- Email: [email protected]
The Breakdown of Preformed Advanced Glycation End Products Reverses Erectile Dysfunction in Streptozotocin‐Induced Diabetic Rats: Preventive Versus Curative Treatment
jsm_217.fm Page 242 Monday, February 13, 2006 8:17 PM Blackwell Publishing IncMalden, USAJSMJournal of Sexual Medicine1743-6095© 2006 International Society for Sexual Medicine200632242252Original ArticleALT-711 Reverses Diabetic EDUsta et al.
242
The Breakdown of Preformed Advanced Glycation End Products Reverses Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats: Preventive Versus Curative Treatment Mustafa F. Usta, MD,1* Muammer Kendirci, MD,* Serap Gur, PhD,* Neale A. Foxwell, BSc,† Trinity J. Bivalacqua, MD, PhD,2* Selim Cellek, MD, PhD,3† and Wayne J.G. Hellstrom, MD* *Tulane University Health Sciences Center, Department of Urology, Section of Andrology, New Orleans, LA, USA; † Wolfson Institute for Biomedical Research, University College London, London, UK DOI: 10.1111/j.1743-6109.2006.00217.x
ABSTRACT
Objectives. Accumulation of advanced glycation end products (AGEs) has been linked to many of the complications of diabetes mellitus, including erectile dysfunction (ED). Furthermore, it has been demonstrated that inhibitors of AGE formation, such as aminoguanidine, can prevent ED in diabetic animals. However, it is unknown whether late administration of a putative cross-link breaker, ALT-711, can reverse diabetic ED. We therefore compared ALT711 and aminoguanidine in their ability to reverse ED in diabetic rats. Materials and Methods. Male Sprague–Dawley rats were randomly divided into four groups: (i) age-matched controls; (ii) streptozotocin (STZ)-induced diabetic rats (60 mg/kg; intraperitoneal injection); (iii) STZ diabetic rats treated with ALT-711 (3 mg/kg/day, intraperitoneal injection); and (iv) STZ diabetic rats treated with aminoguanidine (1 gm/L in drinking water) during the final 6 weeks of 12 weeks of induced diabetes. At the end of 12 weeks, erectile response to cavernous nerve stimulation (CNS) was determined. Neuronal nitric oxide synthase (nNOS) contents were measured in all penises, and AGE levels were determined both in penile tissues and in serum samples. Results. Erectile responses to CNS and penile nNOS protein content were significantly reduced, while AGE levels were elevated in the penises and serum of untreated diabetic animals. Treatment with ALT-711, but not with aminoguanidine, reversed ED and nNOS depletion and reduced serum and penile tissue AGE levels. Conclusions. These results suggest that cross-link breakers, such as ALT-711, are the optimal therapeutic approach, compared with treatment with inhibitors of AGE formation, in the reversal of diabetes-related ED. Usta MF, Kendirci M, Gur S, Foxwell NA, Bivalacqua TJ, Cellek S, and Hellstrom WJG. The breakdown of preformed advanced glycation end products reverses erectile dysfunction in streptozotocin-induced diabetic rats: Preventive versus curative treatment. J Sex Med 2006;3:242–252. Key Words. Advanced Glycation End Products; Diabetes; Nitric Oxide; Nitrergic; Erectile Dysfunction; ALT-711; Diabetic Complications; Cross-Link Breaker
Introduction
E
rectile dysfunction (ED) is defined as the inability to attain or maintain a sufficient penile erection for satisfactory sexual intercourse. It has been reported that 50–75% of diabetic men Present addresses: 1Akdeniz University, School of Medicine, Department of Urology, Section of Andrology, Antalya, Turkey; 2Johns Hopkins Hospital, Brady Urological Institute, Baltimore, MD, USA; 3The Neurology and Gastrointestinal Center of Excellence in Drug Discovery, GlaxoSmithKline, Harlow, Essex, UK.
J Sex Med 2006;3:242–252
have some degree of ED [1,2]. Although the exact pathophysiology of diabetes-related ED remains undetermined, it is widely accepted that diabetes impairs both neurogenic- and endotheliumdependent relaxation of the penile cavernosal smooth muscle [3,4]. A relationship between increased levels of advanced glycation end products (AGEs) in penile tissue and diabetes-related ED has been entertained [5]. It has been reported that AGE can quench endothelial and neuronal derived nitric oxide (NO), decreasing cavernosal smooth muscle © 2006 International Society for Sexual Medicine
jsm_217.fm Page 243 Monday, February 13, 2006 8:17 PM
ALT-711 Reverses Diabetic ED relaxation. It is believed that when AGEs are increased, NO cannot interact with guanylate cyclase, resulting in decreased cyclic guanosine monophosphate levels and ultimately functional ED. In a recent study we demonstrated a significant increase in glycation, AGEs, Galectin-3, and inducible NO synthase (iNOS) protein levels in streptozotocin (STZ) diabetic rat penises. Immunohistochemical staining for AGE and its receptor (Galectin-3) were predominantly localized to the cavernous sinusoids and vascular endothelium in diabetic rats [6]. Advanced glycation end products are purported to be involved in numerous complications of diabetes, such as retinopathy, nephropathy, and neuropathy [7]. Inhibitors of AGE formation can prevent formation of a range of complications in experimental diabetic animals, including ED [6]. Recently, AGE cross-link breakers, such as ALT711, have demonstrated protection against many recognized diabetic complications [8]. The salient difference between AGE formation inhibitors and AGE cross-link breakers is that the latter can cleave established AGEs, thus possessing the potential to reverse established diabetic complications [8]. Indeed, late administration (i.e., after development of the complication) of ALT-711 has reversed AGE-mediated large artery stiffness and distensibility [9], cardiac abnormalities [10], and renal injury [11] in diabetic animal models. To our knowledge, late administration of an AGE formation inhibitor and an AGE cross-link breaker has not been compared in their ability to reverse diabetes-related ED. In our present study, STZ-induced diabetic animals were treated with either aminoguanidine (AGE formation inhibitor) or ALT-711 (AGE cross-link breaker) during the final 6 weeks of a 12-week course of induced diabetes in terms of in vivo erectile function, and tissue and serum AGE levels. Materials and Methods
Induction of Diabetes Male Sprague–Dawley (Charles River Laboratories, Wilmington, MA) rats (18–20 weeks old) were randomly divided into four groups. Group 1 served as age-matched controls and received an intraperitoneal (i.p.) injection of citrate buffer. Groups 2, 3, and 4 received an i.p. injection of STZ in citrate buffer (60 mg/kg) as previously described [6,12–14]. Throughout the study, animals were considered diabetic when blood glucose levels were >10 mmol/L. Six weeks after the STZ
243 injection, groups 3 and 4 were initiated on ALT711 (3 mg/kg/day, i.p. injection) or aminoguanidine (1 g/L, in drinking water), respectively, for another 6 weeks. Body weight and blood glucose levels (Accu-Check, Roche Diagnostics, Basel, Switzerland) were documented at the initiation and after completion of the treatments. All procedures were approved by the Institutional Animal Care and Use Committee of Tulane University. The animals were maintained under controlled temperature and lighting.
In Vivo Erectile Function Twelve weeks after induction of diabetes, erectile responses to cavernosal nerve stimulation (CNS) were measured in all rats as previously described [6,12–14]. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p. injection) and placed on a thermoregulated surgical table. The trachea was cannulated using PE-240 polyethylene tubing to maintain a patent airway. The animals breathed room air enriched with 95% O2 and 5% CO2. One of the carotid arteries was cannulated with PE-50 tubing for the measurement of systemic blood pressure using a pressure transducer (Viggo-Spectramed, Oxnard, CA, USA). A 25-gauge needle filled with 250 U/mL heparin and connected to another pressure transducer via polyethylene tubing was inserted into the right penile crura. Continuous simultaneous recordings of the systemic mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) were performed using a data acquisition system (BioPac Systems, Santa Barbara, CA, USA). The bladder and prostate were exposed through a midline abdominal incision. The right cavernosal nerve and major pelvic ganglion were identified posterolateral to the prostate. An electrical stimulator with a stainless steel bipolar hook was placed around the cavernosal nerve. The nerve was stimulated for 1 minute with square pulses of 15 Hz and 2.5–7.5 V using a stimulator (Grass Instruments, Quincy, MA, USA). ICP/MAP and total ICP (area under the erectile curve [AUC; mm Hg/ second]) values were determined. After completing functional studies, the penises were removed for molecular studies. The blood was collected for AGE analysis. Western Blotting The frozen tissues were pulverized using a stainless steel mortar and pestle on dry ice, homogenized in homogenization buffer (HEPES 20 mM, pH 7.2, ethylenediaminetetraacetic acid [EDTA] J Sex Med 2006;3:242–252
jsm_217.fm Page 244 Monday, February 13, 2006 8:17 PM
244 1 mM, sucrose 0.2 M, dithiothreitol 5 mM, phenymethylsulFonyl fluoride [PMSF] 0.1 mM, leupeptin and soya bean trypsin inhibitor 20 µg/ mL, pepstatin, E-64, bestatin, aprotinin, and 3,4dichloroisocoumarin 5 µg/mL) and centrifuged at 13,000 × g for 30 minutes at 4°C as previously described [15,16]. The protein concentration in the supernatant was measured and equal amounts of protein (50 µg) were run on 7.5% polyacrylamide sodium dodecyl sulfate (SDS) gels, and then transferred to nitrocellulose membranes. The blots were incubated overnight with monoclonal neuronal NO synthase (nNOS) antibody (Transduction Laboratories, Oxford, UK; 1:2000) followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) (Vector Laboratories, Burlingame, CA, USA, 1:2,000) for 2 hours. The reactive bands were detected with a luminol-based kit. The optimal X-ray exposure was selected, scanned and the density of each band corresponding to nNOS was measured using Scion Image software (Scion Corp., Frederick, MD). Nondiabetic rat brain cytosol was used as a positive control. The density of each band was expressed as percentage of control (nondiabetic rat) band within the same blot.
Measurement of AGEs in the Serum Using Fluorometry Advanced glycation end products were measured in the whole serum as described previously [17]. The samples were analyzed using high-performance liquid chromatography (HPLC) to measure fluorescence (λex = 370 nm, λem = 440 nm) and to measure peptides (λ = 214 nm). AGE fluorescence was expressed as the ratio of the area under the fluorescent curve to the area under the peptide curve. Measurement of AGEs in the Serum Using Enzyme-Linked Immunosorbent Assay (ELISA) The wells (96-well Maxisorp enzyme-linked immunosorbent assay [ELISA] plate, NUNC, Paisley, UK) were coated with polyclonal antiAGE antibody (AGE102; 10 µg/mL; Biologo, Kronshagen, Germany) in 50 mmol/L carbonate buffer (pH 9.6) overnight at room temperature as previously described [17]. The wells were then washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked at room temperature with PBS containing 0.25% bovine serum albumin. After washing, the wells were incubated with the standards (AGE-human serum albumin as described below; diluted 1:10– 1:100,000) or samples (rat serum diluted in PBS J Sex Med 2006;3:242–252
Usta et al. 1:10–1:10,000) at room temperature for 3 hours. After washing, the wells were incubated with monoclonal anti-AGE antibody (clone 6D12; 0.5 µg/ mL; Biologo) for 2 hours at room temperature followed by anti-mouse IgG-HRP (1:7,500; Autogen Bioclear, Calne, UK) for 1 hour at room temperature. The wells were washed again and developed with peroxidase substrate (Autogen Bioclear). The absorbance at 650 nm was measured after a 20-minute reaction time. After adding the stopping solution (Autogen Bioclear), the reading was repeated at 450 nm. The absorbance obtained was compared with AGE-human serum albumin used as standard. Because of the high interassay variability, the results were expressed as the percentage of the control (nondiabetic) rat serum within the same plate.
Production of AGE-Human Serum Albumin Human serum albumin (50 mg/mL) was incubated with 1 M glucose in PBS in sterile conditions at 37°C for 12 weeks. Excess unbound glucose was then removed using dialysis against a high volume of PBS. AGE Immunofluorescence The halved penises were fixed in 4% paraformaldehyde for 48 hours at room temperature as described previously [15,17]. The fixed tissues were transferred into 30% sucrose in phosphate buffer and kept at 4°C overnight. The samples were then frozen in OCT compound (BDH, Poole, UK) and serial transverse cryosections at 10–20 µm intervals were obtained using a cryostat (−18°C; Leica, 2800 Frigocut-E, Bensheim, Germany). The sections were dried on gelatine-coated slides for 2 hours at room temperature and then incubated with PBS containing 0.1% Triton X100 and 5% goat serum. The slides were subsequently incubated with an anti-AGE monoclonal antibody (clone 6D12; 2 µg/mL; Biologo) for 2 hours at room temperature, followed by detection with fluorescent conjugated secondary antibody (goat anti-mouse IgG-fluorescein isothiocyanate [FITC], 1:1,000, Chemicon, Temecula, CA). The images were obtained using a laser-scanning confocal microscope (Leica TCSDMRE, Bensheim, Germany). Image analysis was performed using Leica Confocal Software (Version 2.00, build0871). No immunostaining was observed when the primary antibody was omitted. The immunostaining density was measured in a blinded fashion as the mean amplitude of fluorescence per µm2 in randomly selected areas. In
jsm_217.fm Page 245 Monday, February 13, 2006 8:17 PM
245
ALT-711 Reverses Diabetic ED
Table 1 Body weight (BW) and blood glucose (BG) concentrations of each group at the initiation of the study (initial values) and at the end of 12 weeks (end values)
Control (N = 10) Diabetic (N = 10) Diabetic + ALT (N = 12) Diabetic + AG (N = 11)
Initial BW (g)
End BW (g)
Initial BG (mmol/L)
End BG (mmol/L)
348.6 ± 5.9 358.0 ± 7.0 357.9 ± 5.1 360.0 ± 3.6
414.3 ± 10.7* 302.4 ± 24.2*† 300.4 ± 12.1*† 330.3 ± 35.8*†
5.1 ± 0.1 5.1 ± 0.2 5.0 ± 0.2 5.0 ± 0.2
5.0 ± 0.3 19.9 ± 1.4*† 23.2 ± 1.3*† 20.3 ± 1.9*†
*P < 0.05 vs. initial value in the same group; †P < 0.05 vs. end value in the control group. AG = aminoguanidine.
order to avoid day-to-day variation in fluorescence intensity, several sections from different experimental groups were immunostained and analyzed in the same batch on the same day. The laser intensity and gain functions were set according to the control tissue; thereafter, these settings were applied to all sections from all experimental groups within the same batch.
ICP values (Figure 1). A representative ICP tracing after CNS at the 5 V setting for 1 minute in all groups is given in Figure 2.
NNOS Protein Levels Western blotting of the crude extract of the penises using an anti-nNOS antibody revealed a
Statistical Analysis Results are expressed as mean ± standard error of the mean from a number of independent experiments. The differences between the groups were compared by two-tailed Mann–Whitney U-test or one-way ANOVA followed by Dunnett’s test as indicated. Level of significance less than 0.05 was considered significant. Statistical analyses were performed using a software program for statistics (SPSS 10.0 Version, Chicago, IL, USA). Results
Effect of Diabetes and Drug Treatment on Body Weight and Blood Glucose Body weight and blood glucose levels are summarized in Table 1. During the 12-week period, control rats gained weight, while diabetic rats lost weight. The blood glucose concentrations were higher in the diabetic group than in the control group. The animals treated with ALT-711 and aminoguanidine in the last 6 weeks of the study did not differ from the diabetic group in body weight or serum glucose levels. In Vivo Erectile Function Erectile responses to CNS are given in Figures 1 and 2. The mean ICP values were significantly lower in the diabetic group. Treatment of diabetic animals with ALT-711 in the last 6 weeks of the study resulted in significant restoration of erectile function, leading to ICP levels comparable to those in the control group. Treatment with aminoguanidine did not alter the effect of diabetes. A similar trend was observed in ICP/MAP and total
Figure 1 Treatment with ALT-711, not with aminoguanidine (AG), reversed diabetes-induced reduction in the intracavernous pressure (ICP) rise to nerve stimulation. (a) The ratio of ICP to mean arterial pressure (MAP) is calculated and plotted against different voltages of cavernous nerve stimulation for each group. (b) Total ICP increase measured as the area under the curve is plotted against different voltages of cavernous nerve stimulation for each group. Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group, Mann– Whitney U-test. DM = diabetes mellitus.
J Sex Med 2006;3:242–252
jsm_217.fm Page 246 Monday, February 13, 2006 8:17 PM
246
Usta et al.
band at 160 kDa corresponding to nNOS. The density of the band was lower in the nontreated and aminoguanidine-treated diabetic groups than in the control and ALT-711-treated diabetic groups (Figure 3).
Serum AGEs Glycation end products were measured in the serum of the animals using fluorometry and
Figure 3 Diabetes-induced decrease in the nNOS protein content was reversed by ALT-711 treatment but not with aminoguanidine. (a) A representative Western blot showing the bands corresponding to nNOS. 1, rat brain; 2–3, control group; 4–5, diabetic group; 6–7, diabetic group with ALT711 treatment; 8–9, diabetic group with aminoguanidine treatment. (b) Densitometry of the bands in the Western blots from all of the animals (N = 8 per each group) as expressed as percentage of control. Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group, ANOVA followed by Dunnett’s test.
ELISA. Both techniques revealed an increase in serum AGE concentrations in the diabetic group, which was reversed by treatment with ALT-711, but not with aminoguanidine (Figure 4).
AGE Immunofluorescence in the Penis Accumulation of AGEs in the penis was assessed with immunofluorescence using a monoclonal antibody raised against AGEs. The fluorescence intensity was significantly higher in the diabetic group than in the control group. The highest staining was observed at the nerve bundles with lesser degrees of staining in cavernosal smooth muscle, connective tissue, and endothelium. The increase in AGE immunostaining was reversed partially by ALT-711 treatment, but not with aminoguanidine treatment (Figures 5 and 6). Figure 2 Diabetes induced a significant reduction in intracavernous pressure (ICP) increase to cavernous nerve stimulation that is reversed with ALT-711 treatment. Typical tracings of ICP from an age-matched nondiabetic control rat (Control), a 12-week diabetic rat (Diabetic), a 12-week diabetic rat treated with ALT-711 in the last 6 weeks (Diabetic + ALT-711), and a 12-week diabetic rat treated with aminoguanidine in the last 6 weeks (Diabetic + AG). The cavernous nerve was stimulated for 1 minute at the 5 V setting. Vertical scale corresponds to 25 mm Hg of ICP.
J Sex Med 2006;3:242–252
Discussion
This study documents that STZ-induced diabetes causes significant ED in rats within 12 weeks. The diabetic animals treated in the final 6 weeks with ALT-711 did not have ED; their ICP responses to CNS were similar to nondiabetic control animals. Treatment with aminoguanidine for the final 6 weeks did not reverse ED. These data demon-
jsm_217.fm Page 247 Monday, February 13, 2006 8:17 PM
ALT-711 Reverses Diabetic ED
Figure 4 The increase in serum AGEs by diabetes was reversed with ALT-711, but not with aminoguanidine. Serum AGE concentrations were measured using fluorometry as the ratio of AGE-specific fluorescence to total peptide content (a) or using ELISA (b). Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group, ANOVA followed by Dunnett’s test. AU = arbitrary units.
strate that the AGE cross-link breaker, ALT-711, is successful in reversing diabetes-induced ED in this animal model vs. the AGE formation inhibitor, aminoguanidine. To our knowledge, this is the first comparative study between the two anti-AGE agents in a delayed administration setting. The reason for selecting this animal model is twofold. First, the STZ rat model is well established for studying diabetic ED, and irreversible structural damage to the nerves has been shown to occur by 12 weeks [15]. Hence, we initiated drug treatment at the sixth week of a total of 12 weeks of induced diabetes. Previous studies have already shown that this animal model develops ED within the first 4–6 weeks of induced diabetes [6,12–17]. Additionally, it has previously been reported that insulin-dependent diabetes affects endothelial NO synthase (eNOS) and nNOS isoforms, and leads
247 to ED in diabetic animals [13]. Second, ED in diabetic men usually presents later in life compared with other complications necessitating a therapeutic approach rather than a preventive one. It should be noted that the inability to assess erectile function at the time point when ALT-711 treatment was instituted is a partial limitation of this study. However, previous studies have clearly documented that ED is observed within the first 6 weeks of STZ-induced diabetes [6,12–17]. Our laboratory has demonstrated that treatment with aminoguanidine in the last 4 weeks of an 8-week course of diabetes reversed ED without any effect on penile tissue AGE levels [14]. In this study, however, aminoguanidine failed to reverse ED when administered in the last 6 weeks of 12week diabetes. Although it is uncertain what transpires between 4 and 6 weeks that makes aminoguanidine ineffective, several possibilities exist. (i) Inhibition of AGE formation between 4 and 6 weeks may be sufficient to arrest any further increase in AGE levels, to the extent that this prevents loss in erectile function. However, there were no changes in penile tissue AGE levels with aminoguanidine treatment in our previous study [14], suggesting that the ability of aminoguanidine to reverse ED may not be related to AGEs. Because aminoguanidine improves tissue collagen turnover in diabetic penises, this preservation of normal collagen content may be the reason for normal erectile function in these rats. This hypothesis needs further evaluation. (ii) Another hypothesis is that aminoguanidine protects cavernosal eNOS/nNOS levels only during the first 4–6 weeks. Nevertheless, the failure of aminoguanidine to reverse ED after a longer duration of diabetes is documented in this study, and suggests a difference in action between AGE formation inhibitors and cross-link breakers. The reversal of diabetes-induced ED with ALT711 in this study together with earlier investigations revealing aminoguanidine to be protective [6,18] further highlights the importance of AGEs in the pathogenesis of diabetic complication. AGEs are hypothesized to quench NO, and reduce available NO levels in the penis during sexual stimulation [5,6]. AGEs also interfere with the L-arginine–NO balance, by increasing iNOS and reducing eNOS expression in the penis [5]. This increased oxidative stress in the cell bodies of nNOS-containing neurons leads to apoptosis and irreversible nNOS depletion in the penis [15,17]. Because NO derived from nNOS within the autonomic nerves is the major initiator of erection, we J Sex Med 2006;3:242–252
jsm_217.fm Page 248 Monday, February 13, 2006 8:17 PM
248
Usta et al.
Control
Diabetic
Diabetic + ALT
Diabetic + AG
Figure 5 AGE immunostaining in the penis was increased in the diabetic group. Accumulation of AGEs was measured using immunofluorescence with a monoclonal antibody. Note the distinctive staining in the nerve bundles (white arrows). Scale bar applies to all panels.
assessed the nNOS protein levels in diabetic animals with and without delayed treatment. Previously, we demonstrated that penile nNOS protein is depleted throughout 20 weeks of induced diabe-
Figure 6 ALT-711, not aminoguanidine, reversed diabetesinduced AGE accumulation in the penis. Immunostaining intensity of AGEs in the penile nerve bundles is expressed as mean amplitude per µm2 area. Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group; †P < 0.05 significantly different from DM or DM + AG groups, ANOVA followed by Dunnett’s test.
J Sex Med 2006;3:242–252
tes in two phases. In the first phase (approximately first 10–12 weeks), nNOS-containing (nitrergic) nerve axons in the penis remain intact, while their nNOS content gradually decreases. This may be caused by a deficient antegrade axonal transport of nNOS from the cell bodies in the pelvic ganglia to the axon in the penis. In the second phase, accumulated AGEs synergize with NO, leading to oxidative stress and apoptosis of the nitrergic neurons [15]. In the present study, we again demonstrated a significant reduction in nNOS levels in the penis of 12-week diabetic rats. Interestingly, treatment with ALT-711, but not with aminoguanidine, normalized the nNOS levels, suggesting that AGEs might be interfering with the axonal transport of nNOS. Our results postulate a novel mechanism by which AGEs may interfere with NO production in the autonomic nervous system. Another possibility suggests that eNOS has glycation sites located at base pairs 599–622 and AGEs may affect eNOS activity either transcriptionally or posttranslationally [5]. Furthermore, hyperglycemia
jsm_217.fm Page 249 Monday, February 13, 2006 8:17 PM
249
ALT-711 Reverses Diabetic ED causing O-linked eNOS glycosylation at the Akt target S1177 may lead to decreased NO production [19,20]. This plausible mechanism suggests a possible relationship between AGEs and eNOS. The role of eNOS in AGE-related diabetic ED still needs further clarification. Studies investigating the levels of eNOS in diabetic rat penile tissue and the effect of ALT-711 on its expression are ongoing. We have demonstrated in this study that AGE concentrations in the serum are significantly increased during 12 weeks of diabetes. Parallel to serum AGEs, we have also observed an increase in the immunostaining of AGEs in cross-sections of the penises, especially in the nerve bundles. This suggests that the axons within these bundles are targets for AGEs. Treatment with ALT-711, but not with aminoguanidine, reduced the AGE concentrations in the serum to control levels. Although AGE immunostaining in the penises was significantly lowered with ALT-711 treatment, it still remained higher than the control group. This suggests that although ALT-711 can reduce circulating AGEs, perhaps by facilitating their disposal from the body, its effect on AGEs already formed within the tissues may be determined by rates of tissue turnover and other unknown variables related to diabetes. Phase 2 clinical trials of ALT-711 (alagebrium) were initiated in 1998. Four of these trials have been completed and ALT-711 was well tolerated [8]. A recent Phase 2a clinical trial demonstrated ALT-711 improving total arterial compliance in aged humans with vascular stiffening [21]. Another study revealed significant reductions in left ventricular mass, left ventricular diastolic filling, and a positive effect on three key quality of life measurements in elderly patients with isolated diastolic heart failure [22]. These results hint that ALT-711 may in the future be employed to treat AGE-dependent ED in diabetic and/or aged patients. Conclusions
Delayed administration of the AGE cross-link breaker, ALT-711, but not the AGE formation inhibitor, aminoguanidine, reduced elevated serum and penile AGE levels, and reverses nNOS depletion in the diabetic penis. ALT-711 can restore erectile function in STZ-induced diabetic rats. More detailed studies investigating the AGE mechanisms in relation to erectile physiology will catalyze novel concepts about diabetic ED. This
knowledge will undoubtedly provide the basis for further therapeutic approaches for diabetic men with ED. Acknowledgments
ALT-711 was kindly provided by Alteon Inc., Parsippany, NJ, USA. This study was funded by the Juvenile Diabetes Research Foundation (S.C.), a scholarship from the Sexual Medicine Society of North America, Bayer/GlaxoSmithKline (M.F.U.), the American Medical Association (T.J.B.), and Young Investigator Award from the International Society for Sexual and Impotence Research (T.J.B.). The authors thank Dr. Kemal H. Gülkesen for assistance in statistical analyses. Corresponding Author: Wayne J.G. Hellstrom, MD, FACS, Tulane University Health Sciences Center, Department of Urology, Section of Andrology, 1430 Tulane Avenue SL-42, New Orleans, LA 70112, USA. Tel: (504) 988-7308; Fax: (504) 988-5059; E-mail: [email protected] or [email protected] Conflict of Interest: None declared.
References
1 Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB. Impotence and its medical and psychosocial correlates: Results of the Massachusetts Male Aging Study. J Urol 1994;151:54–61. 2 Lewis RW, Fugl-Meyer KS, Bosch R, Fugl-Meyer AR, Laumann EO, Lizza E, Morales AM. Epidemiology/risk factors of sexual dysfunction. J Sex Med 2004;1:35–9. 3 Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen RA. Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men with impotence. N Engl J Med 1989;320:1025–30. 4 Saenz de Tejada I, Angulo J, Cellek S, GonzalesCadavid N, Heaton J, Pickard R, Simonsen U. Pathophysiology of erectile dysfunction. J Sex Med 2005;2:26–39. 5 Seftel AD, Vaziri ND, Ni Z, Razmjouei K, Fogarty J, Hampel N, Polak J, Wang RZ, Ferguson K, Block C, Hass C. Advanced glycation end products in human penis: Elevation in diabetic tissue, site of deposition, and possible effect through iNOS or eNOS. Urology 1997;50:1016–26. 6 Usta MF, Bivalacqua TJ, Yang DY, Ramanihatharan A, Sell D, Viswanathan A, Monnier VM, Hellsttrom WJ. The protective effect of aminoguanidine on erectile function in streptozotocin diabetic rats. J Urol 2003;170:1437–42. 7 Stitt AW, Jenkins AJ, Cooper ME. Advanced glycation end products and diabetic complications. Expert Opin Investig Drugs 2002;11:1205–23. J Sex Med 2006;3:242–252
jsm_217.fm Page 250 Monday, February 13, 2006 8:17 PM
250 8 Vasan S, Foiles P, Founds H. Therapeutic potential of breakers of advanced glycation end product– protein crosslinks. Arch Biochem Biophys 2003; 419:89–96. 9 Wolffenbuttel BH, Boulanger CM, Crijns FR, Huijberts MS, Poitevin P, Swennen GN, Vasan S, Egan JJ, Ulrich P, Cerami A, Levy BI. Breakers of advanced glycation end products restore large artery properties in experimental diabetes. Proc Natl Acad Sci USA 1998;95:4630–4. 10 Candido R, Forbes JM, Thomas MC, Thallas V, Dean RG, Burns WC, Tikellis C, Ritchie RH, Twigg SM, Cooper ME, Burrell LM. A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 2003;92:785–92. 11 Forbes JM, Thallas V, Thomas MC, Founds HW, Burns WC, Jerums G, Cooper ME. The breakdown of preexisting advanced glycation end products is associated with reduced renal fibrosis in experimental diabetes. FASEB J 2003;17:1762–4. 12 Bivalacqua TJ, Usta MF, Kendirci M, Pradhan L, Alvarez X, Champion HC, Kadowitz PJ, Hellstrom WJ. Superoxide anion production in the rat penis impairs erectile function in diabetes: Influence of in vivo extracellular superoxide dismutase gene therapy. J Sex Med 2005;2:187–98. 13 Bivalacqua TJ, Champion HC, Usta MF, Cellek S, Chitaley K, Webb RC, Lewis RL, Mills TM, Hellstrom WJ, Kadowitz PJ. RhoA/Rho kinase suppresses endothelial nitric oxide synthase in the penis: A mechanism for diabetes-associated erectile dysfunction. Proc Natl Acad Sci USA 2004; 101:9121–6. 14 Usta MF, Bivalacqua TJ, Koksal IT, Toptas B, Surmen S, Hellstrom WJ. The protective effect of aminoguanidine on erectile function in diabetic rats is not related to the timing of treatment. BJU Int 2004;94:429–32. 15 Cellek S, Foxwell NA, Moncada S. Two phases of nitrergic neuropathy in streptozotocin-induced diabetic rats. Diabetes 2003;52:2353–62. 16 Cellek S, Rodrigo J, Lobos E, Fernandez P, Serrano J, Moncada S. Selective nitrergic neurodegeneration in diabetes mellitus—A nitric oxide-dependent phenomenon. Br J Pharmacol 1999;128:1804–12. 17 Cellek S, Qu W, Schmidt AM, Moncada S. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes. Diabetologia 2004;47:331–9. 18 Cartledge JJ, Eardley I, Morrison JF. Advanced glycation end-products are responsible for the impairment of corpus cavernosal smooth muscle relaxation seen in diabetes. BJU Int 2001;87:402–7. 19 Musicki B, Kramer MF, Becker RE, Burnett AL. Age-related changes in phosphorylation of endothelial nitric oxide synthase in the rat penis. J Sex Med 2005;2:347–57. J Sex Med 2006;3:242–252
Usta et al. 20 Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest 2001;108:1341–8. 21 Kass DA, Shapiro EP, Kawaguchi M, Capriotti AR, Scuteri A, deGroof RC, Lakatta EG. Improved arterial compliance by a novel advanced glycation end-product crosslink breaker. Circulation 2001; 104:1464–70. 22 Little WC, Zile MR, Kitzman DW, Hundley WG, O’Brien TX, Degroof RC. The effect of alagebrium chloride (ALT-711), a novel glucose cross-link breaker, in the treatment of elderly patients with diastolic heart failure. J Card Fail 2005;11:191–5.
Commentary
Erectile dysfunction (ED) is highly prevalent among diabetic men. In addition, diabetic patients with ED are particularly resistant to standard treatments for ED. Thus, the understanding of pathophysiological mechanisms leading to diabetic ED, as well as the localization of specific therapeutic targets, are priorities for research in the field of sexual medicine. Relevant information is available from other fields of research regarding diabetes-associated complications, in particular studies on the pathophysiology of diabetesinduced vascular dysfunctions. In this regard, the formation of advanced glycosylation end products (AGEs) has been suggested as one of the mechanisms contributing to the vascular alterations caused by diabetes. Nonenzymatic glycosylation of proteins (glycation), also known as Maillard reaction, depends on the glucose concentration and consists of the reaction of the carboxyl group of reducing sugars with the free amino groups of proteins. After the formation of a Schiff base, the adduct of sugar and protein is converted to an Amadori product that undergoes sequential rearrangements that lead to the formation of AGEs in the course of weeks–months. It is well known that AGEs increased in diabetes and interfere with endothelium-dependent relaxation [1]. AGEs can modify intracellular and plasmatic proteins or interact with specific receptors, but also modify extracellular matrix proteins by increasing covalent cross-linking that inhibits matrix degradation. All these actions could contribute to the development of diabetic complications. The present study by Hellstrom and collaborators demonstrates that AGEs are involved in the impairment of erectile responses caused by diabetes and confirms the important role of AGEs in dia-
jsm_217.fm Page 251 Monday, February 13, 2006 8:17 PM
ALT-711 Reverses Diabetic ED betic complications. Previous studies have suggested the contribution of AGEs to the development of ED and cavernosal dysfunction in diabetes [2–4]. The most important finding from the present study is that AGE accumulation may be reversed with the recovery of erectile function. The same effects on erectile function in diabetic rats were previously obtained following preventive treatment with aminoguanidine [4], but the present results show that this therapeutic approach (aminoguanidine treatment) does not reverse the established AGE accumulation. In this regard, the use of ALT-711 has a significant advantage, as this compound is able to reduce the level of pre-existing AGEs. Theoretically, aminoguanidine impedes de novo formation of AGEs [1], while ALT-711, as other AGE cross-link breakers, cleaves the covalent binding of AGE-modified proteins with other amino groups, favoring the elimination of AGEs. In addition, aminoguanidine could have other effects unrelated to AGE inhibition, as it is a known inhibitor of the inducible isoform of nitric oxide synthase, making this compound a poor pharmacological tool. In contrast, the use of AGE crosslink breakers could have a better therapeutic profile and has been shown to produce beneficial cardiovascular effects in elderly patients [5]. Usta and collaborators do not demonstrate an increase of AGEs before ALT-711 was administered, but an increase of serum AGEs after 6 weeks of diabetes in this model [6]. Thus, ALT711 likely facilitated the elimination of these preexisting AGEs, having more impact in serum AGEs, where levels are normalized, while in the cavernosal tissue, a higher amount of AGEs remained, suggesting that these correspond to structural proteins with slow turnover. ALT-711 could interact with other signaling pathways relevant to diabetic complications. ALT711 inhibits protein kinase C (PKC) activation in kidneys from diabetic rats, an effect not completely related to its capacity to reduce AGEs [7]. As PKC overactivity has been proposed as a mechanism contributing to the development of diabetic complications, the putative effect of ALT-711 on PKC activation would enhance the beneficial impact of this agent on diabetic complications and hence, on diabetic ED. Probably, several altered pathways are responsible for complications associated with diabetes, including increase of aldose reductase pathway, PKC overactivity, overflow through hexosamine pathway, and glycation and AGE formation. Individual actions on each one of these pathways have been shown to be effective in
251 animal models of diabetic complications, making difficult to differentiate the relative roles of the respective pathways. However, in addition to the likely interaction among the different pathways, it has been proposed that these pathways share a common link as all of them could be influenced by hyperglycemia-induced excess of superoxide production by the respiratory chain in the mitochondria [8], providing a possible unifying target for preventing alterations in the previously mentioned pathways. This study also shows that elimination of AGEs contribute to the recovery of nitrergic nerve fibers and neuronal nitric oxide synthase expression. Further studies evaluating the effects of ALT-711 on endothelial function, the other main player in the regulation of penile smooth muscle contractility and erectile function, should be of great interest. However, some considerations should be noted. When we talk about AGEs we are referring to a very heterogeneous group of molecules. This is because the macromolecule modified by the sugar, and the sugar itself, can be diverse. In addition, the microenvironment where the glycation takes place (oxidants, antioxidants) could influence the chemical structure of the adduct and the velocity of the process. The heterogeneity in the process of AGE formation could be more marked in the case of human diabetes. Finally, the glycation process is not interrupted by the AGE breaker and adducts at different time points of the process can be found simultaneously. In this regard, there is little information on the effects of these agents on early and intermediate glycation products, which have been found to contribute to diabetic vascular dysfunction [6] and cause endothelial dysfunction in corpus cavernosum from normal rats [9]. In summary, this interesting study suggests that glycation plays an important role in the development of diabetes-associated ED and, although human diabetes is more complex and varied, provides a rationale for considering AGE elimination as a therapeutic approach for treating diabetic ED. This will be dependent on further understanding of the time-course, pharmacokinetics, and longterm side-effects of cross-link breakers. In addition, this study confirms that intervention on the specific mechanisms involved in diabetes-induced vascular and/or neurological alterations has a beneficial impact on erectile function in diabetes. Javier Angulo, MD, and Iñigo Sáenz de Tejada, MD Institute of Sexual Medicine, Madrid, Spain J Sex Med 2006;3:242–252
jsm_217.fm Page 252 Monday, February 13, 2006 8:17 PM
252
Usta et al.
References
1 Bucala R, Tracey KJ, Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest 1991;87:432–8. 2 Seftel AD, Vaziri ND, Ni Z, Razmjouci K, Fogarty J, Hampel N, Polak J, Wang RZ, Ferguson K, Block C, Haas C. Advanced glycation end products in human penis: Elevation in diabetic tissue, site of deposition and possible effect through iNOS or eNOS. Urology 1997;50:1016–26. 3 Cartledge JJ, Eardley I, Morrison JF. Advanced glycation end-products are responsible for the impairment of corpus cavernosum smooth muscle relaxation seen in diabetes. BJU Int 2001;87:402–7. 4 Usta MF, Bivalacqua TJ, Yang DY, Ramanitharan A, Sell DR, Viswanathan A, Monnier VM, Hellstrom WJ. The protective effect of aminoguanidine on erectile function in streptozotocin diabetic rats. J Urol 2003;170:1437–42. 5 Little WC, Zile MR, Kitzman DW, Hundley WG, O’Brien TX, Degroof RC. The effect of alagebrium chloride (ALT-711), a novel glucose cross-
J Sex Med 2006;3:242–252
6
7
8 9
link breaker, in the treatment of elderly patients with diastolic heart failure. J Card Fail 2005;11: 196–9. Rodríguez-Mañas L, Angulo J, Vallejo S, Peiró C, Sánchez Ferrer A, Cercas E, López-Dóriga P, Sánchez-Ferrer CF. Early and intermediate Amadori glycosylation adducts, oxidative stress, and endothelial dysfunction in the streptozotocin-induced diabetic rats vasculature. Diabetologia 2003;46:556– 66. Thallas-Bonke V, Lindschau C, Rizkalla B, Bach LA, Boner G, Meier M, Haller H, Cooper ME, Forbes JM. Attenuation of extracellular matrix accumulation in diabetic nephropathy by the advanced glycation end product cross-link breaker ALT-711 via protein kinase C-α-dependent pathway. Diabetes 2004;53: 2921–30. Brownlee M. The pathobiology of diabetic complications. A unifying mechanism. Diabetes 2005;54: 1615–25. Cartledge JJ, Eardley I, Morrison JFB. Impairment of corpus cavernosal smooth muscle relaxation by glycosylated human hemoglobin. BJU Int 2000;85: 735–41.
242
The Breakdown of Preformed Advanced Glycation End Products Reverses Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats: Preventive Versus Curative Treatment Mustafa F. Usta, MD,1* Muammer Kendirci, MD,* Serap Gur, PhD,* Neale A. Foxwell, BSc,† Trinity J. Bivalacqua, MD, PhD,2* Selim Cellek, MD, PhD,3† and Wayne J.G. Hellstrom, MD* *Tulane University Health Sciences Center, Department of Urology, Section of Andrology, New Orleans, LA, USA; † Wolfson Institute for Biomedical Research, University College London, London, UK DOI: 10.1111/j.1743-6109.2006.00217.x
ABSTRACT
Objectives. Accumulation of advanced glycation end products (AGEs) has been linked to many of the complications of diabetes mellitus, including erectile dysfunction (ED). Furthermore, it has been demonstrated that inhibitors of AGE formation, such as aminoguanidine, can prevent ED in diabetic animals. However, it is unknown whether late administration of a putative cross-link breaker, ALT-711, can reverse diabetic ED. We therefore compared ALT711 and aminoguanidine in their ability to reverse ED in diabetic rats. Materials and Methods. Male Sprague–Dawley rats were randomly divided into four groups: (i) age-matched controls; (ii) streptozotocin (STZ)-induced diabetic rats (60 mg/kg; intraperitoneal injection); (iii) STZ diabetic rats treated with ALT-711 (3 mg/kg/day, intraperitoneal injection); and (iv) STZ diabetic rats treated with aminoguanidine (1 gm/L in drinking water) during the final 6 weeks of 12 weeks of induced diabetes. At the end of 12 weeks, erectile response to cavernous nerve stimulation (CNS) was determined. Neuronal nitric oxide synthase (nNOS) contents were measured in all penises, and AGE levels were determined both in penile tissues and in serum samples. Results. Erectile responses to CNS and penile nNOS protein content were significantly reduced, while AGE levels were elevated in the penises and serum of untreated diabetic animals. Treatment with ALT-711, but not with aminoguanidine, reversed ED and nNOS depletion and reduced serum and penile tissue AGE levels. Conclusions. These results suggest that cross-link breakers, such as ALT-711, are the optimal therapeutic approach, compared with treatment with inhibitors of AGE formation, in the reversal of diabetes-related ED. Usta MF, Kendirci M, Gur S, Foxwell NA, Bivalacqua TJ, Cellek S, and Hellstrom WJG. The breakdown of preformed advanced glycation end products reverses erectile dysfunction in streptozotocin-induced diabetic rats: Preventive versus curative treatment. J Sex Med 2006;3:242–252. Key Words. Advanced Glycation End Products; Diabetes; Nitric Oxide; Nitrergic; Erectile Dysfunction; ALT-711; Diabetic Complications; Cross-Link Breaker
Introduction
E
rectile dysfunction (ED) is defined as the inability to attain or maintain a sufficient penile erection for satisfactory sexual intercourse. It has been reported that 50–75% of diabetic men Present addresses: 1Akdeniz University, School of Medicine, Department of Urology, Section of Andrology, Antalya, Turkey; 2Johns Hopkins Hospital, Brady Urological Institute, Baltimore, MD, USA; 3The Neurology and Gastrointestinal Center of Excellence in Drug Discovery, GlaxoSmithKline, Harlow, Essex, UK.
J Sex Med 2006;3:242–252
have some degree of ED [1,2]. Although the exact pathophysiology of diabetes-related ED remains undetermined, it is widely accepted that diabetes impairs both neurogenic- and endotheliumdependent relaxation of the penile cavernosal smooth muscle [3,4]. A relationship between increased levels of advanced glycation end products (AGEs) in penile tissue and diabetes-related ED has been entertained [5]. It has been reported that AGE can quench endothelial and neuronal derived nitric oxide (NO), decreasing cavernosal smooth muscle © 2006 International Society for Sexual Medicine
jsm_217.fm Page 243 Monday, February 13, 2006 8:17 PM
ALT-711 Reverses Diabetic ED relaxation. It is believed that when AGEs are increased, NO cannot interact with guanylate cyclase, resulting in decreased cyclic guanosine monophosphate levels and ultimately functional ED. In a recent study we demonstrated a significant increase in glycation, AGEs, Galectin-3, and inducible NO synthase (iNOS) protein levels in streptozotocin (STZ) diabetic rat penises. Immunohistochemical staining for AGE and its receptor (Galectin-3) were predominantly localized to the cavernous sinusoids and vascular endothelium in diabetic rats [6]. Advanced glycation end products are purported to be involved in numerous complications of diabetes, such as retinopathy, nephropathy, and neuropathy [7]. Inhibitors of AGE formation can prevent formation of a range of complications in experimental diabetic animals, including ED [6]. Recently, AGE cross-link breakers, such as ALT711, have demonstrated protection against many recognized diabetic complications [8]. The salient difference between AGE formation inhibitors and AGE cross-link breakers is that the latter can cleave established AGEs, thus possessing the potential to reverse established diabetic complications [8]. Indeed, late administration (i.e., after development of the complication) of ALT-711 has reversed AGE-mediated large artery stiffness and distensibility [9], cardiac abnormalities [10], and renal injury [11] in diabetic animal models. To our knowledge, late administration of an AGE formation inhibitor and an AGE cross-link breaker has not been compared in their ability to reverse diabetes-related ED. In our present study, STZ-induced diabetic animals were treated with either aminoguanidine (AGE formation inhibitor) or ALT-711 (AGE cross-link breaker) during the final 6 weeks of a 12-week course of induced diabetes in terms of in vivo erectile function, and tissue and serum AGE levels. Materials and Methods
Induction of Diabetes Male Sprague–Dawley (Charles River Laboratories, Wilmington, MA) rats (18–20 weeks old) were randomly divided into four groups. Group 1 served as age-matched controls and received an intraperitoneal (i.p.) injection of citrate buffer. Groups 2, 3, and 4 received an i.p. injection of STZ in citrate buffer (60 mg/kg) as previously described [6,12–14]. Throughout the study, animals were considered diabetic when blood glucose levels were >10 mmol/L. Six weeks after the STZ
243 injection, groups 3 and 4 were initiated on ALT711 (3 mg/kg/day, i.p. injection) or aminoguanidine (1 g/L, in drinking water), respectively, for another 6 weeks. Body weight and blood glucose levels (Accu-Check, Roche Diagnostics, Basel, Switzerland) were documented at the initiation and after completion of the treatments. All procedures were approved by the Institutional Animal Care and Use Committee of Tulane University. The animals were maintained under controlled temperature and lighting.
In Vivo Erectile Function Twelve weeks after induction of diabetes, erectile responses to cavernosal nerve stimulation (CNS) were measured in all rats as previously described [6,12–14]. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p. injection) and placed on a thermoregulated surgical table. The trachea was cannulated using PE-240 polyethylene tubing to maintain a patent airway. The animals breathed room air enriched with 95% O2 and 5% CO2. One of the carotid arteries was cannulated with PE-50 tubing for the measurement of systemic blood pressure using a pressure transducer (Viggo-Spectramed, Oxnard, CA, USA). A 25-gauge needle filled with 250 U/mL heparin and connected to another pressure transducer via polyethylene tubing was inserted into the right penile crura. Continuous simultaneous recordings of the systemic mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) were performed using a data acquisition system (BioPac Systems, Santa Barbara, CA, USA). The bladder and prostate were exposed through a midline abdominal incision. The right cavernosal nerve and major pelvic ganglion were identified posterolateral to the prostate. An electrical stimulator with a stainless steel bipolar hook was placed around the cavernosal nerve. The nerve was stimulated for 1 minute with square pulses of 15 Hz and 2.5–7.5 V using a stimulator (Grass Instruments, Quincy, MA, USA). ICP/MAP and total ICP (area under the erectile curve [AUC; mm Hg/ second]) values were determined. After completing functional studies, the penises were removed for molecular studies. The blood was collected for AGE analysis. Western Blotting The frozen tissues were pulverized using a stainless steel mortar and pestle on dry ice, homogenized in homogenization buffer (HEPES 20 mM, pH 7.2, ethylenediaminetetraacetic acid [EDTA] J Sex Med 2006;3:242–252
jsm_217.fm Page 244 Monday, February 13, 2006 8:17 PM
244 1 mM, sucrose 0.2 M, dithiothreitol 5 mM, phenymethylsulFonyl fluoride [PMSF] 0.1 mM, leupeptin and soya bean trypsin inhibitor 20 µg/ mL, pepstatin, E-64, bestatin, aprotinin, and 3,4dichloroisocoumarin 5 µg/mL) and centrifuged at 13,000 × g for 30 minutes at 4°C as previously described [15,16]. The protein concentration in the supernatant was measured and equal amounts of protein (50 µg) were run on 7.5% polyacrylamide sodium dodecyl sulfate (SDS) gels, and then transferred to nitrocellulose membranes. The blots were incubated overnight with monoclonal neuronal NO synthase (nNOS) antibody (Transduction Laboratories, Oxford, UK; 1:2000) followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) (Vector Laboratories, Burlingame, CA, USA, 1:2,000) for 2 hours. The reactive bands were detected with a luminol-based kit. The optimal X-ray exposure was selected, scanned and the density of each band corresponding to nNOS was measured using Scion Image software (Scion Corp., Frederick, MD). Nondiabetic rat brain cytosol was used as a positive control. The density of each band was expressed as percentage of control (nondiabetic rat) band within the same blot.
Measurement of AGEs in the Serum Using Fluorometry Advanced glycation end products were measured in the whole serum as described previously [17]. The samples were analyzed using high-performance liquid chromatography (HPLC) to measure fluorescence (λex = 370 nm, λem = 440 nm) and to measure peptides (λ = 214 nm). AGE fluorescence was expressed as the ratio of the area under the fluorescent curve to the area under the peptide curve. Measurement of AGEs in the Serum Using Enzyme-Linked Immunosorbent Assay (ELISA) The wells (96-well Maxisorp enzyme-linked immunosorbent assay [ELISA] plate, NUNC, Paisley, UK) were coated with polyclonal antiAGE antibody (AGE102; 10 µg/mL; Biologo, Kronshagen, Germany) in 50 mmol/L carbonate buffer (pH 9.6) overnight at room temperature as previously described [17]. The wells were then washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked at room temperature with PBS containing 0.25% bovine serum albumin. After washing, the wells were incubated with the standards (AGE-human serum albumin as described below; diluted 1:10– 1:100,000) or samples (rat serum diluted in PBS J Sex Med 2006;3:242–252
Usta et al. 1:10–1:10,000) at room temperature for 3 hours. After washing, the wells were incubated with monoclonal anti-AGE antibody (clone 6D12; 0.5 µg/ mL; Biologo) for 2 hours at room temperature followed by anti-mouse IgG-HRP (1:7,500; Autogen Bioclear, Calne, UK) for 1 hour at room temperature. The wells were washed again and developed with peroxidase substrate (Autogen Bioclear). The absorbance at 650 nm was measured after a 20-minute reaction time. After adding the stopping solution (Autogen Bioclear), the reading was repeated at 450 nm. The absorbance obtained was compared with AGE-human serum albumin used as standard. Because of the high interassay variability, the results were expressed as the percentage of the control (nondiabetic) rat serum within the same plate.
Production of AGE-Human Serum Albumin Human serum albumin (50 mg/mL) was incubated with 1 M glucose in PBS in sterile conditions at 37°C for 12 weeks. Excess unbound glucose was then removed using dialysis against a high volume of PBS. AGE Immunofluorescence The halved penises were fixed in 4% paraformaldehyde for 48 hours at room temperature as described previously [15,17]. The fixed tissues were transferred into 30% sucrose in phosphate buffer and kept at 4°C overnight. The samples were then frozen in OCT compound (BDH, Poole, UK) and serial transverse cryosections at 10–20 µm intervals were obtained using a cryostat (−18°C; Leica, 2800 Frigocut-E, Bensheim, Germany). The sections were dried on gelatine-coated slides for 2 hours at room temperature and then incubated with PBS containing 0.1% Triton X100 and 5% goat serum. The slides were subsequently incubated with an anti-AGE monoclonal antibody (clone 6D12; 2 µg/mL; Biologo) for 2 hours at room temperature, followed by detection with fluorescent conjugated secondary antibody (goat anti-mouse IgG-fluorescein isothiocyanate [FITC], 1:1,000, Chemicon, Temecula, CA). The images were obtained using a laser-scanning confocal microscope (Leica TCSDMRE, Bensheim, Germany). Image analysis was performed using Leica Confocal Software (Version 2.00, build0871). No immunostaining was observed when the primary antibody was omitted. The immunostaining density was measured in a blinded fashion as the mean amplitude of fluorescence per µm2 in randomly selected areas. In
jsm_217.fm Page 245 Monday, February 13, 2006 8:17 PM
245
ALT-711 Reverses Diabetic ED
Table 1 Body weight (BW) and blood glucose (BG) concentrations of each group at the initiation of the study (initial values) and at the end of 12 weeks (end values)
Control (N = 10) Diabetic (N = 10) Diabetic + ALT (N = 12) Diabetic + AG (N = 11)
Initial BW (g)
End BW (g)
Initial BG (mmol/L)
End BG (mmol/L)
348.6 ± 5.9 358.0 ± 7.0 357.9 ± 5.1 360.0 ± 3.6
414.3 ± 10.7* 302.4 ± 24.2*† 300.4 ± 12.1*† 330.3 ± 35.8*†
5.1 ± 0.1 5.1 ± 0.2 5.0 ± 0.2 5.0 ± 0.2
5.0 ± 0.3 19.9 ± 1.4*† 23.2 ± 1.3*† 20.3 ± 1.9*†
*P < 0.05 vs. initial value in the same group; †P < 0.05 vs. end value in the control group. AG = aminoguanidine.
order to avoid day-to-day variation in fluorescence intensity, several sections from different experimental groups were immunostained and analyzed in the same batch on the same day. The laser intensity and gain functions were set according to the control tissue; thereafter, these settings were applied to all sections from all experimental groups within the same batch.
ICP values (Figure 1). A representative ICP tracing after CNS at the 5 V setting for 1 minute in all groups is given in Figure 2.
NNOS Protein Levels Western blotting of the crude extract of the penises using an anti-nNOS antibody revealed a
Statistical Analysis Results are expressed as mean ± standard error of the mean from a number of independent experiments. The differences between the groups were compared by two-tailed Mann–Whitney U-test or one-way ANOVA followed by Dunnett’s test as indicated. Level of significance less than 0.05 was considered significant. Statistical analyses were performed using a software program for statistics (SPSS 10.0 Version, Chicago, IL, USA). Results
Effect of Diabetes and Drug Treatment on Body Weight and Blood Glucose Body weight and blood glucose levels are summarized in Table 1. During the 12-week period, control rats gained weight, while diabetic rats lost weight. The blood glucose concentrations were higher in the diabetic group than in the control group. The animals treated with ALT-711 and aminoguanidine in the last 6 weeks of the study did not differ from the diabetic group in body weight or serum glucose levels. In Vivo Erectile Function Erectile responses to CNS are given in Figures 1 and 2. The mean ICP values were significantly lower in the diabetic group. Treatment of diabetic animals with ALT-711 in the last 6 weeks of the study resulted in significant restoration of erectile function, leading to ICP levels comparable to those in the control group. Treatment with aminoguanidine did not alter the effect of diabetes. A similar trend was observed in ICP/MAP and total
Figure 1 Treatment with ALT-711, not with aminoguanidine (AG), reversed diabetes-induced reduction in the intracavernous pressure (ICP) rise to nerve stimulation. (a) The ratio of ICP to mean arterial pressure (MAP) is calculated and plotted against different voltages of cavernous nerve stimulation for each group. (b) Total ICP increase measured as the area under the curve is plotted against different voltages of cavernous nerve stimulation for each group. Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group, Mann– Whitney U-test. DM = diabetes mellitus.
J Sex Med 2006;3:242–252
jsm_217.fm Page 246 Monday, February 13, 2006 8:17 PM
246
Usta et al.
band at 160 kDa corresponding to nNOS. The density of the band was lower in the nontreated and aminoguanidine-treated diabetic groups than in the control and ALT-711-treated diabetic groups (Figure 3).
Serum AGEs Glycation end products were measured in the serum of the animals using fluorometry and
Figure 3 Diabetes-induced decrease in the nNOS protein content was reversed by ALT-711 treatment but not with aminoguanidine. (a) A representative Western blot showing the bands corresponding to nNOS. 1, rat brain; 2–3, control group; 4–5, diabetic group; 6–7, diabetic group with ALT711 treatment; 8–9, diabetic group with aminoguanidine treatment. (b) Densitometry of the bands in the Western blots from all of the animals (N = 8 per each group) as expressed as percentage of control. Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group, ANOVA followed by Dunnett’s test.
ELISA. Both techniques revealed an increase in serum AGE concentrations in the diabetic group, which was reversed by treatment with ALT-711, but not with aminoguanidine (Figure 4).
AGE Immunofluorescence in the Penis Accumulation of AGEs in the penis was assessed with immunofluorescence using a monoclonal antibody raised against AGEs. The fluorescence intensity was significantly higher in the diabetic group than in the control group. The highest staining was observed at the nerve bundles with lesser degrees of staining in cavernosal smooth muscle, connective tissue, and endothelium. The increase in AGE immunostaining was reversed partially by ALT-711 treatment, but not with aminoguanidine treatment (Figures 5 and 6). Figure 2 Diabetes induced a significant reduction in intracavernous pressure (ICP) increase to cavernous nerve stimulation that is reversed with ALT-711 treatment. Typical tracings of ICP from an age-matched nondiabetic control rat (Control), a 12-week diabetic rat (Diabetic), a 12-week diabetic rat treated with ALT-711 in the last 6 weeks (Diabetic + ALT-711), and a 12-week diabetic rat treated with aminoguanidine in the last 6 weeks (Diabetic + AG). The cavernous nerve was stimulated for 1 minute at the 5 V setting. Vertical scale corresponds to 25 mm Hg of ICP.
J Sex Med 2006;3:242–252
Discussion
This study documents that STZ-induced diabetes causes significant ED in rats within 12 weeks. The diabetic animals treated in the final 6 weeks with ALT-711 did not have ED; their ICP responses to CNS were similar to nondiabetic control animals. Treatment with aminoguanidine for the final 6 weeks did not reverse ED. These data demon-
jsm_217.fm Page 247 Monday, February 13, 2006 8:17 PM
ALT-711 Reverses Diabetic ED
Figure 4 The increase in serum AGEs by diabetes was reversed with ALT-711, but not with aminoguanidine. Serum AGE concentrations were measured using fluorometry as the ratio of AGE-specific fluorescence to total peptide content (a) or using ELISA (b). Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group, ANOVA followed by Dunnett’s test. AU = arbitrary units.
strate that the AGE cross-link breaker, ALT-711, is successful in reversing diabetes-induced ED in this animal model vs. the AGE formation inhibitor, aminoguanidine. To our knowledge, this is the first comparative study between the two anti-AGE agents in a delayed administration setting. The reason for selecting this animal model is twofold. First, the STZ rat model is well established for studying diabetic ED, and irreversible structural damage to the nerves has been shown to occur by 12 weeks [15]. Hence, we initiated drug treatment at the sixth week of a total of 12 weeks of induced diabetes. Previous studies have already shown that this animal model develops ED within the first 4–6 weeks of induced diabetes [6,12–17]. Additionally, it has previously been reported that insulin-dependent diabetes affects endothelial NO synthase (eNOS) and nNOS isoforms, and leads
247 to ED in diabetic animals [13]. Second, ED in diabetic men usually presents later in life compared with other complications necessitating a therapeutic approach rather than a preventive one. It should be noted that the inability to assess erectile function at the time point when ALT-711 treatment was instituted is a partial limitation of this study. However, previous studies have clearly documented that ED is observed within the first 6 weeks of STZ-induced diabetes [6,12–17]. Our laboratory has demonstrated that treatment with aminoguanidine in the last 4 weeks of an 8-week course of diabetes reversed ED without any effect on penile tissue AGE levels [14]. In this study, however, aminoguanidine failed to reverse ED when administered in the last 6 weeks of 12week diabetes. Although it is uncertain what transpires between 4 and 6 weeks that makes aminoguanidine ineffective, several possibilities exist. (i) Inhibition of AGE formation between 4 and 6 weeks may be sufficient to arrest any further increase in AGE levels, to the extent that this prevents loss in erectile function. However, there were no changes in penile tissue AGE levels with aminoguanidine treatment in our previous study [14], suggesting that the ability of aminoguanidine to reverse ED may not be related to AGEs. Because aminoguanidine improves tissue collagen turnover in diabetic penises, this preservation of normal collagen content may be the reason for normal erectile function in these rats. This hypothesis needs further evaluation. (ii) Another hypothesis is that aminoguanidine protects cavernosal eNOS/nNOS levels only during the first 4–6 weeks. Nevertheless, the failure of aminoguanidine to reverse ED after a longer duration of diabetes is documented in this study, and suggests a difference in action between AGE formation inhibitors and cross-link breakers. The reversal of diabetes-induced ED with ALT711 in this study together with earlier investigations revealing aminoguanidine to be protective [6,18] further highlights the importance of AGEs in the pathogenesis of diabetic complication. AGEs are hypothesized to quench NO, and reduce available NO levels in the penis during sexual stimulation [5,6]. AGEs also interfere with the L-arginine–NO balance, by increasing iNOS and reducing eNOS expression in the penis [5]. This increased oxidative stress in the cell bodies of nNOS-containing neurons leads to apoptosis and irreversible nNOS depletion in the penis [15,17]. Because NO derived from nNOS within the autonomic nerves is the major initiator of erection, we J Sex Med 2006;3:242–252
jsm_217.fm Page 248 Monday, February 13, 2006 8:17 PM
248
Usta et al.
Control
Diabetic
Diabetic + ALT
Diabetic + AG
Figure 5 AGE immunostaining in the penis was increased in the diabetic group. Accumulation of AGEs was measured using immunofluorescence with a monoclonal antibody. Note the distinctive staining in the nerve bundles (white arrows). Scale bar applies to all panels.
assessed the nNOS protein levels in diabetic animals with and without delayed treatment. Previously, we demonstrated that penile nNOS protein is depleted throughout 20 weeks of induced diabe-
Figure 6 ALT-711, not aminoguanidine, reversed diabetesinduced AGE accumulation in the penis. Immunostaining intensity of AGEs in the penile nerve bundles is expressed as mean amplitude per µm2 area. Error bars represent the standard error of the mean. *P < 0.05 significantly different from control group; †P < 0.05 significantly different from DM or DM + AG groups, ANOVA followed by Dunnett’s test.
J Sex Med 2006;3:242–252
tes in two phases. In the first phase (approximately first 10–12 weeks), nNOS-containing (nitrergic) nerve axons in the penis remain intact, while their nNOS content gradually decreases. This may be caused by a deficient antegrade axonal transport of nNOS from the cell bodies in the pelvic ganglia to the axon in the penis. In the second phase, accumulated AGEs synergize with NO, leading to oxidative stress and apoptosis of the nitrergic neurons [15]. In the present study, we again demonstrated a significant reduction in nNOS levels in the penis of 12-week diabetic rats. Interestingly, treatment with ALT-711, but not with aminoguanidine, normalized the nNOS levels, suggesting that AGEs might be interfering with the axonal transport of nNOS. Our results postulate a novel mechanism by which AGEs may interfere with NO production in the autonomic nervous system. Another possibility suggests that eNOS has glycation sites located at base pairs 599–622 and AGEs may affect eNOS activity either transcriptionally or posttranslationally [5]. Furthermore, hyperglycemia
jsm_217.fm Page 249 Monday, February 13, 2006 8:17 PM
249
ALT-711 Reverses Diabetic ED causing O-linked eNOS glycosylation at the Akt target S1177 may lead to decreased NO production [19,20]. This plausible mechanism suggests a possible relationship between AGEs and eNOS. The role of eNOS in AGE-related diabetic ED still needs further clarification. Studies investigating the levels of eNOS in diabetic rat penile tissue and the effect of ALT-711 on its expression are ongoing. We have demonstrated in this study that AGE concentrations in the serum are significantly increased during 12 weeks of diabetes. Parallel to serum AGEs, we have also observed an increase in the immunostaining of AGEs in cross-sections of the penises, especially in the nerve bundles. This suggests that the axons within these bundles are targets for AGEs. Treatment with ALT-711, but not with aminoguanidine, reduced the AGE concentrations in the serum to control levels. Although AGE immunostaining in the penises was significantly lowered with ALT-711 treatment, it still remained higher than the control group. This suggests that although ALT-711 can reduce circulating AGEs, perhaps by facilitating their disposal from the body, its effect on AGEs already formed within the tissues may be determined by rates of tissue turnover and other unknown variables related to diabetes. Phase 2 clinical trials of ALT-711 (alagebrium) were initiated in 1998. Four of these trials have been completed and ALT-711 was well tolerated [8]. A recent Phase 2a clinical trial demonstrated ALT-711 improving total arterial compliance in aged humans with vascular stiffening [21]. Another study revealed significant reductions in left ventricular mass, left ventricular diastolic filling, and a positive effect on three key quality of life measurements in elderly patients with isolated diastolic heart failure [22]. These results hint that ALT-711 may in the future be employed to treat AGE-dependent ED in diabetic and/or aged patients. Conclusions
Delayed administration of the AGE cross-link breaker, ALT-711, but not the AGE formation inhibitor, aminoguanidine, reduced elevated serum and penile AGE levels, and reverses nNOS depletion in the diabetic penis. ALT-711 can restore erectile function in STZ-induced diabetic rats. More detailed studies investigating the AGE mechanisms in relation to erectile physiology will catalyze novel concepts about diabetic ED. This
knowledge will undoubtedly provide the basis for further therapeutic approaches for diabetic men with ED. Acknowledgments
ALT-711 was kindly provided by Alteon Inc., Parsippany, NJ, USA. This study was funded by the Juvenile Diabetes Research Foundation (S.C.), a scholarship from the Sexual Medicine Society of North America, Bayer/GlaxoSmithKline (M.F.U.), the American Medical Association (T.J.B.), and Young Investigator Award from the International Society for Sexual and Impotence Research (T.J.B.). The authors thank Dr. Kemal H. Gülkesen for assistance in statistical analyses. Corresponding Author: Wayne J.G. Hellstrom, MD, FACS, Tulane University Health Sciences Center, Department of Urology, Section of Andrology, 1430 Tulane Avenue SL-42, New Orleans, LA 70112, USA. Tel: (504) 988-7308; Fax: (504) 988-5059; E-mail: [email protected] or [email protected] Conflict of Interest: None declared.
References
1 Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB. Impotence and its medical and psychosocial correlates: Results of the Massachusetts Male Aging Study. J Urol 1994;151:54–61. 2 Lewis RW, Fugl-Meyer KS, Bosch R, Fugl-Meyer AR, Laumann EO, Lizza E, Morales AM. Epidemiology/risk factors of sexual dysfunction. J Sex Med 2004;1:35–9. 3 Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen RA. Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men with impotence. N Engl J Med 1989;320:1025–30. 4 Saenz de Tejada I, Angulo J, Cellek S, GonzalesCadavid N, Heaton J, Pickard R, Simonsen U. Pathophysiology of erectile dysfunction. J Sex Med 2005;2:26–39. 5 Seftel AD, Vaziri ND, Ni Z, Razmjouei K, Fogarty J, Hampel N, Polak J, Wang RZ, Ferguson K, Block C, Hass C. Advanced glycation end products in human penis: Elevation in diabetic tissue, site of deposition, and possible effect through iNOS or eNOS. Urology 1997;50:1016–26. 6 Usta MF, Bivalacqua TJ, Yang DY, Ramanihatharan A, Sell D, Viswanathan A, Monnier VM, Hellsttrom WJ. The protective effect of aminoguanidine on erectile function in streptozotocin diabetic rats. J Urol 2003;170:1437–42. 7 Stitt AW, Jenkins AJ, Cooper ME. Advanced glycation end products and diabetic complications. Expert Opin Investig Drugs 2002;11:1205–23. J Sex Med 2006;3:242–252
jsm_217.fm Page 250 Monday, February 13, 2006 8:17 PM
250 8 Vasan S, Foiles P, Founds H. Therapeutic potential of breakers of advanced glycation end product– protein crosslinks. Arch Biochem Biophys 2003; 419:89–96. 9 Wolffenbuttel BH, Boulanger CM, Crijns FR, Huijberts MS, Poitevin P, Swennen GN, Vasan S, Egan JJ, Ulrich P, Cerami A, Levy BI. Breakers of advanced glycation end products restore large artery properties in experimental diabetes. Proc Natl Acad Sci USA 1998;95:4630–4. 10 Candido R, Forbes JM, Thomas MC, Thallas V, Dean RG, Burns WC, Tikellis C, Ritchie RH, Twigg SM, Cooper ME, Burrell LM. A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 2003;92:785–92. 11 Forbes JM, Thallas V, Thomas MC, Founds HW, Burns WC, Jerums G, Cooper ME. The breakdown of preexisting advanced glycation end products is associated with reduced renal fibrosis in experimental diabetes. FASEB J 2003;17:1762–4. 12 Bivalacqua TJ, Usta MF, Kendirci M, Pradhan L, Alvarez X, Champion HC, Kadowitz PJ, Hellstrom WJ. Superoxide anion production in the rat penis impairs erectile function in diabetes: Influence of in vivo extracellular superoxide dismutase gene therapy. J Sex Med 2005;2:187–98. 13 Bivalacqua TJ, Champion HC, Usta MF, Cellek S, Chitaley K, Webb RC, Lewis RL, Mills TM, Hellstrom WJ, Kadowitz PJ. RhoA/Rho kinase suppresses endothelial nitric oxide synthase in the penis: A mechanism for diabetes-associated erectile dysfunction. Proc Natl Acad Sci USA 2004; 101:9121–6. 14 Usta MF, Bivalacqua TJ, Koksal IT, Toptas B, Surmen S, Hellstrom WJ. The protective effect of aminoguanidine on erectile function in diabetic rats is not related to the timing of treatment. BJU Int 2004;94:429–32. 15 Cellek S, Foxwell NA, Moncada S. Two phases of nitrergic neuropathy in streptozotocin-induced diabetic rats. Diabetes 2003;52:2353–62. 16 Cellek S, Rodrigo J, Lobos E, Fernandez P, Serrano J, Moncada S. Selective nitrergic neurodegeneration in diabetes mellitus—A nitric oxide-dependent phenomenon. Br J Pharmacol 1999;128:1804–12. 17 Cellek S, Qu W, Schmidt AM, Moncada S. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes. Diabetologia 2004;47:331–9. 18 Cartledge JJ, Eardley I, Morrison JF. Advanced glycation end-products are responsible for the impairment of corpus cavernosal smooth muscle relaxation seen in diabetes. BJU Int 2001;87:402–7. 19 Musicki B, Kramer MF, Becker RE, Burnett AL. Age-related changes in phosphorylation of endothelial nitric oxide synthase in the rat penis. J Sex Med 2005;2:347–57. J Sex Med 2006;3:242–252
Usta et al. 20 Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest 2001;108:1341–8. 21 Kass DA, Shapiro EP, Kawaguchi M, Capriotti AR, Scuteri A, deGroof RC, Lakatta EG. Improved arterial compliance by a novel advanced glycation end-product crosslink breaker. Circulation 2001; 104:1464–70. 22 Little WC, Zile MR, Kitzman DW, Hundley WG, O’Brien TX, Degroof RC. The effect of alagebrium chloride (ALT-711), a novel glucose cross-link breaker, in the treatment of elderly patients with diastolic heart failure. J Card Fail 2005;11:191–5.
Commentary
Erectile dysfunction (ED) is highly prevalent among diabetic men. In addition, diabetic patients with ED are particularly resistant to standard treatments for ED. Thus, the understanding of pathophysiological mechanisms leading to diabetic ED, as well as the localization of specific therapeutic targets, are priorities for research in the field of sexual medicine. Relevant information is available from other fields of research regarding diabetes-associated complications, in particular studies on the pathophysiology of diabetesinduced vascular dysfunctions. In this regard, the formation of advanced glycosylation end products (AGEs) has been suggested as one of the mechanisms contributing to the vascular alterations caused by diabetes. Nonenzymatic glycosylation of proteins (glycation), also known as Maillard reaction, depends on the glucose concentration and consists of the reaction of the carboxyl group of reducing sugars with the free amino groups of proteins. After the formation of a Schiff base, the adduct of sugar and protein is converted to an Amadori product that undergoes sequential rearrangements that lead to the formation of AGEs in the course of weeks–months. It is well known that AGEs increased in diabetes and interfere with endothelium-dependent relaxation [1]. AGEs can modify intracellular and plasmatic proteins or interact with specific receptors, but also modify extracellular matrix proteins by increasing covalent cross-linking that inhibits matrix degradation. All these actions could contribute to the development of diabetic complications. The present study by Hellstrom and collaborators demonstrates that AGEs are involved in the impairment of erectile responses caused by diabetes and confirms the important role of AGEs in dia-
jsm_217.fm Page 251 Monday, February 13, 2006 8:17 PM
ALT-711 Reverses Diabetic ED betic complications. Previous studies have suggested the contribution of AGEs to the development of ED and cavernosal dysfunction in diabetes [2–4]. The most important finding from the present study is that AGE accumulation may be reversed with the recovery of erectile function. The same effects on erectile function in diabetic rats were previously obtained following preventive treatment with aminoguanidine [4], but the present results show that this therapeutic approach (aminoguanidine treatment) does not reverse the established AGE accumulation. In this regard, the use of ALT-711 has a significant advantage, as this compound is able to reduce the level of pre-existing AGEs. Theoretically, aminoguanidine impedes de novo formation of AGEs [1], while ALT-711, as other AGE cross-link breakers, cleaves the covalent binding of AGE-modified proteins with other amino groups, favoring the elimination of AGEs. In addition, aminoguanidine could have other effects unrelated to AGE inhibition, as it is a known inhibitor of the inducible isoform of nitric oxide synthase, making this compound a poor pharmacological tool. In contrast, the use of AGE crosslink breakers could have a better therapeutic profile and has been shown to produce beneficial cardiovascular effects in elderly patients [5]. Usta and collaborators do not demonstrate an increase of AGEs before ALT-711 was administered, but an increase of serum AGEs after 6 weeks of diabetes in this model [6]. Thus, ALT711 likely facilitated the elimination of these preexisting AGEs, having more impact in serum AGEs, where levels are normalized, while in the cavernosal tissue, a higher amount of AGEs remained, suggesting that these correspond to structural proteins with slow turnover. ALT-711 could interact with other signaling pathways relevant to diabetic complications. ALT711 inhibits protein kinase C (PKC) activation in kidneys from diabetic rats, an effect not completely related to its capacity to reduce AGEs [7]. As PKC overactivity has been proposed as a mechanism contributing to the development of diabetic complications, the putative effect of ALT-711 on PKC activation would enhance the beneficial impact of this agent on diabetic complications and hence, on diabetic ED. Probably, several altered pathways are responsible for complications associated with diabetes, including increase of aldose reductase pathway, PKC overactivity, overflow through hexosamine pathway, and glycation and AGE formation. Individual actions on each one of these pathways have been shown to be effective in
251 animal models of diabetic complications, making difficult to differentiate the relative roles of the respective pathways. However, in addition to the likely interaction among the different pathways, it has been proposed that these pathways share a common link as all of them could be influenced by hyperglycemia-induced excess of superoxide production by the respiratory chain in the mitochondria [8], providing a possible unifying target for preventing alterations in the previously mentioned pathways. This study also shows that elimination of AGEs contribute to the recovery of nitrergic nerve fibers and neuronal nitric oxide synthase expression. Further studies evaluating the effects of ALT-711 on endothelial function, the other main player in the regulation of penile smooth muscle contractility and erectile function, should be of great interest. However, some considerations should be noted. When we talk about AGEs we are referring to a very heterogeneous group of molecules. This is because the macromolecule modified by the sugar, and the sugar itself, can be diverse. In addition, the microenvironment where the glycation takes place (oxidants, antioxidants) could influence the chemical structure of the adduct and the velocity of the process. The heterogeneity in the process of AGE formation could be more marked in the case of human diabetes. Finally, the glycation process is not interrupted by the AGE breaker and adducts at different time points of the process can be found simultaneously. In this regard, there is little information on the effects of these agents on early and intermediate glycation products, which have been found to contribute to diabetic vascular dysfunction [6] and cause endothelial dysfunction in corpus cavernosum from normal rats [9]. In summary, this interesting study suggests that glycation plays an important role in the development of diabetes-associated ED and, although human diabetes is more complex and varied, provides a rationale for considering AGE elimination as a therapeutic approach for treating diabetic ED. This will be dependent on further understanding of the time-course, pharmacokinetics, and longterm side-effects of cross-link breakers. In addition, this study confirms that intervention on the specific mechanisms involved in diabetes-induced vascular and/or neurological alterations has a beneficial impact on erectile function in diabetes. Javier Angulo, MD, and Iñigo Sáenz de Tejada, MD Institute of Sexual Medicine, Madrid, Spain J Sex Med 2006;3:242–252
jsm_217.fm Page 252 Monday, February 13, 2006 8:17 PM
252
Usta et al.
References
1 Bucala R, Tracey KJ, Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest 1991;87:432–8. 2 Seftel AD, Vaziri ND, Ni Z, Razmjouci K, Fogarty J, Hampel N, Polak J, Wang RZ, Ferguson K, Block C, Haas C. Advanced glycation end products in human penis: Elevation in diabetic tissue, site of deposition and possible effect through iNOS or eNOS. Urology 1997;50:1016–26. 3 Cartledge JJ, Eardley I, Morrison JF. Advanced glycation end-products are responsible for the impairment of corpus cavernosum smooth muscle relaxation seen in diabetes. BJU Int 2001;87:402–7. 4 Usta MF, Bivalacqua TJ, Yang DY, Ramanitharan A, Sell DR, Viswanathan A, Monnier VM, Hellstrom WJ. The protective effect of aminoguanidine on erectile function in streptozotocin diabetic rats. J Urol 2003;170:1437–42. 5 Little WC, Zile MR, Kitzman DW, Hundley WG, O’Brien TX, Degroof RC. The effect of alagebrium chloride (ALT-711), a novel glucose cross-
J Sex Med 2006;3:242–252
6
7
8 9
link breaker, in the treatment of elderly patients with diastolic heart failure. J Card Fail 2005;11: 196–9. Rodríguez-Mañas L, Angulo J, Vallejo S, Peiró C, Sánchez Ferrer A, Cercas E, López-Dóriga P, Sánchez-Ferrer CF. Early and intermediate Amadori glycosylation adducts, oxidative stress, and endothelial dysfunction in the streptozotocin-induced diabetic rats vasculature. Diabetologia 2003;46:556– 66. Thallas-Bonke V, Lindschau C, Rizkalla B, Bach LA, Boner G, Meier M, Haller H, Cooper ME, Forbes JM. Attenuation of extracellular matrix accumulation in diabetic nephropathy by the advanced glycation end product cross-link breaker ALT-711 via protein kinase C-α-dependent pathway. Diabetes 2004;53: 2921–30. Brownlee M. The pathobiology of diabetic complications. A unifying mechanism. Diabetes 2005;54: 1615–25. Cartledge JJ, Eardley I, Morrison JFB. Impairment of corpus cavernosal smooth muscle relaxation by glycosylated human hemoglobin. BJU Int 2000;85: 735–41.