of the eNOS/cGMP pathway. Our present work reproduced previous findings demonstrating improved NO production and/or bioavailability, and extended that knowledge by suggesting an antifibrosis effect of this amino acid in the penis. Fibrosis is involved in most diabetic complications, such as cardiomyopathy and nephropathy. Corporal fibrosis also has emerged as the predominant underlying cause of DED, especially in poor responders to PDE5 inhibitors. Although it differs in organs and cells, fibrosis shares some common characteristics. In patients with diabetes, hyperglycemic conditions may serve as the initial insult throughout the corpora and penile arteries, which is typically followed by an inflammatory response.24 Profibrotic factors are subsequently released. It has been demonstrated in animal models that diabetes leads to increased expression of TGF-b1 and penile fibrosis.25e26 These studies suggest that upregulation of TGF-b1 and its downstream effectors might play a pivotal role in diabetes-induced structural changes.25e27 In line with these previous studies, our work suggests that in diabetic J Sex Med 2016;-:1e8
rats, TGF-b1 and PAI-1 are both upregulated. It is well documented that TGF-b1 has multiple biologic functions, including cell proliferation, differentiation, apoptosis, autophagy, and ECM protein production.28 The role of TGF-b1 in penile fibrosis has been confirmed by measuring mRNA expression in penile tissues of young and aging rats.29 TGF-b1 expression was higher in older rats than in young rats. In diabetes models, accumulation of myofibroblasts and the switch to a synthetic phenotype producing ECM proteins due to TGF-b1 activation have been shown to exacerbate penile fibrosis. As such, collagen and ECM proteins accumulate in the penis, accompanied by loss of functional cells. PAI-1, the most potent cellular inhibitor of the urokinase-type plasminogen activator/tissue-type plasminogen activator/plasmin fibrinolytic system, plays a prominent role in wound healing. However, under certain circumstances (eg, diabetes), excessive PAI-1 expression contributes to induced fibrogenesis by suppressing cellular proteolytic activity and increasing ECM protein stability.30 Taken together, elevated TGF-b1 and PAI-1 expression promotes excessive deposition of collagen and ECM proteins in cavernous tissue. However, both in vivo and in vitro studies have demonstrated that taurine can inhibit expression of TGF-b1.31e32 In addition, Lee et al reported that taurine significantly reduced PAI-1 expression in rats with streptozotocin-induced diabetes at the mRNA and protein level.33 Here, taurine administration reversed the elevated expression of TGF-b1 and PAI-1. As a result, collagens and ECM proteins, including collagen I and collagen IV, were reduced.
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Figure 4. Semiquantitative analyses of smooth muscle and collagen ratio and a-smooth muscle actin (a-SMA) expression in all groups. (A) Masson trichrome and immunohistochemical staining with antibody to a-SMA (magnification 100). (B) Representative Western blot analysis of a-SMA expression. (C) Data are expressed as a ratio of smooth muscle to collagen. (D) Quantitative analysis of a-SMA-positive area in cavernous tissue was performed with an image analyzer. (E) Data are presented as the relative density values of a-SMA to b-actin loading control. The relative ratio measures in the control group is arbitrarily presented as 1. *P < .05 compared with the control group. † P < .05 compared with the DED þ Taurine group. Cavernous smooth muscle cells compose the predominant structure of erectile tissue and are responsible for blood flow control into the corpus cavernosum. In patients with DED, loss of smooth muscle cells further aggravates penile fibrosis. Increased apoptosis and reduced a-SMA expression qualifies this view. Moreover, insulted smooth muscle cells may serve as fibrogenic effector cells, which in return, produce a variety of mediators, cytokines, and other factors involved in inflammation.24 Preservation of taurine for smooth muscle cells can break this vicious circle. In summary, taurine, as an antifibrosis agent, not only reduced ECM protein deposition but also protected functional cells from apoptosis. As DED is closely related to hypogonadism,34 we determined plasma testosterone levels. In line with the results of previous studies,35 DED rats had a marked decrease in plasma testosterone levels. Moreover, taurine affects hormonal parameters in aged rats.11 In the present study, the levels of testosterone were increased in DED rats treated with taurine but were still lower compared to those of normal controls. Accordingly, the increase in plasma testosterone levels might have an indirect effect on erectile function. Our previous work (unpublished, doctorate dissertation of Dr Jun Yang) noted that APO-positive rats had only mild to moderate ED, whereas APO-negative rats’ sexual function was
impaired severely and often had a poorer response to PDE5 inhibitors. In this study, we used an APO-negative model to mimic severe ED. Our results indicate that taurine could at least partially ameliorate ED in APO-negative rats. This finding provides evidence for a potential novel therapy for severe ED. Future studies are needed to validate the combination of PDE5 inhibitors and taurine, which could improve the response rate in nonresponders to PDE5 inhibitors. A major limitation of the current research is that our streptozotocin-induced diabetes model reflects type 1 diabetes but may not necessarily represent the pathologic processes that occur in the more common type of diabetes. Furthermore, we did not thoroughly investigate the mechanisms involved in the benefits of taurine for DED. Therefore, future studies should examine the effect of taurine in vitro and determine the correlation with erectile function and penile fibrosis during the course of diabetes.
CONCLUSION Data from the present study suggest that taurine supplementation may improve erectile function in rats with DED and ameliorate penile fibrosis as well as endothelial dysfunction. This J Sex Med 2016;-:1e8
Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis
finding provides evidence that taurine supplementation may be an alternative therapy for nonresponders to PDE5 inhibitors. Corresponding Authors: Mingchao Li, MD, PhD, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel: (86) 027-83663673; Fax: (86) 027 83663673; E-mail: [email protected] Jihong Liu, MD, PhD, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel: (86) 02783663673; Fax: (86) 027 83663673; [email protected] Conflict of interest: The authors report no conflicts of interest. Funding: This work was supported by a grant from the National Natural Sciences Foundation of China (No. 81300477).
STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Yajun Ruan; Mingchao Li; Tao Wang; Jihong Liu (b) Acquisition of Data Yajun Ruan; Mingchao Li; Jun Yang; Ke Rao (c) Analysis and Interpretation of Data Yajun Ruan; Mingchao Li; Shaogang Wang; Weiming Yang Category 2 (a) Drafting the Article Yajun Ruan; Mingchao Li (b) Revising It for Intellectual Content Yajun Ruan; Mingchao Li; Tao Wang; Jihong Liu; Zhangqun Ye Category 3 (a) Final Approval of the Completed Article Yajun Ruan; Mingchao Li; Tao Wang; Jun Yang; Ke Rao; Shaogang Wang; Weiming Yang; Jihong Liu; Zhangqun Ye
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33. Lee EA, Seo JY, Jiang Z, et al. Reactive oxygen species mediate high glucose-induced plasminogen activator inhibitor1 up-regulation in mesangial cells and in diabetic kidney. Kidney Int 2005;67:1762-1771. 34. Chung E, De Young L, Brock GB. Investigative models in erectile dysfunction: A state-of-the-art review of current animal models. J Sex Med 2011;8:3291-3305. 35. Kataoka T, Hotta Y, Maeda Y, et al. Assessment of androgen replacement therapy for erectile function in rats with type 2 diabetes mellitus by examining nitric oxide-related and inflammatory factors. J Sex Med 2014;11:920-929.
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SUPPLEMENTARY DATA
30. Ghosh AK, Vaughan DE. Pai-1 in tissue fibrosis. J Cell Physiol 2012;227:493-507.
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jsxm.2016.02.164.
J Sex Med 2016;-:1e8
ABSTRACT
Introduction: For patients with diabetes, erectile dysfunction (ED) is common and greatly affects quality of life. However, these patients often exhibit a poor response to first-line oral phosphodiesterase type 5 inhibitors. Aim: To investigate whether taurine, a sulfur-containing amino acid, affects diabetic ED (DED). Methods: Type 1 diabetes mellitus was induced in male rats by using streptozotocin. After 12 weeks, an apomorphine test was conducted to confirm DED. Only rats with DED were administered taurine or vehicle for 4 weeks. Age-matched nondiabetic rats were administered saline intraperitoneally for 4 weeks. Main Outcome Measures: Erectile function was evaluated by electrical stimulation of the cavernous nerve. Histologic and molecular alterations of the corpus cavernosum also were analyzed. Results: Erectile function was significantly reduced in the diabetic rats compared with in the nondiabetic rats, and was improved in the diabetic rats treated with taurine. The corpus cavernosum of the rats with DED exhibited severe fibrosis and decreased smooth muscle content. Deposition of extracellular matrix proteins was increased in the diabetic rats, while expression of endothelial nitric oxide synthase/cyclic guanosine monophosphate/nitric oxide pathwayerelated proteins was reduced. Taurine supplementation ameliorated erectile response as well as histologic and molecular alterations. Conclusion: Taurine supplementation improves erectile function in rats with DED probably by potential antifibrotic activity. This finding provides evidence for a potential new therapy for DED. J Sex Med 2016;-:1e8. Copyright 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. Key Words: Taurine; Diabetes; Erectile Dysfunction; Fibrosis; Extracellular Matrix
INTRODUCTION Erectile dysfunction (ED), defined as the inability to attain or maintain a penile erection sufficient for successful vaginal intercourse, is becoming more common worldwide.1 Approximately 15% to 20% of the general male population experiences ED.2,3 Patients with diabetes are nearly 3 times more likely to suffer from ED compared to patients without diabetes. In addition, ED appears to arise 10 years earlier in patients with diabetes and tends to be more severe, significantly decreasing health-related quality of life.4 Furthermore, the prevalence of ED is 95% in patients with diabetes above the age of 60.3 Received September 25, 2015. Accepted February 17, 2016. 1
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China;
2
Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China
Copyright ª 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsxm.2016.02.164
J Sex Med 2016;-:1e8
Introduction of phosphodiesterase type 5 (PDE5) inhibitors revolutionized the treatment of ED. PDE5 inhibitors enhance nitric oxide (NO)-mediated relaxation of the corpus cavernosum by preventing cyclic guanosine monophosphate (cGMP) degradation. Currently, PDE5 inhibitors are the first-line therapy for ED.5e6 However, because ED is caused by multiple factors, a certain proportion of patients, especially those with diabetes, exhibit a poor response to PDE5 inhibitors.7 Moreover, the failure rate for all PDE5 inhibitors is higher in men with diabetes than in men without.8 In addition, diabetes could induce a series of pathophysiologic changes that could contribute to the decreased response to PDE5 inhibitors. Therefore, it is urgent to develop novel treatment modalities for men with diabetic ED (DED). Taurine (2-aminoethanesulfonic acid), a sulfur-containing amino acid, is one of the most abundant free amino acids in the human body. It plays a role in several physiologic functions, including neuromodulation or neurotransmission, modulation of calcium flow, osmoregulation, stabilization of membranes, and bile formation in the liver.9 Data from existing studies have 1
2
indicated that taurine might decrease cholesterol level, control high blood pressure, and have protective potential in the cardiovascular system.10 Previous studies also have shown a beneficial effect of taurine on diabetes-associated complications. Importantly, it has been demonstrated that taurine supplementation can enhance sexual response and mating ability in aged rats.11 However, whether taurine could mitigate DED has not been investigated. In the present study, we evaluated the effect of taurine in rats with DED in vivo. Our results show that taurine improves erectile function in diabetic rats and could serve as an alternative therapy for ED.
METHODS Treatment of Animals This study was approved by the Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China. Forty-five 8-week-old male Sprague-Dawley rats were used in the present study. After overnight fasting, the rats were injected with freshly prepared streptozotocin (60 mg/kg; Sigma-Aldrich, St. Louis, MO, USA; n ¼ 37) or vehicle (0.1 mol/L citratephosphate buffer; pH, 4.2; n ¼ 8; nondiabetic control group) intraperitoneally. Blood glucose levels were measured 72 hours after streptozotocin injection using a blood glucose meter (ACCU-CHEK Performa; Roche Diagnostics, Shanghai, China). Only rats with a fasting glucose concentration 16.7 mmol/L were considered as diabetic rats (n ¼ 35). At 12 weeks, 30 diabetic rats survived. An apomorphine (APO) test was conducted to confirm DED. Eighteen APOnegative rats were used for further experiments.12,13 Rats with DED were administered taurine (400 mg/kg; Sigma-Aldrich; DED þ taurine group) or vehicle (saline, DED group) intraperitoneally daily for another 4 weeks (n ¼ 9 per group). This dose of taurine is within the range used in previous studies.14e16
In Vivo Erection Studies At 12 weeks after diabetes was induced, erectile function was evaluated by observing APO-induced erection in all surviving rats. APO was injected into the loose skin of the cervical vertebra subcutaneously (80 mg/kg). Then a camera was set in the bottom of the cage for 30 minutes, and the number of erections was recorded. The criteria for penile erection were based on previous studies as follows: stretching of the penis with a congested glans, repeated pelvic thrusts immediately followed by an upright position, and complete emergence of an engorged glans penis and distal penile shaft; otherwise, the result was negative.13,17 At week 16, following a 2-day washout of taurine, intracavernosal pressure (ICP) was measured in all groups as described previously.18 Under proper anesthesia (pentobarbital sodium, 40 mg/kg, i.p.), the rats were fixed on the table, and the left carotid artery was exposed. A PE-50 tube filled with heparinized saline
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(100 IU/mL) was cannulated into the artery and connected to a pressure transducer to monitor mean arterial pressure (MAP) continuously. A 25-G needle was inserted into the left penile crura to monitor ICP. Pressure data were recorded by a data acquisition system (PowerLab/4SP; AD Instruments, Bella Vista, Australia). Erectile response was elicited by electrical stimulation using a bipolar electrode hooked to the cavernous nerve. Stimulation parameters were as follows: 7.5 v, 12 Hz, and 1.2 ms; this was judged to cause a maximal reaction.18e20 The ratio between maximum ICP and MAP (ICP max/MAP) was calculated to normalize for variations in systemic blood pressure. Total ICP was determined by the area under the curve (AUC, mmHg $ s) during stimulation. After finishing these in vivo studies, the rats were sacrificed by intraperitoneal overdose of pentobarbital. Penile midshafts were maintained in 4% paraformaldehyde overnight and embedded in paraffin for subsequent histologic studies. Remaining penile tissues were snap frozen in liquid nitrogen and stored at 80 C for subsequent molecular detection. Blood samples were obtained from rats via the vena cava. The total testosterone levels were measured in the central laboratory of our institution.
Western Blot Analysis Equal amounts (40 mg/lane) of protein extracts were electrophoresed on 10% sodium dodecylsulfateepolyacrylamide gels, transferred to polyvinylidene fluoride membranes (Immobilon-P Transfer Membrane; Millipore Corporation, Billerica, MA, USA), and probed with antibodies against endothelial nitric oxide synthase (eNOS; 1:500; BD Biosciences, San Jose, CA, USA), plasminogen activator inhibitor type 1 (PAI-1; 1:2500; BD Biosciences), collagen I (1:500; Boster, Wuhan, China), collagen IV (1:500; Proteintech, Wuhan, China), transforming growth factor b1 (TGF-b1; 1:500; Abcam, Cambridge, UK), aesmooth muscle actin (a-SMA; 1:1000; Abcam), or b-actin (1:500; Boster). Primary antibodies were incubated overnight at 4 C followed by incubation with horseradish peroxidase-labeled secondary antibody (1:10,000; Jackson ImmunoResearch, West Grove, PA, USA), then detected with Bio-Rad Clarity Western ECL Substrate (Bio-Rad Laboratories, CA, USA). The resulting image was analyzed using ImageJ software (http://rsb.info.nih.gov/ij) to determine the integrated density value of each protein band.
cGMP Measurement cGMP was measured in extracts from penile samples using a commercial cGMP enzyme immunoassay kit (Cayman Chemicals Inc., Ann Arbor, MI, USA) following the manufacturer’s instructions. Protein content in each sample was measured using the BCA assay and expressed as range of pmol cGMP/mg protein. All samples were analyzed in triplicate.20
Histologic Assessment Penile tissue sections (5 mm thickness) were processed for immunohistochemistry as described previously.21 The primary antibody was mouse antiea-SMA (1:150; Boster). Masson’s J Sex Med 2016;-:1e8
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Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis
trichrome staining was performed according to the standard protocol. Semiquantitative analysis was performed to evaluate intensity using Image-Pro plus software (Media Cybernetics, Silver Spring, MD, USA).
Statistical Analysis Results were analyzed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard error of the mean. Statistical analyses were performed using 1-way analysis of variance followed by the Tukey-Kramer test for post hoc comparisons. Differences among groups were considered significant at P < .05.
RESULTS Metabolic Variables and ICP/MAP Assessment There were no significant differences in initial body weight or serum glucose concentration among the 3 groups. At 16 weeks after diabetes was induced, fasting glucose concentration was significantly higher, and body weight along with total testosterone levels were significantly lower in the diabetic rats than in the age-matched normal controls. No significant differences in body weight or glucose concentration were found between the DED and DED þ taurine groups (Table 1). Erectile function was evaluated by measuring maximum ICP, ICP max/MAP, and total ICP. All erectile function variables were significantly lower in the diabetic rats than in the control rats (Figure 1). Partial but significant recovery of erectile function was seen in the DED þ taurine group. MAP did not differ significantly among the groups (Table 1, Figure 1).
EXTRACELLULAR MATRIX PROTEIN PRODUCTION We performed Western blot analysis to evaluate production of extracellular matrix (ECM) proteins, including PAI-1, collagen I, and collagen IV. Cavernous ECM protein production was
significantly higher in the DED group than in the normal controls (P < .05); however, taurine reversed this change (Figure 2). We also performed Western blot analysis to evaluate expression of a-SMA and TGF-b1 in each group. The diabetic rats showed lower a-SMA expression than the normal controls. Expression of a-SMA was higher in the DED þ taurine group than in the DED group (all P < .05; Figures 4B and 4E). Meanwhile, expression of TGF-b1 was higher in the DED group than in the normal controls; however, taurine partially reversed its upregulation.
eNOS Expression We performed eNOS Western blot analysis to quantify endothelial function in all groups. Penile tissue in the rats with DED showed significantly lower eNOS immunoexpression compared with the normal controls (P < .05). This decline was attenuated in part by taurine (Figures 3A and 3B).
cGMP Concentration Cavernous tissue cGMP concentration in the DED group was significantly lower than that in the normal controls (P < .05). The DED þ taurine group had a higher concentration of cGMP than the DED group (P < .05; Figure 3C).
Histologic Changes As displayed in Figure 4, corporal tissue in the diabetic rats showed a significantly lower smooth muscle/collagen ratio compared with tissue in the normal controls (0.13 and 0.30, respectively; P < .05). Taurine increased the ratio in the DED þ taurine group compared with that in the DED group. Immunohistochemical staining showed that taurine-treated DED group had a significant increase of a-SMA in corpus cavernosum compared with DED group (13.53% and 7.73%; P < .05).
DISCUSSION In this study, we demonstrated that administration of taurine, not as a food supplement, in rats with streptozotocin-induced
Table 1. Metabolic and Physiological Variables Variable
Controla (n ¼ 8)
Initial weight (g) Final weight (g) Initial fasting glucose (mmol/L) Final fasting glucose (mmol/L) Testosterone (ng/mL) MAP (mm Hg) ICP (mm Hg) AUC (mm Hg $ s)
240.90 522.90 6.25 6.23 2.55 112.50 97.58 5125
± ± ± ± ± ± ± ±
11.47 60.31 0.41 0.47 0.61 9.73 9.04 723.6
DEDa (n ¼ 9) 233.10 243.00 6.12 30.54 0.73 114.10 43.28 1504
MAP ¼ mean arterial pressure; ICP ¼ intracavernous pressure; AUC ¼ area under the curve. a Data are presented as mean value ± standard deviation. * P< .05 compared with the control group. † P< .05 compared with the DED þ taurine group. J Sex Med 2016;-:1e8
± ± ± ± ± ± ± ±
11.09 42.66* 0.44 2.48* 0.05* 10.30 12.10*,† 505.2*,†
DED þ taurinea (n ¼ 9) 233.00 257.50 6.11 30.86 1.60 115.80 71.99 3687
± ± ± ± ± ± ± ±
12.06 33.35* 0.48 2.11* 0.25*,† 10.56 16.08* 1308*
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Figure 1. Increase of erectile function in taurine-treated diabetic rats. (A) Representative tracing of intracavernosal pressure (ICP) and mean arterial pressure (MAP) for age-matched normal control, diabetic erectile dysfunction (DED) and taurine-treated DED groups. (B, C) Erectile function presented as maximum ICP/MAP and total ICP (AUC) in each group. *P < .05 compared with the control group. †P < .05 compared with the DED þ Taurine group. diabetes significantly decreased penile fibrosis; significantly decreased production of ECM proteins, including PAI-1, collagen I, and collagen IV; and upregulated eNOS expression; and that these changes were associated with physiologically relevant changes in erectile function. Taurine is known to play a role in a variety of cellular processes, including calcium regulation, inflammation, osmoregulation, oxidative stress, and cysteine detoxification, all of which may
contribute to cardiovascular homeostasis. Taurine has become a potential therapeutic target for cardiovascular disease, preventing stroke, atherosclerosis, and hypertension.22 Maia et al reported that taurine supplementation was beneficial to control high blood pressure and vascular remodeling induced via postweaning protein restriction.16 Besides, the protective effect of taurine on endothelial function in diabetic rats already has been proved.14 As a benchmark for cardiovascular disease,23 erectile function also has been
Figure 2. Taurine reduces cavernous extracellular matrix protein deposition. (A) Representative Western blot for plasminogen activator inhibitor type1 (PAI-1, 47 kDa), transforming growth factor beta 1 (TGF-b1, 37 kDa), collagen I (139 kDa), and collagen IV (161 kDa) in the control group, DED group and DED rats treated with taurine group. (B) Data are presented as the relative density of each protein compared with that of b-actin. *P < .05 compared with the control group. †P < .05 compared with the DED þ Taurine group. J Sex Med 2016;-:1e8
Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis
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Figure 3. Taurine treatment restores cavernous endothelial nitric oxide synthase (eNOS) expression. (A) Representative Western blot for eNOS (140 kDa) in each group. (B) Data are presented as the relative density values of eNOS to the b-actin loading control. The relative ratio measures in the control group is arbitrarily presented as 1. (C) ELISA demonstrating quantity of cGMP. *P < .05 compared with the control group. †P < .05 compared with the DED þ Taurine group. investigated. A previous ex vivo study indicated that chronic treatment with taurine could prevent development of corpus cavernosum dysfunction after induction of diabetes.15 Dalaklioglu et al found that the protective effect of taurine might be associated with suppression of nicotinamide adenine dinucleotide phosphate oxidase and Rho-kinase activity. However, the NO/cGMP pathway, which is the most classic pathway in the erectile process, has not been discussed. Our results suggest that taurine may partially restore endothelial function via upregulation of the eNOS/cGMP pathway. Our present work reproduced previous findings demonstrating improved NO production and/or bioavailability, and extended that knowledge by suggesting an antifibrosis effect of this amino acid in the penis. Fibrosis is involved in most diabetic complications, such as cardiomyopathy and nephropathy. Corporal fibrosis also has emerged as the predominant underlying cause of DED, especially in poor responders to PDE5 inhibitors. Although it differs in organs and cells, fibrosis shares some common characteristics. In patients with diabetes, hyperglycemic conditions may serve as the initial insult throughout the corpora and penile arteries, which is typically followed by an inflammatory response.24 Profibrotic factors are subsequently released. It has been demonstrated in animal models that diabetes leads to increased expression of TGF-b1 and penile fibrosis.25e26 These studies suggest that upregulation of TGF-b1 and its downstream effectors might play a pivotal role in diabetes-induced structural changes.25e27 In line with these previous studies, our work suggests that in diabetic J Sex Med 2016;-:1e8
rats, TGF-b1 and PAI-1 are both upregulated. It is well documented that TGF-b1 has multiple biologic functions, including cell proliferation, differentiation, apoptosis, autophagy, and ECM protein production.28 The role of TGF-b1 in penile fibrosis has been confirmed by measuring mRNA expression in penile tissues of young and aging rats.29 TGF-b1 expression was higher in older rats than in young rats. In diabetes models, accumulation of myofibroblasts and the switch to a synthetic phenotype producing ECM proteins due to TGF-b1 activation have been shown to exacerbate penile fibrosis. As such, collagen and ECM proteins accumulate in the penis, accompanied by loss of functional cells. PAI-1, the most potent cellular inhibitor of the urokinase-type plasminogen activator/tissue-type plasminogen activator/plasmin fibrinolytic system, plays a prominent role in wound healing. However, under certain circumstances (eg, diabetes), excessive PAI-1 expression contributes to induced fibrogenesis by suppressing cellular proteolytic activity and increasing ECM protein stability.30 Taken together, elevated TGF-b1 and PAI-1 expression promotes excessive deposition of collagen and ECM proteins in cavernous tissue. However, both in vivo and in vitro studies have demonstrated that taurine can inhibit expression of TGF-b1.31e32 In addition, Lee et al reported that taurine significantly reduced PAI-1 expression in rats with streptozotocin-induced diabetes at the mRNA and protein level.33 Here, taurine administration reversed the elevated expression of TGF-b1 and PAI-1. As a result, collagens and ECM proteins, including collagen I and collagen IV, were reduced.
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Figure 4. Semiquantitative analyses of smooth muscle and collagen ratio and a-smooth muscle actin (a-SMA) expression in all groups. (A) Masson trichrome and immunohistochemical staining with antibody to a-SMA (magnification 100). (B) Representative Western blot analysis of a-SMA expression. (C) Data are expressed as a ratio of smooth muscle to collagen. (D) Quantitative analysis of a-SMA-positive area in cavernous tissue was performed with an image analyzer. (E) Data are presented as the relative density values of a-SMA to b-actin loading control. The relative ratio measures in the control group is arbitrarily presented as 1. *P < .05 compared with the control group. † P < .05 compared with the DED þ Taurine group. Cavernous smooth muscle cells compose the predominant structure of erectile tissue and are responsible for blood flow control into the corpus cavernosum. In patients with DED, loss of smooth muscle cells further aggravates penile fibrosis. Increased apoptosis and reduced a-SMA expression qualifies this view. Moreover, insulted smooth muscle cells may serve as fibrogenic effector cells, which in return, produce a variety of mediators, cytokines, and other factors involved in inflammation.24 Preservation of taurine for smooth muscle cells can break this vicious circle. In summary, taurine, as an antifibrosis agent, not only reduced ECM protein deposition but also protected functional cells from apoptosis. As DED is closely related to hypogonadism,34 we determined plasma testosterone levels. In line with the results of previous studies,35 DED rats had a marked decrease in plasma testosterone levels. Moreover, taurine affects hormonal parameters in aged rats.11 In the present study, the levels of testosterone were increased in DED rats treated with taurine but were still lower compared to those of normal controls. Accordingly, the increase in plasma testosterone levels might have an indirect effect on erectile function. Our previous work (unpublished, doctorate dissertation of Dr Jun Yang) noted that APO-positive rats had only mild to moderate ED, whereas APO-negative rats’ sexual function was
impaired severely and often had a poorer response to PDE5 inhibitors. In this study, we used an APO-negative model to mimic severe ED. Our results indicate that taurine could at least partially ameliorate ED in APO-negative rats. This finding provides evidence for a potential novel therapy for severe ED. Future studies are needed to validate the combination of PDE5 inhibitors and taurine, which could improve the response rate in nonresponders to PDE5 inhibitors. A major limitation of the current research is that our streptozotocin-induced diabetes model reflects type 1 diabetes but may not necessarily represent the pathologic processes that occur in the more common type of diabetes. Furthermore, we did not thoroughly investigate the mechanisms involved in the benefits of taurine for DED. Therefore, future studies should examine the effect of taurine in vitro and determine the correlation with erectile function and penile fibrosis during the course of diabetes.
CONCLUSION Data from the present study suggest that taurine supplementation may improve erectile function in rats with DED and ameliorate penile fibrosis as well as endothelial dysfunction. This J Sex Med 2016;-:1e8
Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis
finding provides evidence that taurine supplementation may be an alternative therapy for nonresponders to PDE5 inhibitors. Corresponding Authors: Mingchao Li, MD, PhD, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel: (86) 027-83663673; Fax: (86) 027 83663673; E-mail: [email protected] Jihong Liu, MD, PhD, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel: (86) 02783663673; Fax: (86) 027 83663673; [email protected] Conflict of interest: The authors report no conflicts of interest. Funding: This work was supported by a grant from the National Natural Sciences Foundation of China (No. 81300477).
STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Yajun Ruan; Mingchao Li; Tao Wang; Jihong Liu (b) Acquisition of Data Yajun Ruan; Mingchao Li; Jun Yang; Ke Rao (c) Analysis and Interpretation of Data Yajun Ruan; Mingchao Li; Shaogang Wang; Weiming Yang Category 2 (a) Drafting the Article Yajun Ruan; Mingchao Li (b) Revising It for Intellectual Content Yajun Ruan; Mingchao Li; Tao Wang; Jihong Liu; Zhangqun Ye Category 3 (a) Final Approval of the Completed Article Yajun Ruan; Mingchao Li; Tao Wang; Jun Yang; Ke Rao; Shaogang Wang; Weiming Yang; Jihong Liu; Zhangqun Ye
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Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jsxm.2016.02.164.
J Sex Med 2016;-:1e8