World J Mens Health.  2019 May;37(2):138-147. 10.5534/wjmh.180026.

Obesity and Erectile Dysfunction: From Bench to Clinical Implication

• Moon KH
• Park SY
• Kim YW

• KMID: 2443229
• DOI: http://doi.org/10.5534/wjmh.180026

Abstract

Obesity is a major public health issue worldwide and is frequently associated with erectile dysfunction (ED). Both conditions may share an internal pathologic environment, also known as common soil. Their main pathophysiologic processes are oxidative stress, inflammation, and resultant insulin and leptin resistance. Moreover, the severity of ED is correlated with comorbid medical conditions, including obesity. Therefore, amelioration of these comorbidities may increase the efficacy of ED treatment with phosphodiesterase 5 inhibitors, the first-line medication for patients with ED. Although metformin was originally developed as an insulin sensitizer six decades ago, it has also been shown to improve leptin resistance. In addition, metformin has been reported to reduce oxidative stress, inflammatory response, and body weight, as well as improve ED, in animal and human studies. Moreover, administration of a combination of metformin and phosphodiesterase 5 inhibitors improves erectile function in patients with ED who have a poor response to sildenafil and are insulin resistant. Thus, concomitant treatment of metabolic derangements associated with obesity in patients with ED who are obese would improve the efficacy and reduce the refractory response to penile vasodilators. In this review, we discuss the connecting factors between obesity and ED and the possible combined treatment modalities.

Keyword

Erectile dysfunction; Metformin; Obesity; Phosphodiesterase 5 inhibitors

MeSH Terms

Animals
Body Weight
Comorbidity
Erectile Dysfunction*
Humans
Inflammation
Insulin
Leptin
Male
Metformin
Obesity*
Oxidative Stress
Phosphodiesterase 5 Inhibitors
Public Health
Sildenafil Citrate
Soil
Vasodilator Agents
Insulin
Leptin
Metformin
Phosphodiesterase 5 Inhibitors
Sildenafil Citrate
Soil
Vasodilator Agents

Reference

1. Malik S, Wong ND, Franklin SS, Kamath TV, L'Italien GJ, Pio JR, et al. Impact of the metabolic syndrome on mortality from coronary heart disease, cardiovascular disease, and all causes in United States adults. Circulation. 2004; 110:1245–1250. PMID: 15326067.

2. Cersosimo E, DeFronzo RA. Insulin resistance and endothelial dysfunction: the road map to cardiovascular diseases. Diabetes Metab Res Rev. 2006; 22:423–436. PMID: 16506274.

3. Park J, Kwon OS, Cho SY, Paick JS, Kim SW. Chronic administration of atorvastatin could partially ameliorate erectile function in streptozotocin-induced diabetic rats. PLoS One. 2017; 12:e0172751. PMID: 28245261.

4. Corona G, Rastrelli G, Morelli A, Vignozzi L, Mannucci E, Maggi M. Hypogonadism and metabolic syndrome. J Endocrinol Invest. 2011; 34:557–567. PMID: 21720206.

5. de Souza ILL, Barros BC, de Oliveira GA, Queiroga FR, Toscano LT, Silva AS, et al. Hypercaloric diet establishes erectile dysfunction in rat: mechanisms underlying the endothelial damage. Front Physiol. 2017; 8:760. PMID: 29085300.

6. Bacon CG, Mittleman MA, Kawachi I, Giovannucci E, Glasser DB, Rimm EB. A prospective study of risk factors for erectile dysfunction. J Urol. 2006; 176:217–221. PMID: 16753404.

7. Fillo J, Levcikova M, Ondrusova M, Breza J, Labas P. Importance of different grades of abdominal obesity on testosterone level, erectile dysfunction, and clinical coincidence. Am J Mens Health. 2017; 11:240–245. PMID: 27184064.

8. Demir O, Akgul K, Akar Z, Cakmak O, Ozdemir I, Bolukbasi A, et al. Association between severity of lower urinary tract symptoms, erectile dysfunction and metabolic syndrome. Aging Male. 2009; 12:29–34. PMID: 19326294.

9. Aleid M, Muneer A, Renshaw S, George J, Jenkinson AD, Adamo M, et al. Early effect of bariatric surgery on urogenital function in morbidly obese men. J Sex Med. 2017; 14:205–214. PMID: 28087357.

10. Choi YS, Lee SK, Bae WJ, Kim SJ, Cho HJ, Hong SH, et al. Bariatric surgery improves the cavernosal neuronal, vasorelaxation, and contraction mechanisms for erectile dysfunction as result of amelioration of glucose homeostasis in a diabetic rat model. PLoS One. 2014; 9:e104042. PMID: 25137300.

11. Kun L, Pin Z, Jianzhong D, Xiaodong H, Haoyong Y, Yuqian B, et al. Significant improvement of erectile function after Roux-en-Y gastric bypass surgery in obese Chinese men with erectile dysfunction. Obes Surg. 2015; 25:838–844. PMID: 25361762.

12. Hannan JL, Maio MT, Komolova M, Adams MA. Beneficial impact of exercise and obesity interventions on erectile function and its risk factors. J Sex Med. 2009; (6 Suppl 3):254–261. PMID: 19170860.

13. Meldrum DR, Gambone JC, Morris MA, Esposito K, Giugliano D, Ignarro LJ. Lifestyle and metabolic approaches to maximizing erectile and vascular health. Int J Impot Res. 2012; 24:61–68. PMID: 22072232.

14. Maiorino MI, Bellastella G, Esposito K. Lifestyle modifications and erectile dysfunction: what can be expected? Asian J Androl. 2015; 17:5–10. PMID: 25248655.

15. Patel JP, Lee EH, Mena CI, Walker CN. Effects of metformin on endothelial health and erectile dysfunction. Transl Androl Urol. 2017; 6:556–565. PMID: 28725599.

16. Roh E, Song DK, Kim MS. Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism. Exp Mol Med. 2016; 48:e216. PMID: 26964832.

17. Kim YW, Choi DW, Park YH, Huh JY, Won KC, Choi KH, et al. Leptin-like effects of MTII are augmented in MSG-obese rats. Regul Pept. 2005; 127:63–70. PMID: 15680471.

18. Kim YW, Kim JY, Park YH, Park SY, Won KC, Choi KH, et al. Metformin restores leptin sensitivity in high-fat-fed obese rats with leptin resistance. Diabetes. 2006; 55:716–724. PMID: 16505235.

19. Small CJ, Stanley SA, Bloom SR. Appetite control and reproduction: leptin and beyond. Semin Reprod Med. 2002; 20:389–398. PMID: 12536362.

20. Shimobayashi M, Albert V, Woelnerhanssen B, Frei IC, Weissenberger D, Meyer-Gerspach AC, et al. Insulin resistance causes inflammation in adipose tissue. J Clin Invest. 2018; 128:1538–1550. PMID: 29528335.

21. de Git KC, Adan RA. Leptin resistance in diet-induced obesity: the role of hypothalamic inflammation. Obes Rev. 2015; 16:207–224. PMID: 25589226.

22. Egan OK, Inglis MA, Anderson GM. Leptin signaling in AgRP neurons modulates puberty onset and adult fertility in mice. J Neurosci. 2017; 37:3875–3886. PMID: 28275162.

23. Costanzo PR, Knoblovits P. Male gonadal axis function in patients with type 2 diabetes. Horm Mol Biol Clin Investig. 2016; 26:129–134.

24. Nakao K, Iwata K, Takeshita T, Ozawa H. Expression of hypothalamic kisspeptin, neurokinin B, and dynorphin A neurons attenuates in female Zucker fatty rats. Neurosci Lett. 2018; 665:135–139. PMID: 29203206.

25. Quennell JH, Mulligan AC, Tups A, Liu X, Phipps SJ, Kemp CJ, et al. Leptin indirectly regulates gonadotropin-releasing hormone neuronal function. Endocrinology. 2009; 150:2805–2812. PMID: 19179437.

26. Isidori AM, Caprio M, Strollo F, Moretti C, Frajese G, Isidori A, et al. Leptin and androgens in male obesity: evidence for leptin contribution to reduced androgen levels. J Clin Endocrinol Metab. 1999; 84:3673–3680. PMID: 10523013.

27. Williams G. Aromatase up-regulation, insulin and raised intracellular oestrogens in men, induce adiposity, metabolic syndrome and prostate disease, via aberrant ER-α and GPER signalling. Mol Cell Endocrinol. 2012; 351:269–278. PMID: 22233684.

28. Knoblovits P, Costanzo PR, Valzacchi GJ, Gueglio G, Layus AO, Kozak AE, et al. Erectile dysfunction, obesity, insulin resistance, and their relationship with testosterone levels in eugonadal patients in an andrology clinic setting. J Androl. 2010; 31:263–270. PMID: 19834133.

29. Saad F, Yassin A, Doros G, Haider A. Effects of long-term treatment with testosterone on weight and waist size in 411 hypogonadal men with obesity classes I-III: observational data from two registry studies. Int J Obes (Lond). 2016; 40:162–170. PMID: 26219417.

30. Ghosh A, Gao L, Thakur A, Siu PM, Lai CWK. Role of free fatty acids in endothelial dysfunction. J Biomed Sci. 2017; 24:50. PMID: 28750629.

31. Kim YW, Park SY, Kim JY, Huh JY, Jeon WS, Yoon CJ, et al. Metformin restores the penile expression of nitric oxide synthase in high-fat-fed obese rats. J Androl. 2007; 28:555–560. PMID: 17314235.

32. Kim JE, Kim YW, Lee IK, Kim JY, Kang YJ, Park SY. AMP-activated protein kinase activation by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) inhibits palmitate-induced endothelial cell apoptosis through reactive oxygen species suppression. J Pharmacol Sci. 2008; 106:394–403. PMID: 18360094.

33. Kim F, Tysseling KA, Rice J, Pham M, Haji L, Gallis BM, et al. Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta. Arterioscler Thromb Vasc Biol. 2005; 25:989–994. PMID: 15731493.

34. Ryu SY, Choi YJ, Park SY, Kim JY, Kim YD, Kim YW. Udenafil, a phosphodiesterase 5 inhibitor, reduces body weight in high-fat-fed mice. World J Mens Health. 2018; 36:41–49. PMID: 29164831.

35. Li S, Xu J, Yao W, Li H, Liu Q, Xiao F, et al. Sevoflurane pretreatment attenuates TNF-α-induced human endothelial cell dysfunction through activating eNOS/NO pathway. Biochem Biophys Res Commun. 2015; 460:879–886. PMID: 25838201.

36. Giugliano F, Esposito K, Di Palo C, Ciotola M, Giugliano G, Marfella R, et al. Erectile dysfunction associates with endothelial dysfunction and raised proinflammatory cytokine levels in obese men. J Endocrinol Invest. 2004; 27:665–669. PMID: 15505991.

37. Hafez G, Gonulalan U, Kosan M, Arioglu E, Ozturk B, Cetinkaya M, et al. Acetylsalicylic acid protects erectile function in diabetic rats. Andrologia. 2014; 46:997–1003. PMID: 24428436.

38. Bayraktar Z, Albayrak S. Antiplatelet (aspirin) therapy as a new option in the treatment of vasculogenic erectile dysfunction: a prospective randomized double-blind placebo-controlled study. Int Urol Nephrol. 2018; 50:411–418. PMID: 29344879.

39. Mong PY, Petrulio C, Kaufman HL, Wang Q. Activation of Rho kinase by TNF-alpha is required for JNK activation in human pulmonary microvascular endothelial cells. J Immunol. 2008; 180:550–558. PMID: 18097057.

40. Roumeguère T, Van Antwerpen P, Fathi H, Rousseau A, Vanhamme L, Franck T, et al. Relationship between oxidative stress and erectile function. Free Radic Res. 2017; 51:924–931. PMID: 29041845.

41. Carneiro FS, Zemse S, Giachini FR, Carneiro ZN, Lima VV, Webb RC, et al. TNF-alpha infusion impairs corpora cavernosa reactivity. J Sex Med. 2009; (6 Suppl 3):311–319. PMID: 19267854.

42. Carneiro FS, Sturgis LC, Giachini FR, Carneiro ZN, Lima VV, Wynne BM, et al. TNF-alpha knockout mice have increased corpora cavernosa relaxation. J Sex Med. 2009; 6:115–125. PMID: 19170842.

43. Van de Voorde J, Pauwels B, Boydens C, Decaluwé K. Adipocytokines in relation to cardiovascular disease. Metabolism. 2013; 62:1513–1521. PMID: 23866981.

44. Matos G, Hirotsu C, Alvarenga TA, Cintra F, Bittencourt L, Tufik S, et al. The association between TNF-α and erectile dysfunction complaints. Andrology. 2013; 1:872–878. PMID: 24123864.

45. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun. 1999; 257:79–83. PMID: 10092513.

46. Achari AE, Jain SK. Adiponectin, a therapeutic target for obesity, diabetes, and endothelial dysfunction. Int J Mol Sci. 2017; 18:E1321. PMID: 28635626.

47. Kim JE, Song SE, Kim YW, Kim JY, Park SC, Park YK, et al. Adiponectin inhibits palmitate-induced apoptosis through suppression of reactive oxygen species in endothelial cells: involvement of cAMP/protein kinase A and AMP-activated protein kinase. J Endocrinol. 2010; 207:35–44. PMID: 20675307.

48. Ouchi N, Ohishi M, Kihara S, Funahashi T, Nakamura T, Nagaretani H, et al. Association of hypoadiponectinemia with impaired vasoreactivity. Hypertension. 2003; 42:231–234. PMID: 12860835.

49. Gentili A, Zaibi MS, Alomar SY, De Vuono S, Ricci MA, Alaeddin A, et al. Circulating levels of the adipokines monocyte chemotactic protein-4 (MCP-4), macrophage inflammatory protein-1β (MIP-1β), and eotaxin-3 in severe obesity and following bariatric surgery. Horm Metab Res. 2016; 48:847–853. PMID: 27300476.

50. Arent SM, Walker AJ, Pellegrino JK, Sanders DJ, McFadden BA, Ziegenfuss TN, et al. The combined effects of exercise, diet, and a multi-ingredient dietary supplement on body composition and adipokine changes in overweight adults. J Am Coll Nutr. 2018; 37:111–120. PMID: 29111889.

51. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, et al. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation. 2000; 101:1539–1545. PMID: 10747347.

52. El Assar M, Angulo J, Santos-Ruiz M, Ruiz de Adana JC, Pindado ML, Sánchez-Ferrer A, et al. Asymmetric dimethylarginine (ADMA) elevation and arginase up-regulation contribute to endothelial dysfunction related to insulin resistance in rats and morbidly obese humans. J Physiol. 2016; 594:3045–3060. PMID: 26840628.

53. El Assar M, Ruiz de Adana JC, Angulo J, Pindado Martínez ML, Hernández Matías A, Rodríguez-Mañas L. Preserved endothelial function in human obesity in the absence of insulin resistance. J Transl Med. 2013; 11:263. PMID: 24138787.

54. Chen S, Wu R, Huang Y, Zheng F, Ou Y, Tu X, et al. Insulin resistance is an independent determinate of ED in young adult men. PLoS One. 2013; 8:e83951. PMID: 24391852.

55. Pierce GL, Lesniewski LA, Lawson BR, Beske SD, Seals DR. Nuclear factor-{kappa}B activation contributes to vascular endothelial dysfunction via oxidative stress in overweight/obese middle-aged and older humans. Circulation. 2009; 119:1284–1292. PMID: 19237660.

56. Hwang MH, Lee S. Insulin resistance: vascular function and exercise. Integr Med Res. 2016; 5:198–203. PMID: 28462118.

57. Dozio E, Barassi A, Dogliotti G, Malavazos AE, Colpi GM, D'Eril GV, et al. Adipokines, hormonal parameters, and cardiovascular risk factors: similarities and differences between patients with erectile dysfunction of arteriogenic and nonarteriogenic origin. J Sex Med. 2012; 9:2370–2377. PMID: 22616653.

58. Zhang WH, Zhang XH. Clinical and preclinical treatment of urologic diseases with phosphodiesterase isoenzymes 5 inhibitors: an update. Asian J Androl. 2016; 18:723–731. PMID: 26620458.

59. Kapoor MS, Khan SA, Gupta SK, Choudhary R, Bodakhe SH. Animal models of erectile dysfunction. J Pharmacol Toxicol Methods. 2015; 76:43–54. PMID: 26279495.

60. Fillo J, Breza J, Levčíkova M, Luha J, Vachulova A, Durdík Š, et al. Occurrence of erectile dysfunction, testosterone deficiency syndrome and metabolic syndrome in patients with abdominal obesity. Where is a sufficient level of testosterone? Int Urol Nephrol. 2012; 44:1113–1120. PMID: 22315154.

61. Kaya E, Sikka SC, Gur S. A comprehensive review of metabolic syndrome affecting erectile dysfunction. J Sex Med. 2015; 12:856–875. PMID: 25675988.

62. McMahon CN, Smith CJ, Shabsigh R. Treating erectile dysfunction when PDE5 inhibitors fail. BMJ. 2006; 332:589–592. PMID: 16528082.

63. Faulkner LD, Dowling AR, Stuart RC, Nillni EA, Hill JW. Reduced melanocortin production causes sexual dysfunction in male mice with POMC neuronal insulin and leptin insensitivity. Endocrinology. 2015; 156:1372–1385. PMID: 25590244.

64. Malin SK, Kashyap SR. Effects of metformin on weight loss: potential mechanisms. Curr Opin Endocrinol Diabetes Obes. 2014; 21:323–329. PMID: 25105996.

65. Pastor-Villaescusa B, Cañete MD, Caballero-Villarraso J, Hoyos R, Latorre M, Vázquez-Cobela R, et al. Metformin for obesity in prepubertal and pubertal children: a randomized controlled trial. Pediatrics. 2017; 140:e20164285. PMID: 28759403.

66. Saisho Y. Metformin and inflammation: its potential beyond glucose-lowering effect. Endocr Metab Immune Disord Drug Targets. 2015; 15:196–205. PMID: 25772174.

67. Rey-Valzacchi GJ, Costanzo PR, Finger LA, Layus AO, Gueglio GM, Litwak LE, 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. PMID: 22016348.

68. Majithiya JB, Balaraman R. Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocin-induced diabetic rats. Life Sci. 2006; 78:2615–2624. PMID: 16318863.

69. García-Prieto CF, Gil-Ortega M, Aránguez I, Ortiz-Besoain M, Somoza B, Fernández-Alfonso MS. Vascular AMPK as an attractive target in the treatment of vascular complications of obesity. Vascul Pharmacol. 2015; 67-69:10–20. PMID: 25869500.

70. Chantler PD, Shrader CD, Tabone LE, d'Audiffret AC, Huseynova K, Brooks SD, et al. Cerebral cortical microvascular rarefaction in metabolic syndrome is dependent on insulin resistance and loss of nitric oxide bioavailability. Microcirculation. 2015; 22:435–445. PMID: 26014499.

71. 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. PMID: 23889981.

72. Jing Y, Wu F, Li D, Yang L, Li Q, Li R. Metformin improves obesity-associated inflammation by altering macrophages polarization. Mol Cell Endocrinol. 2018; 461:256–264. PMID: 28935544.

73. Goldberg RB, Aroda VR, Bluemke DA, Barrett-Connor E, Budoff M, Crandall JP, et al. Effect of long-term metformin and lifestyle in the diabetes prevention program and its outcome study on coronary artery calcium. Circulation. 2017; 136:52–64. PMID: 28476766.

74. Silva FH, Alexandre EC, Calmasini FB, Calixto MC, Antunes E. Treatment with metformin improves erectile dysfunction in a murine model of obesity associated with insulin resistance. Urology. 2015; 86:423.e1–423.e6. PMID: 26199165.

75. Vignozzi L, Filippi S, Comeglio P, Cellai I, Morelli A, Rastrelli G, et al. Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa. J Sex Med. 2014; 11:1694–1708. PMID: 24844317.

76. Vitale C, Mercuro G, Cornoldi A, Fini M, Volterrani M, Rosano GM. Metformin improves endothelial function in patients with metabolic syndrome. J Intern Med. 2005; 258:250–256. PMID: 16115299.

77. Giagulli VA, Carbone MD, Ramunni MI, Licchelli B, De Pergola G, Sabbà C, et al. Adding liraglutide to lifestyle changes, metformin and testosterone therapy boosts erectile function in diabetic obese men with overt hypogonadism. Andrology. 2015; 3:1094–1103. PMID: 26447645.

78. Moon KH, Kim SW, Moon du G, Kim JJ, Park NC, Lee SW, et al. A phase 3 study to evaluate the 1-year efficacy and safety of udenafil 75 mg once daily in patients with erectile dysfunction. J Sex Med. 2016; 13:1263–1269. PMID: 27319276.

79. Varma A, Das A, Hoke NN, Durrant DE, Salloum FN, Kukreja RC. Anti-inflammatory and cardioprotective effects of tadalafil in diabetic mice. PLoS One. 2012; 7:e45243. PMID: 23028874.

80. Nunes AK, Rapôso C, Rocha SW, Barbosa KP, Luna RL, da Cruz-Höfling MA, et al. Involvement of AMPK, IKβα-NFκB and eNOS in the sildenafil anti-inflammatory mechanism in a demyelination model. Brain Res. 2015; 1627:119–133. PMID: 26404052.

81. Santi D, Granata AR, Guidi A, Pignatti E, Trenti T, Roli L, et al. Six months of daily treatment with vardenafil improves parameters of endothelial inflammation and of hypogonadism in male patients with type 2 diabetes and erectile dysfunction: a randomized, double-blind, prospective trial. Eur J Endocrinol. 2016; 174:513–522. PMID: 26792933.

82. De Toni L, Strapazzon G, Gianesello L, Caretta N, Pilon C, Bruttocao A, et al. Effects of type 5-phosphodiesterase inhibition on energy metabolism and mitochondrial biogenesis in human adipose tissue ex vivo. J Endocrinol Invest. 2011; 34:738–741. PMID: 22234177.

83. Fiore D, Gianfrilli D, Giannetta E, Galea N, Panio G, di Dato C, et al. PDE5 inhibition ameliorates visceral adiposity targeting the miR-22/SIRT1 pathway: evidence from the CECSID trial. J Clin Endocrinol Metab. 2016; 101:1525–1534. PMID: 26964730.

84. Mammi C, Pastore D, Lombardo MF, Ferrelli F, Caprio M, Consoli C, et al. Sildenafil reduces insulin-resistance in human endothelial cells. PLoS One. 2011; 6:e14542. PMID: 21297971.

85. Ramirez CE, Nian H, Yu C, Gamboa JL, Luther JM, Brown NJ, et al. Treatment with sildenafil improves insulin sensitivity in prediabetes: a randomized, controlled trial. J Clin Endocrinol Metab. 2015; 100:4533–4540. PMID: 26580240.

86. Ho JE, Arora P, Walford GA, Ghorbani A, Guanaga DP, Dhakal BP, et al. Effect of phosphodiesterase inhibition on insulin resistance in obese individuals. J Am Heart Assoc. 2014; 3:e001001. PMID: 25213566.

87. Anderson SG, Hutchings DC, Woodward M, Rahimi K, Rutter MK, Kirby M, et al. Phosphodiesterase type-5 inhibitor use in type 2 diabetes is associated with a reduction in all-cause mortality. Heart. 2016; 102:1750–1756. PMID: 27465053.

88. Peuler JD, Phelps LE. Sildenafil does not enhance but rather attenuates vasorelaxant effects of antidiabetic agents. J Smooth Muscle Res. 2015; 51:22–36. PMID: 26004378.

89. Cho MC, Paick JS. Udenafil for the treatment of erectile dysfunction. Ther Clin Risk Manag. 2014; 10:341–354. PMID: 24868160.