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Treatment With Metformin Improves Erectile Dysfunction in a Murine Model of Obesity Associated With Insulin Resistance
Basic and Translational Science Treatment With Metformin Improves Erectile Dysfunction in a Murine Model of Obesity Associated With Insulin Resistance bio H. Silva, Eduardo C. Alexandre, Fabiano B. Calmasini, Marina C. Calixto, and Fa Edson Antunes OBJECTIVE MATERIAL AND METHODS
RESULTS
CONCLUSION
To evaluate the effects of treatment with metformin on a murine model of obesity-associated erectile dysfunction. C57BL/6 male mice were fed for 10 weeks with standard chow or high-fat diet. Lean and obese mice were treated with the insulin sensitizer metformin (300 mg/kg/day, 2 weeks). Intracavernosal pressure (ICP) and in vitro corpus cavernosum (CC) relaxations to both acetylcholine and electrical field stimulation, as well as phenylephrine-induced contractions, were obtained. Levels of cyclic guanosine monophosphate in CC were detected by enzyme immunoassay. High-fat-fed mice exhibited higher body weight and insulin resistance. Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P <.05). Two-week therapy with metformin reversed the decreased ICP in obese group. The maximal response to acetylcholine in CC was 35% lower (P <.05) in the obese compared to the lean group, which were restored by metformin treatment. Likewise, the impaired electrical field stimulationeinduced CC relaxations in obese mice were also partly restored by metformin. Contractile responses to phenylephrine were significantly greater (P <.05) in obese compared to lean mice, which were fully restored by metformin. Basal and stimulated cyclic guanosine monophosphate productions in the erectile tissues were significantly lower (P <.05) in the obese group, an effect fully restored by metformin. Treatment with metformin restored the erectile function in obese mice, through improvement of in vitro endothelial and nitrergic cavernosal relaxations. Therefore, use of metformin may be a good pharmacologic approach to treat insulin resistanceeassociated erectile dysfunction. UROLOGY -: 1.e1–1.e6, 2015. 2015 Elsevier Inc.
E
rectile dysfunction (ED) is characterized by a persistent inability to achieve and/or maintain an erection sufficient for satisfactory sexual performance and has been associated with an abnormal function in the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway and hence, lower NO bioavailability in the erectile tissue.1 Epidemiologic studies show that obesity is an important risk factor for both ED and insulin resistance (IR).2-5 Evidence supports a strong causal link between IR and ED.6-10 In fact, ED has been considered an early clinical manifestation of risk factors for cardiovascular events including acute myocardial infarction.11 In addition, ED may Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, Sao Paulo, Brazil Address correspondence to: Fabio Henrique da Silva, Ph.D., Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, Sao Paulo 13084-971, Brazil. E-mail: [email protected] Submitted: January 26, 2015, accepted (with revisions): April 28, 2015
ª 2015 Elsevier Inc. All Rights Reserved
be the first clinical sign of IR in young men.8 A recent randomized controlled trial in patients with ED with poor response to sildenafil reported that treatment with metformin improves erectile function.7 IR is a state of dysregulation of glucose-insulin homeostasis, in which the ability of insulin to stimulate glucose uptake in peripheral tissues is reduced.2 Insulin is well known for inducing vascular relaxation through a mechanism that involves endothelium-dependent NO production.12 Vasodilator action of insulin is mediated by the phosphatidylinositol 3 kinase (PI3K)-Akt pathway that phosphorylates endothelial NO synthase at Ser1177.12 IR has been strongly associated with decreased NO bioavailability and endothelial dysfunction.13 Previous studies showed that high-fat-fed obese mice display ED, as demonstrated by reductions of intracavernous pressure (ICP) by cavernosal nerve electrostimulation and impaired endothelial and nitrergic cavernosal relaxations, as well as by increase of contractile response elicited by a1-adrenergic receptor activation.14,15 http://dx.doi.org/10.1016/j.urology.2015.04.035 0090-4295/15
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Table 1. Body weight, epididymal fat mass, and insulin sensitivity of lean and obese mice treated with vehicle (water) or metformin (300 mg/kg/day, 14 days) by gavage
Body weight (g) Epididymal fat mass (g) Kitt (min1)
Lean þ V
Obese þ V
Lean þ Met
Obese þ Met
31 0.6 0.32 0.06 4.34 0.30
43 2* 1.89 0.13* 2.06 0.37*
30 0.7 0.39 0.02 4.38 0.29
42 1* 1.86 0.10* 4.24 0.45y
Kitt, constant rate for blood glucose disappearance; Met, metformin; V, vehicle. Data are mean standard error of mean of 5 mice. * P <.05 in comparison with lean þ vehicle group. y P <.05 in comparison with obese þ vehicle group.
The insulin sensitizer metformin is a first-line pharmacologic treatment for patients with type 2 diabetes mellitus, which acts by decreasing hepatic glucose production and increasing insulin sensitivity in skeletal muscle and adipose tissue.16 Recently, metformin was shown to increase the adenosine signaling in corpus cavernosum from the high-fat-fed rabbits via increases in NO production.17 However, no detailed functional study has explored the effect of metformin treatment on a murine model of obesity-associated ED. Our hypothesis is that metformin reverses ED in the murine model of obesity associated with IR through improvement of NO bioavailability in the erectile tissue. Therefore, in the present study, in a continuing effort to investigate the effect of metformin treatment on ED, we have treated insulinresistant obese mice with the insulin sensitizer metformin and evaluated in vivo and in vitro functional responses of the mice corpus cavernosum.
MATERIAL AND METHODS Animals All animal procedures and experimental protocols are in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College for Animal Experimentation and followed the Guide for the Care and Use of Laboratory Animals. Four-week-old male C57BL/6J mice were provided by Central Animal House Services of the University of Campinas.
Diet-induced Obesity and Treatment Mice were housed 3 per cage on a 12-hour light-dark cycle and fed for 10 weeks with either a standard chow diet (carbohydrate, 70%; protein, 20%; fat, 10%) or a high-fat diet that induces obesity (carbohydrate, 29%; protein, 16%; fat, 55%), as previously described. Lean and obese mice were treated with vehicle (water) or the insulin sensitizer metformin (300 mg/kg/day) by gavage from the eighth to the 10th week. In our protocols, we used a total of 70 mice divided into 4 experimental groups, namely lean plus vehicle (N ¼ 16), lean plus metformin (n ¼ 19), obese plus vehicle (n ¼ 16), and obese plus metformin (n ¼ 19). Tail-cuff pressure was measured by using a modified tail-cuff method in conscious animals in a quiet room.
In Vivo Insulin Sensitivity After 6 hours of fasting, systemic insulin sensitivity was analyzed by the insulin tolerance test. Tail blood samples were collected before (0 min) and at 5, 10, 15, 25, 30, and 60 minutes after an intraperitoneal injection of 1.00 U/kg of regular insulin (Novolin R; NovoNordisk, Bagsværd, Denmark). Glucose concentrations were measured using a glucometer (ACCUCHEK Performa; Roche Diagnostics, Indianapolis, IN), and the values were used to 1.e2
calculate the constant rate for blood glucose disappearance (Kitt), based on the linear regression of the Napierian logarithm of glucose concentrations obtained from 0 to 30 minutes of the test. Kitt was calculated using the formula 0.693/(t1/2)x 1 100.
ICP Measurement Mice were anesthetized with an intraperitoneal injection of urethane (1.8 g/kg). To perform ICP measurement, right penile crus was exposed and cannulated using a 26-gauge needle. The cannula was filled with sterile heparinized saline solution and attached to a pressure detector for continuous ICP monitoring. The bladder and prostate were exposed through a midline abdominal incision. The right major pelvic ganglion and cavernous nerve were identified posterolateral to the prostate on one side. The cavernous nerve was electrically stimulated with 2 platinum electrodes connected to a Grass S48 stimulator (Astro-Med Industrial Park, West Warwick, RI). The cavernous nerve stimulation was conducted at 6 V, with 1 ms pulse width and trains of stimuli lasting 60 seconds at varying frequencies, with intervals of 3 minutes between the stimulation trains. Changes in ICP were recorded using a PowerLab 400 data acquisition system (LabChart software, version 7.0; ADInstruments, Colorado Springs, CO). Mice undergoing ICP measurements were not used for other experimental protocols.15
Functional Studies in Cavernosal Strips and Concentration-Response Curves Mice were anesthetized with isoflurane and exsanguinated. Strips of mice CC were mounted in a 10-mL organ system containing Krebs solution at 37 C continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4) and vertically suspended between 2 metal hooks. One hook was connected to a force transducer and the other acted as a fixed attachment point. Tissues were allowed to equilibrate for 60 minutes under a resting tension of 2.5 mN. Isometric force was recorded using a PowerLab 400 data acquisition system (LabChart, version 7.0; ADInstruments, MA). Cumulative concentration-response curve was constructed for the muscarinic agonist acetylcholine (ACh; 109 to 3 105 M) in cavernosal strips precontracted with the a1-adrenergic receptor agonist phenylephrine (PE; 105 M). Cumulative concentrationresponse curves of PE (108 to 3 104 M) were also obtained in the cavernosal tissue. Nonlinear regression analysis to determine the pEC50 was carried out using GraphPad Prism (GraphPad Software, San Diego, CA) with the constraint that V ¼ 0. All concentration-response data were evaluated for a fit to a logistics function in the form: E ¼ Emax/[1 þ (10c/10x)n] þ V, where E is the maximum response produced by agonists; c is the logarithm of the EC50, the concentration of drug that produces a half-maximal response; x is the logarithm of the concentration of the drug; the exponential term, n, is a curve-fitting parameter that defines the slope of the concentration-response line, and F is the response observed in the absence of added drug. UROLOGY
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Drugs and Chemicals ACh, atropine, guanethidine, metformin, and PE were purchased from Sigma-Aldrich (St Louis, MO). All reagents used were of analytical grade. Stock solutions were prepared in deionized water and stored in aliquots at 20 C. Dilutions were prepared immediately before use.
Statistical Analysis The program InStat (GraphPad Software Inc.) was used for statistical analysis. Data are expressed as the mean standard error of mean of N experiments. Statistical comparisons were made using 1-way analysis of variance, and the Tukey method was chosen as the post test. A value of P <.05 was considered statistically significant.
RESULTS
Figure 1. (A) Intracavernous pressure (ICP) obtained by stimulation of cavernous nerve at 4-12 Hz in anesthezised mice and (B) in vitro corpus cavernosum relaxations to electrical field stimulation (2-32 Hz) from lean and obese mice, treated or not with metformin (300 mg/kg/day, 2 weeks). Data represent the mean standard error of mean for lean þ vehicle (n ¼ 5), obese þ vehicle (n ¼ 5); lean þ metformin (n ¼ 6) and obese þ metformin (n ¼ 6) groups. *P <.05 in comparison with lean þ vehicle group; # P <.05 in comparison with obese þ vehicle group.
Electrical Field Stimulation Electrical field stimulation (EFS) was applied in strips placed between two platinum ring electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park). EFS was conducted at 50 V, with 1 ms pulse width and trains of stimuli lasting 10 seconds at varying frequencies. Frequency-response relationships were investigated at supra maximum voltage in all preparations stimulated electrically. To study the nitrergic relaxations, cavernosal tissues were pretreated with both guanethidine (3 105 M; to deplete the catecholamine stores of adrenergic fibers) and atropine (106 M; to produce muscarinic antagonism) before precontraction with PE. When a stable contraction level was attained, a series of EFS-induced relaxations were constructed (4-32 Hz).
Morphometric Characteristics and Insulin Tolerance Test The high-fat-fed mice displayed a significant increase (P <.05) in body weight and epididymal fat mass compared to lean mice. Treatment with metformin (300 mg/kg/day, 2 weeks) did not significantly affect these parameters (Table 1). Obese mice also displayed IR as demonstrated by the lower Kitt values compared to the lean group. Metformin treatment fully restored the insulin sensitivity in the obese group, without changing the insulin sensitivity in the lean group (Table 1). Tail-cuff pressure was not modified in any experimental group (82 3, 84 2, 82 3, and 83 4 mm Hg for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin groups, respectively). The average weights of dry cavernosal strips were 10 0.2, 10.1 0.3, 10.1 0.4, and 10.2 0.4 mg for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin mice, respectively. Erectile Function The cavernous nervous stimulation (4-12 Hz) caused frequency-dependent increases of ICP in all groups (Fig. 1A). However, ICPs in obese mice were significantly lower compared to those in lean mice at all frequencies studied (P <.05; Fig. 1A). Treatment with metformin fully restored the ICP in obese mice at all frequencies studied, without changing the ICP in lean group (P <.05; Fig. 1A).
Determination of cGMP Levels Mice were anesthetized with isoflurane and exsanguinated. Cavernosal segments were equilibrated for 30 minutes in Krebs solution at 37 C continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4). Tissues were stimulated for 1.5 minutes with ACh (105 M). Next, tissues were immediately frozen in liquid nitrogen. Frozen tissues were pulverized, homogenized in trichloroacetic acid (5% wt/vol), and centrifuged for 10 minutes at 4 C at 1500 g and the supernatant was collected. The pellet was dried and weighed. Trichloroacetic acid was extracted from the supernatant with 3 washes of water-saturated ether. Preparation of tracer, samples, standards, and incubation with antibody were performed as described in commercially available kits (Cayman Chemical Cyclic GMP EIA kit, Ann Arbor, MI). The assays were performed in duplicates, and the pellet weight was used to normalize the data that were expressed as pmol/mg tissue. UROLOGY
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EFS-induced Cavernosal Relaxations The addition of PE (105 M) to the cavernosal tissues pretreated with guanethidine (3 105 M) and atropine (106 M) caused a submaximal contraction of cavernosal segments that did not significantly differ between groups (0.33 0.04, 0.37 0.05, 0.31 0.04, and 0.31 0.04 mN for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin groups, respectively; n ¼ 10-12). EFS of PE-contracted tissues caused frequency-dependent relaxations in both lean and obese groups. A marked decrease in the relaxations of cavernosal tissues from obese in comparison with lean mice was observed at all frequencies studied (P <.05; Fig. 1B). Treatment with metformin partly restored the impaired EFS-induced relaxant response in the obese 1.e3
Figure 2. (A) Concentration-response curves to acetylcholine (ACh) in corpus cavernosum (CC) strips from lean and obese mice, treated or not with metformin (300 mg/kg/day, 2 weeks). Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (105 M) in each tissue, which was taken as 100%. (B) Emax values for ACh in all groups. Data represent the mean standard error of mean (SEM) for lean þ vehicle (n ¼ 5), obese þ vehicle (n ¼ 5); lean þ metformin (n ¼ 6), and obese þ metformin (n ¼ 6) groups. (C) Cyclic guanosine monophosphate GMP (cGMP) contents of mice CC from lean and obese mice, treated or not with metformin. CC strips were incubated with ACh (105 M). cGMP levels represent the mean SEM for 4 mice in each group. *P <.05 in comparison with lean þ vehicle group; #P <.05 in comparison with obese þ vehicle group.
group by about 83%, 82%, and 81% at frequencies 8, 16, and 32 Hz, respectively (Fig. 1B). In lean mice, no significant changes after metformin treatment were observed in EFSinduced CC relaxations (Fig. 1B). ACh-induced Cavernosal Relaxations and cGMP Levels in Corpus Cavernosum The cumulative addition of ACh (109-105 M) to PEcontracted tissues produced concentration-dependent relaxations in all groups (Fig. 2A). However, the potency (pEC50) for ACh was 3.7-fold lower (P <.001) in cavernosal 1.e4
Figure 3. (A) Concentration-response curves to phenylephrine in corpus cavernosum strips from lean and obese mice, treated or not with metformin (300 mg/kg/day, 2 weeks). (B) Emax values for all groups. Data are shown in mN and represent the mean SEM for lean þ vehicle (n ¼ 7), obese þ vehicle (n ¼ 7), lean þ metformin (n ¼ 9), and obese þ metformin (n ¼ 9) groups. *P <.05 in comparison with lean þ vehicle group; #P <.05 in comparison with obese þ vehicle group.
tissue of obese mice compared to lean mice (6.28 0.07 and 6.85 0.05, respectively; Fig. 2A). The maximal response (Emax) elicited by ACh was also significantly lower (P <.05) in cavernosal strips from obese compared to lean mice (50 3% and 77 3%, respectively; Fig. 2B). Treatment with metformin partly reversed the lower Emax and pEC50 parameters for ACh in cavernosal tissue of obese mice (66 2% and 6.66 0.05, respectively), with no significant changes in the lean mice (70 4% and 6.77 0.04%, respectively; Fig. 2A,B). The basal cGMP content in the erectile tissue was 40% lower (P <.05) in obese group compared to the lean group (Fig. 2C). Treatment with metformin fully restored (P <.05) the reduced basal cGMP levels in the obese group, with no changes in the lean group (Fig. 2C). In lean UROLOGY
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group, incubation of CC with ACh (105 M) significantly elevated (P <.05) the cGMP above baseline. In the obese group, no elevations in the cGMP levels by ACh were observed compared to the respective baseline. Treatment with metformin fully restored the ACh-induced increases in cGMP levels in CC from obese mice, with no significant changes in the lean group (P <.05; Fig. 2C). PE-induced Cavernosal Contractions PE (109-104 M) induced concentration-dependent contractions in cavernosal preparations in all groups (Fig. 3A). The Emax was significantly greater in obese compared to lean mice (P <.05; Fig. 3B). Metformin treatment prevented the elevation of PE-induced contractile responses in CC of obese mice. No significant changes after metformin treatment were observed in lean mice (Fig. 3A). No significant differences of pEC50 for PE were found in any experimental group (5.23 0.04, 5.42 0.03, 5.39 0.03, and 5.40 0.04 for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin groups, respectively).
COMMENT In the present study, using a murine model of IR-associated obesity, we show that metformin treatment restores the reduced ICP and the impaired in vitro endothelial and nitrergic cavernosal relaxations. Metformin acts by increasing the insulin sensitivity.16 At the molecular level, metformin increases the tyrosine phosphorylation of insulin receptor, phosphatidylinositol 3 kinase activity, phosphorylation of Akt and adenosine monophosphate-activated protein kinase activity.18-20 Previous studies have shown that prolonged treatment with metformin improves ACh- and flow-mediated dilatation of the brachial artery in humans with IR, as well as in vitro vasodilatation induced by ACh in mesenteric and aorta from insulin-resistant rats.21-24 ED in obese Zucker rats was attributed to impaired relaxant responses to insulin in penile arteries.25 We have, therefore, treated obese and lean mice with metformin to investigate the erectile function. In our study, metformin treatment fully reversed the IR in obese mice, which is consistent with previous studies.24,26 Metformin treatment normalized the reduced ICP, as well as the impaired in vitro endothelium-dependent and nitrergic relaxations in obese mice. Decreased basal levels of cGMP were found in obese mice CC that were also normalized by metformin treatment. These findings strongly suggest that IR plays an important role in the development of ED by the impairment of NO bioavailability. In rabbits fed with a high-fat diet, metformin ameliorated the in vitro relaxant responses to ACh in the erectile tissue, along with hyperglycemia and glucose intolerance.17 In the flaccid state, CC and penile vessels are contracted mainly via stimulation of a1-adrenoceptors by norepinephrine derived from the sympathetic nerves.1 Previous studies reported that ED in hypertensive and aging animals has been associated with increased a1-adrenoceptoremediated cavernosal vasoconstriction.27,28 In our study, the maximal UROLOGY
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contractile response to a1-adrenoceptor agonist PE was greater in the obese group, which was also fully restored by metformin treatment. It is likely that enhanced levels of cGMP by metformin in obese mice counteract the contractile responses downstream the a1-adrenoceptor, making penile tumescence more difficult to occur. Recently, in a rat model of hypertension with no accompanying IR, metformin reversed the decreased ICP and increased the relaxing responses to both ACh and EFS and reduced contractile responses to PE in the erectile tissue.29 However, in our study, no alteration in the tail-cuff pressure was observed between the groups, excluding arterial hypertension as a cause for the development of ED.
CONCLUSION Our study shows that treatment of mice with metformin improves the ED in high-fat-fed obese mice. IR associated with obesity seems to contribute to ED development. Therefore, the use of metformin treatment may be a good pharmacologic approach to treat IR-associated ED. Acknowledgments. Fabio H. Silva thanks São Paulo Research Foundation (FAPESP) for the financial support. References 1. Andersson KE. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol Rev. 2011; 63:811-859. 2. Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 2006;444:840-846. 3. Bal K, Oder M, Sahin AS, et al. Prevalence of metabolic syndrome and its association with erectile dysfunction among urologic patients: metabolic backgrounds of erectile dysfunction. Urology. 2007; 69:356-360. 4. Wang F, Dai S, Wang M, Morrison H. Erectile dysfunction and fruit/vegetable consumption among diabetic Canadian men. Urology. 2013;82:1330-1335. 5. Corona G, Rastrelli G, Filippi S, et al. Erectile dysfunction and central obesity: an Italian perspective. Asian J Androl. 2014;16:581-591. 6. Bansal TC, Guay AT, Jacobson J, et al. Incidence of metabolic syndrome and insulin resistance in a population with organic erectile dysfunction. J Sex Med. 2005;2:96-103. 7. Rey-Valzacchi GJ, Costanzo PR, Finger LA, et al. Addition of metformin to sildenafil treatment for erectile dysfunction in eugonadal nondiabetic men with insulin resistance. A prospective, randomized, double-blind pilot study. J Androl. 2012;33:608-614. 8. Yao F, Liu L, Zhang Y, et al. Erectile dysfunction may be the first clinical sign of insulin resistance and endothelial dysfunction in young men. Clin Res Cardiol. 2013;102:645-651. 9. Chen S, Wu R, Huang Y, et al. Insulin resistance is an independent determinate of ED in young adult men. PLoS One. 2013;8:e83951. 10. Russo GI, Cimino S, Fragala E, et al. Insulin resistance is an independent predictor of severe lower urinary tract symptoms and of erectile dysfunction: results from a cross-sectional study. J Sex Med. 2014;11:2074-2082. 11. Thompson IM, Tangen CM, Goodman PJ, et al. Erectile dysfunction and subsequent cardiovascular disease. JAMA. 2005;294:2996-3002. 12. Kim JA, Montagnani M, Koh KK, et al. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation. 2006;113:1888-1904. 13. Zecchin HG, Priviero FB, Souza CT, et al. Defective insulin and acetylcholine induction of endothelial cell-nitric oxide synthase through insulin receptor substrate/Akt signaling pathway in aorta of obese rats. Diabetes. 2007;56:1014-1024.
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14. Toque HA, da Silva FH, Calixto MC, et al. High-fat diet associated with obesity induces impairment of mouse corpus cavernosum responses. BJU Int. 2011;107:1628-1634. 15. Silva FH, Leiria LO, Alexandre EC, et al. Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice. J Sex Med. 2014;11:2661-2670. 16. Pawlyk AC, Giacomini KM, McKeon C, et al. Metformin pharmacogenomics: current status and future directions. Diabetes. 2014; 63:2590-2599. 17. Vignozzi L, Filippi S, Comeglio P, et al. Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa. J Sex Med. 2014;11:1694-1708. 18. Kumar N, Dey CS. Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells. Br J Pharmacol. 2002;137:329-336. 19. Bhamra GS, Hausenloy DJ, Davidson SM, et al. Metformin protects the ischemic heart by the Akt-mediated inhibition of mitochondrial permeability transition pore opening. Basic Res Cardiol. 2008;103: 274-284. 20. Rice S, Pellatt LJ, Bryan SJ, et al. Action of metformin on the insulin-signaling pathway and on glucose transport in human granulosa cells. J Clin Endocrinol Metab. 2011;96:427-435. 21. Katakam PV, Ujhelyi MR, Hoenig M, et al. Metformin improves vascular functioning insulin-resistant rats. Hypertension. 2000;35: 108-112.
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22. Mather KJ, Berma S, Anderson TJ. Improved endothelial function with metformin in type II diabetes mellitus. J Am Coll Cardiol. 2001; 37:1344-1350. 23. Vitale C, Mercuro G, Cornoldi A, et al. Metformin improves endothelial function in patients with metabolic syndrome. J Intern Med. 2005;258:250-256. 24. Sena CM, Matafome P, Louro T, et al. Metformin restores endothelial function in aorta of diabetic rats. Br J Pharmacol. 2011;163: 424-437. 25. Contreras C, Sanchez A, Martınez P, et al. Insulin resistance in penile arteries from a rat model of metabolic syndrome. Br J Pharmacol. 2010;161:350-364. 26. Leiria LO, Sollon C, Calixto MC, et al. Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice. PLoS One. 2012;7:e48507. 27. Carneiro FS, Giachini FR, Lima VV, et al. DOCA-salt treatment enhances responses to endothelin-1 in murine corpus cavernosum. Can J Physiol Pharmacol. 2008;86:320-328. 28. Silva FH, Lanaro C, Leiria LO, et al. Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum. Am J Physiol Heart Circ Physiol. 2014;307:H1393-H1400. 29. Labazi H, Wynne BM, Tostes R, Webb RC. Metformin treatment improves erectile function in an angiotensin II model of erectile dysfunction. J Sex Med. 2013;10:2154-2164.
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RESULTS
CONCLUSION
To evaluate the effects of treatment with metformin on a murine model of obesity-associated erectile dysfunction. C57BL/6 male mice were fed for 10 weeks with standard chow or high-fat diet. Lean and obese mice were treated with the insulin sensitizer metformin (300 mg/kg/day, 2 weeks). Intracavernosal pressure (ICP) and in vitro corpus cavernosum (CC) relaxations to both acetylcholine and electrical field stimulation, as well as phenylephrine-induced contractions, were obtained. Levels of cyclic guanosine monophosphate in CC were detected by enzyme immunoassay. High-fat-fed mice exhibited higher body weight and insulin resistance. Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P <.05). Two-week therapy with metformin reversed the decreased ICP in obese group. The maximal response to acetylcholine in CC was 35% lower (P <.05) in the obese compared to the lean group, which were restored by metformin treatment. Likewise, the impaired electrical field stimulationeinduced CC relaxations in obese mice were also partly restored by metformin. Contractile responses to phenylephrine were significantly greater (P <.05) in obese compared to lean mice, which were fully restored by metformin. Basal and stimulated cyclic guanosine monophosphate productions in the erectile tissues were significantly lower (P <.05) in the obese group, an effect fully restored by metformin. Treatment with metformin restored the erectile function in obese mice, through improvement of in vitro endothelial and nitrergic cavernosal relaxations. Therefore, use of metformin may be a good pharmacologic approach to treat insulin resistanceeassociated erectile dysfunction. UROLOGY -: 1.e1–1.e6, 2015. 2015 Elsevier Inc.
E
rectile dysfunction (ED) is characterized by a persistent inability to achieve and/or maintain an erection sufficient for satisfactory sexual performance and has been associated with an abnormal function in the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway and hence, lower NO bioavailability in the erectile tissue.1 Epidemiologic studies show that obesity is an important risk factor for both ED and insulin resistance (IR).2-5 Evidence supports a strong causal link between IR and ED.6-10 In fact, ED has been considered an early clinical manifestation of risk factors for cardiovascular events including acute myocardial infarction.11 In addition, ED may Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, Sao Paulo, Brazil Address correspondence to: Fabio Henrique da Silva, Ph.D., Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, Sao Paulo 13084-971, Brazil. E-mail: [email protected] Submitted: January 26, 2015, accepted (with revisions): April 28, 2015
ª 2015 Elsevier Inc. All Rights Reserved
be the first clinical sign of IR in young men.8 A recent randomized controlled trial in patients with ED with poor response to sildenafil reported that treatment with metformin improves erectile function.7 IR is a state of dysregulation of glucose-insulin homeostasis, in which the ability of insulin to stimulate glucose uptake in peripheral tissues is reduced.2 Insulin is well known for inducing vascular relaxation through a mechanism that involves endothelium-dependent NO production.12 Vasodilator action of insulin is mediated by the phosphatidylinositol 3 kinase (PI3K)-Akt pathway that phosphorylates endothelial NO synthase at Ser1177.12 IR has been strongly associated with decreased NO bioavailability and endothelial dysfunction.13 Previous studies showed that high-fat-fed obese mice display ED, as demonstrated by reductions of intracavernous pressure (ICP) by cavernosal nerve electrostimulation and impaired endothelial and nitrergic cavernosal relaxations, as well as by increase of contractile response elicited by a1-adrenergic receptor activation.14,15 http://dx.doi.org/10.1016/j.urology.2015.04.035 0090-4295/15
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Table 1. Body weight, epididymal fat mass, and insulin sensitivity of lean and obese mice treated with vehicle (water) or metformin (300 mg/kg/day, 14 days) by gavage
Body weight (g) Epididymal fat mass (g) Kitt (min1)
Lean þ V
Obese þ V
Lean þ Met
Obese þ Met
31 0.6 0.32 0.06 4.34 0.30
43 2* 1.89 0.13* 2.06 0.37*
30 0.7 0.39 0.02 4.38 0.29
42 1* 1.86 0.10* 4.24 0.45y
Kitt, constant rate for blood glucose disappearance; Met, metformin; V, vehicle. Data are mean standard error of mean of 5 mice. * P <.05 in comparison with lean þ vehicle group. y P <.05 in comparison with obese þ vehicle group.
The insulin sensitizer metformin is a first-line pharmacologic treatment for patients with type 2 diabetes mellitus, which acts by decreasing hepatic glucose production and increasing insulin sensitivity in skeletal muscle and adipose tissue.16 Recently, metformin was shown to increase the adenosine signaling in corpus cavernosum from the high-fat-fed rabbits via increases in NO production.17 However, no detailed functional study has explored the effect of metformin treatment on a murine model of obesity-associated ED. Our hypothesis is that metformin reverses ED in the murine model of obesity associated with IR through improvement of NO bioavailability in the erectile tissue. Therefore, in the present study, in a continuing effort to investigate the effect of metformin treatment on ED, we have treated insulinresistant obese mice with the insulin sensitizer metformin and evaluated in vivo and in vitro functional responses of the mice corpus cavernosum.
MATERIAL AND METHODS Animals All animal procedures and experimental protocols are in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College for Animal Experimentation and followed the Guide for the Care and Use of Laboratory Animals. Four-week-old male C57BL/6J mice were provided by Central Animal House Services of the University of Campinas.
Diet-induced Obesity and Treatment Mice were housed 3 per cage on a 12-hour light-dark cycle and fed for 10 weeks with either a standard chow diet (carbohydrate, 70%; protein, 20%; fat, 10%) or a high-fat diet that induces obesity (carbohydrate, 29%; protein, 16%; fat, 55%), as previously described. Lean and obese mice were treated with vehicle (water) or the insulin sensitizer metformin (300 mg/kg/day) by gavage from the eighth to the 10th week. In our protocols, we used a total of 70 mice divided into 4 experimental groups, namely lean plus vehicle (N ¼ 16), lean plus metformin (n ¼ 19), obese plus vehicle (n ¼ 16), and obese plus metformin (n ¼ 19). Tail-cuff pressure was measured by using a modified tail-cuff method in conscious animals in a quiet room.
In Vivo Insulin Sensitivity After 6 hours of fasting, systemic insulin sensitivity was analyzed by the insulin tolerance test. Tail blood samples were collected before (0 min) and at 5, 10, 15, 25, 30, and 60 minutes after an intraperitoneal injection of 1.00 U/kg of regular insulin (Novolin R; NovoNordisk, Bagsværd, Denmark). Glucose concentrations were measured using a glucometer (ACCUCHEK Performa; Roche Diagnostics, Indianapolis, IN), and the values were used to 1.e2
calculate the constant rate for blood glucose disappearance (Kitt), based on the linear regression of the Napierian logarithm of glucose concentrations obtained from 0 to 30 minutes of the test. Kitt was calculated using the formula 0.693/(t1/2)x 1 100.
ICP Measurement Mice were anesthetized with an intraperitoneal injection of urethane (1.8 g/kg). To perform ICP measurement, right penile crus was exposed and cannulated using a 26-gauge needle. The cannula was filled with sterile heparinized saline solution and attached to a pressure detector for continuous ICP monitoring. The bladder and prostate were exposed through a midline abdominal incision. The right major pelvic ganglion and cavernous nerve were identified posterolateral to the prostate on one side. The cavernous nerve was electrically stimulated with 2 platinum electrodes connected to a Grass S48 stimulator (Astro-Med Industrial Park, West Warwick, RI). The cavernous nerve stimulation was conducted at 6 V, with 1 ms pulse width and trains of stimuli lasting 60 seconds at varying frequencies, with intervals of 3 minutes between the stimulation trains. Changes in ICP were recorded using a PowerLab 400 data acquisition system (LabChart software, version 7.0; ADInstruments, Colorado Springs, CO). Mice undergoing ICP measurements were not used for other experimental protocols.15
Functional Studies in Cavernosal Strips and Concentration-Response Curves Mice were anesthetized with isoflurane and exsanguinated. Strips of mice CC were mounted in a 10-mL organ system containing Krebs solution at 37 C continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4) and vertically suspended between 2 metal hooks. One hook was connected to a force transducer and the other acted as a fixed attachment point. Tissues were allowed to equilibrate for 60 minutes under a resting tension of 2.5 mN. Isometric force was recorded using a PowerLab 400 data acquisition system (LabChart, version 7.0; ADInstruments, MA). Cumulative concentration-response curve was constructed for the muscarinic agonist acetylcholine (ACh; 109 to 3 105 M) in cavernosal strips precontracted with the a1-adrenergic receptor agonist phenylephrine (PE; 105 M). Cumulative concentrationresponse curves of PE (108 to 3 104 M) were also obtained in the cavernosal tissue. Nonlinear regression analysis to determine the pEC50 was carried out using GraphPad Prism (GraphPad Software, San Diego, CA) with the constraint that V ¼ 0. All concentration-response data were evaluated for a fit to a logistics function in the form: E ¼ Emax/[1 þ (10c/10x)n] þ V, where E is the maximum response produced by agonists; c is the logarithm of the EC50, the concentration of drug that produces a half-maximal response; x is the logarithm of the concentration of the drug; the exponential term, n, is a curve-fitting parameter that defines the slope of the concentration-response line, and F is the response observed in the absence of added drug. UROLOGY
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Drugs and Chemicals ACh, atropine, guanethidine, metformin, and PE were purchased from Sigma-Aldrich (St Louis, MO). All reagents used were of analytical grade. Stock solutions were prepared in deionized water and stored in aliquots at 20 C. Dilutions were prepared immediately before use.
Statistical Analysis The program InStat (GraphPad Software Inc.) was used for statistical analysis. Data are expressed as the mean standard error of mean of N experiments. Statistical comparisons were made using 1-way analysis of variance, and the Tukey method was chosen as the post test. A value of P <.05 was considered statistically significant.
RESULTS
Figure 1. (A) Intracavernous pressure (ICP) obtained by stimulation of cavernous nerve at 4-12 Hz in anesthezised mice and (B) in vitro corpus cavernosum relaxations to electrical field stimulation (2-32 Hz) from lean and obese mice, treated or not with metformin (300 mg/kg/day, 2 weeks). Data represent the mean standard error of mean for lean þ vehicle (n ¼ 5), obese þ vehicle (n ¼ 5); lean þ metformin (n ¼ 6) and obese þ metformin (n ¼ 6) groups. *P <.05 in comparison with lean þ vehicle group; # P <.05 in comparison with obese þ vehicle group.
Electrical Field Stimulation Electrical field stimulation (EFS) was applied in strips placed between two platinum ring electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park). EFS was conducted at 50 V, with 1 ms pulse width and trains of stimuli lasting 10 seconds at varying frequencies. Frequency-response relationships were investigated at supra maximum voltage in all preparations stimulated electrically. To study the nitrergic relaxations, cavernosal tissues were pretreated with both guanethidine (3 105 M; to deplete the catecholamine stores of adrenergic fibers) and atropine (106 M; to produce muscarinic antagonism) before precontraction with PE. When a stable contraction level was attained, a series of EFS-induced relaxations were constructed (4-32 Hz).
Morphometric Characteristics and Insulin Tolerance Test The high-fat-fed mice displayed a significant increase (P <.05) in body weight and epididymal fat mass compared to lean mice. Treatment with metformin (300 mg/kg/day, 2 weeks) did not significantly affect these parameters (Table 1). Obese mice also displayed IR as demonstrated by the lower Kitt values compared to the lean group. Metformin treatment fully restored the insulin sensitivity in the obese group, without changing the insulin sensitivity in the lean group (Table 1). Tail-cuff pressure was not modified in any experimental group (82 3, 84 2, 82 3, and 83 4 mm Hg for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin groups, respectively). The average weights of dry cavernosal strips were 10 0.2, 10.1 0.3, 10.1 0.4, and 10.2 0.4 mg for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin mice, respectively. Erectile Function The cavernous nervous stimulation (4-12 Hz) caused frequency-dependent increases of ICP in all groups (Fig. 1A). However, ICPs in obese mice were significantly lower compared to those in lean mice at all frequencies studied (P <.05; Fig. 1A). Treatment with metformin fully restored the ICP in obese mice at all frequencies studied, without changing the ICP in lean group (P <.05; Fig. 1A).
Determination of cGMP Levels Mice were anesthetized with isoflurane and exsanguinated. Cavernosal segments were equilibrated for 30 minutes in Krebs solution at 37 C continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4). Tissues were stimulated for 1.5 minutes with ACh (105 M). Next, tissues were immediately frozen in liquid nitrogen. Frozen tissues were pulverized, homogenized in trichloroacetic acid (5% wt/vol), and centrifuged for 10 minutes at 4 C at 1500 g and the supernatant was collected. The pellet was dried and weighed. Trichloroacetic acid was extracted from the supernatant with 3 washes of water-saturated ether. Preparation of tracer, samples, standards, and incubation with antibody were performed as described in commercially available kits (Cayman Chemical Cyclic GMP EIA kit, Ann Arbor, MI). The assays were performed in duplicates, and the pellet weight was used to normalize the data that were expressed as pmol/mg tissue. UROLOGY
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EFS-induced Cavernosal Relaxations The addition of PE (105 M) to the cavernosal tissues pretreated with guanethidine (3 105 M) and atropine (106 M) caused a submaximal contraction of cavernosal segments that did not significantly differ between groups (0.33 0.04, 0.37 0.05, 0.31 0.04, and 0.31 0.04 mN for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin groups, respectively; n ¼ 10-12). EFS of PE-contracted tissues caused frequency-dependent relaxations in both lean and obese groups. A marked decrease in the relaxations of cavernosal tissues from obese in comparison with lean mice was observed at all frequencies studied (P <.05; Fig. 1B). Treatment with metformin partly restored the impaired EFS-induced relaxant response in the obese 1.e3
Figure 2. (A) Concentration-response curves to acetylcholine (ACh) in corpus cavernosum (CC) strips from lean and obese mice, treated or not with metformin (300 mg/kg/day, 2 weeks). Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (105 M) in each tissue, which was taken as 100%. (B) Emax values for ACh in all groups. Data represent the mean standard error of mean (SEM) for lean þ vehicle (n ¼ 5), obese þ vehicle (n ¼ 5); lean þ metformin (n ¼ 6), and obese þ metformin (n ¼ 6) groups. (C) Cyclic guanosine monophosphate GMP (cGMP) contents of mice CC from lean and obese mice, treated or not with metformin. CC strips were incubated with ACh (105 M). cGMP levels represent the mean SEM for 4 mice in each group. *P <.05 in comparison with lean þ vehicle group; #P <.05 in comparison with obese þ vehicle group.
group by about 83%, 82%, and 81% at frequencies 8, 16, and 32 Hz, respectively (Fig. 1B). In lean mice, no significant changes after metformin treatment were observed in EFSinduced CC relaxations (Fig. 1B). ACh-induced Cavernosal Relaxations and cGMP Levels in Corpus Cavernosum The cumulative addition of ACh (109-105 M) to PEcontracted tissues produced concentration-dependent relaxations in all groups (Fig. 2A). However, the potency (pEC50) for ACh was 3.7-fold lower (P <.001) in cavernosal 1.e4
Figure 3. (A) Concentration-response curves to phenylephrine in corpus cavernosum strips from lean and obese mice, treated or not with metformin (300 mg/kg/day, 2 weeks). (B) Emax values for all groups. Data are shown in mN and represent the mean SEM for lean þ vehicle (n ¼ 7), obese þ vehicle (n ¼ 7), lean þ metformin (n ¼ 9), and obese þ metformin (n ¼ 9) groups. *P <.05 in comparison with lean þ vehicle group; #P <.05 in comparison with obese þ vehicle group.
tissue of obese mice compared to lean mice (6.28 0.07 and 6.85 0.05, respectively; Fig. 2A). The maximal response (Emax) elicited by ACh was also significantly lower (P <.05) in cavernosal strips from obese compared to lean mice (50 3% and 77 3%, respectively; Fig. 2B). Treatment with metformin partly reversed the lower Emax and pEC50 parameters for ACh in cavernosal tissue of obese mice (66 2% and 6.66 0.05, respectively), with no significant changes in the lean mice (70 4% and 6.77 0.04%, respectively; Fig. 2A,B). The basal cGMP content in the erectile tissue was 40% lower (P <.05) in obese group compared to the lean group (Fig. 2C). Treatment with metformin fully restored (P <.05) the reduced basal cGMP levels in the obese group, with no changes in the lean group (Fig. 2C). In lean UROLOGY
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group, incubation of CC with ACh (105 M) significantly elevated (P <.05) the cGMP above baseline. In the obese group, no elevations in the cGMP levels by ACh were observed compared to the respective baseline. Treatment with metformin fully restored the ACh-induced increases in cGMP levels in CC from obese mice, with no significant changes in the lean group (P <.05; Fig. 2C). PE-induced Cavernosal Contractions PE (109-104 M) induced concentration-dependent contractions in cavernosal preparations in all groups (Fig. 3A). The Emax was significantly greater in obese compared to lean mice (P <.05; Fig. 3B). Metformin treatment prevented the elevation of PE-induced contractile responses in CC of obese mice. No significant changes after metformin treatment were observed in lean mice (Fig. 3A). No significant differences of pEC50 for PE were found in any experimental group (5.23 0.04, 5.42 0.03, 5.39 0.03, and 5.40 0.04 for lean þ vehicle, obese þ vehicle, lean þ metformin, and obese þ metformin groups, respectively).
COMMENT In the present study, using a murine model of IR-associated obesity, we show that metformin treatment restores the reduced ICP and the impaired in vitro endothelial and nitrergic cavernosal relaxations. Metformin acts by increasing the insulin sensitivity.16 At the molecular level, metformin increases the tyrosine phosphorylation of insulin receptor, phosphatidylinositol 3 kinase activity, phosphorylation of Akt and adenosine monophosphate-activated protein kinase activity.18-20 Previous studies have shown that prolonged treatment with metformin improves ACh- and flow-mediated dilatation of the brachial artery in humans with IR, as well as in vitro vasodilatation induced by ACh in mesenteric and aorta from insulin-resistant rats.21-24 ED in obese Zucker rats was attributed to impaired relaxant responses to insulin in penile arteries.25 We have, therefore, treated obese and lean mice with metformin to investigate the erectile function. In our study, metformin treatment fully reversed the IR in obese mice, which is consistent with previous studies.24,26 Metformin treatment normalized the reduced ICP, as well as the impaired in vitro endothelium-dependent and nitrergic relaxations in obese mice. Decreased basal levels of cGMP were found in obese mice CC that were also normalized by metformin treatment. These findings strongly suggest that IR plays an important role in the development of ED by the impairment of NO bioavailability. In rabbits fed with a high-fat diet, metformin ameliorated the in vitro relaxant responses to ACh in the erectile tissue, along with hyperglycemia and glucose intolerance.17 In the flaccid state, CC and penile vessels are contracted mainly via stimulation of a1-adrenoceptors by norepinephrine derived from the sympathetic nerves.1 Previous studies reported that ED in hypertensive and aging animals has been associated with increased a1-adrenoceptoremediated cavernosal vasoconstriction.27,28 In our study, the maximal UROLOGY
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contractile response to a1-adrenoceptor agonist PE was greater in the obese group, which was also fully restored by metformin treatment. It is likely that enhanced levels of cGMP by metformin in obese mice counteract the contractile responses downstream the a1-adrenoceptor, making penile tumescence more difficult to occur. Recently, in a rat model of hypertension with no accompanying IR, metformin reversed the decreased ICP and increased the relaxing responses to both ACh and EFS and reduced contractile responses to PE in the erectile tissue.29 However, in our study, no alteration in the tail-cuff pressure was observed between the groups, excluding arterial hypertension as a cause for the development of ED.
CONCLUSION Our study shows that treatment of mice with metformin improves the ED in high-fat-fed obese mice. IR associated with obesity seems to contribute to ED development. Therefore, the use of metformin treatment may be a good pharmacologic approach to treat IR-associated ED. Acknowledgments. Fabio H. Silva thanks São Paulo Research Foundation (FAPESP) for the financial support. References 1. Andersson KE. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol Rev. 2011; 63:811-859. 2. Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 2006;444:840-846. 3. Bal K, Oder M, Sahin AS, et al. Prevalence of metabolic syndrome and its association with erectile dysfunction among urologic patients: metabolic backgrounds of erectile dysfunction. Urology. 2007; 69:356-360. 4. Wang F, Dai S, Wang M, Morrison H. Erectile dysfunction and fruit/vegetable consumption among diabetic Canadian men. Urology. 2013;82:1330-1335. 5. Corona G, Rastrelli G, Filippi S, et al. Erectile dysfunction and central obesity: an Italian perspective. Asian J Androl. 2014;16:581-591. 6. Bansal TC, Guay AT, Jacobson J, et al. Incidence of metabolic syndrome and insulin resistance in a population with organic erectile dysfunction. J Sex Med. 2005;2:96-103. 7. Rey-Valzacchi GJ, Costanzo PR, Finger LA, et al. Addition of metformin to sildenafil treatment for erectile dysfunction in eugonadal nondiabetic men with insulin resistance. A prospective, randomized, double-blind pilot study. J Androl. 2012;33:608-614. 8. Yao F, Liu L, Zhang Y, et al. Erectile dysfunction may be the first clinical sign of insulin resistance and endothelial dysfunction in young men. Clin Res Cardiol. 2013;102:645-651. 9. Chen S, Wu R, Huang Y, et al. Insulin resistance is an independent determinate of ED in young adult men. PLoS One. 2013;8:e83951. 10. Russo GI, Cimino S, Fragala E, et al. Insulin resistance is an independent predictor of severe lower urinary tract symptoms and of erectile dysfunction: results from a cross-sectional study. J Sex Med. 2014;11:2074-2082. 11. Thompson IM, Tangen CM, Goodman PJ, et al. Erectile dysfunction and subsequent cardiovascular disease. JAMA. 2005;294:2996-3002. 12. Kim JA, Montagnani M, Koh KK, et al. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation. 2006;113:1888-1904. 13. Zecchin HG, Priviero FB, Souza CT, et al. Defective insulin and acetylcholine induction of endothelial cell-nitric oxide synthase through insulin receptor substrate/Akt signaling pathway in aorta of obese rats. Diabetes. 2007;56:1014-1024.
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14. Toque HA, da Silva FH, Calixto MC, et al. High-fat diet associated with obesity induces impairment of mouse corpus cavernosum responses. BJU Int. 2011;107:1628-1634. 15. Silva FH, Leiria LO, Alexandre EC, et al. Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice. J Sex Med. 2014;11:2661-2670. 16. Pawlyk AC, Giacomini KM, McKeon C, et al. Metformin pharmacogenomics: current status and future directions. Diabetes. 2014; 63:2590-2599. 17. Vignozzi L, Filippi S, Comeglio P, et al. Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa. J Sex Med. 2014;11:1694-1708. 18. Kumar N, Dey CS. Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells. Br J Pharmacol. 2002;137:329-336. 19. Bhamra GS, Hausenloy DJ, Davidson SM, et al. Metformin protects the ischemic heart by the Akt-mediated inhibition of mitochondrial permeability transition pore opening. Basic Res Cardiol. 2008;103: 274-284. 20. Rice S, Pellatt LJ, Bryan SJ, et al. Action of metformin on the insulin-signaling pathway and on glucose transport in human granulosa cells. J Clin Endocrinol Metab. 2011;96:427-435. 21. Katakam PV, Ujhelyi MR, Hoenig M, et al. Metformin improves vascular functioning insulin-resistant rats. Hypertension. 2000;35: 108-112.
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22. Mather KJ, Berma S, Anderson TJ. Improved endothelial function with metformin in type II diabetes mellitus. J Am Coll Cardiol. 2001; 37:1344-1350. 23. Vitale C, Mercuro G, Cornoldi A, et al. Metformin improves endothelial function in patients with metabolic syndrome. J Intern Med. 2005;258:250-256. 24. Sena CM, Matafome P, Louro T, et al. Metformin restores endothelial function in aorta of diabetic rats. Br J Pharmacol. 2011;163: 424-437. 25. Contreras C, Sanchez A, Martınez P, et al. Insulin resistance in penile arteries from a rat model of metabolic syndrome. Br J Pharmacol. 2010;161:350-364. 26. Leiria LO, Sollon C, Calixto MC, et al. Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice. PLoS One. 2012;7:e48507. 27. Carneiro FS, Giachini FR, Lima VV, et al. DOCA-salt treatment enhances responses to endothelin-1 in murine corpus cavernosum. Can J Physiol Pharmacol. 2008;86:320-328. 28. Silva FH, Lanaro C, Leiria LO, et al. Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum. Am J Physiol Heart Circ Physiol. 2014;307:H1393-H1400. 29. Labazi H, Wynne BM, Tostes R, Webb RC. Metformin treatment improves erectile function in an angiotensin II model of erectile dysfunction. J Sex Med. 2013;10:2154-2164.
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