Module 1 Testosterone backgrounder - PowerPoint Presentation

Module 1 Testosterone backgrounder Approval Number : G.MKT.GM.MH.04.2018.0513

Testosterone biosynthesis and its regulation Module 1: Testosterone backgrounder Approval Number : G.MKT.GM.MH.04.2018.0513

Testosterone Testosterone is the most important steroid made by the testis1,2A healthy adult man produces~6–7 mg of testosterone per dayfrom the Leydig cells in the testes1,2Testosterone mainly acts asan androgen, but can also bearomatized into estrogensThis provides ~25% of totaldaily 17β-estradiol production2The half-life of free testosteronein the blood is only ~12 min1 Seminiferous tubules Testosterone-producing Leydig cells 1. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010. 2. O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext . South Dartmouth (MA); 2017.

AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CE, cholesteryl ester; ER, endoplasmic reticulum; HSD, hydroxysteroid dehydrogenase; LH, luteinizing hormone; P450scc, cholesterol side-chain cleavage enzyme; PBR, peripheral-type benzodiazepine receptor; StAR , steroidogenic acute regulatory proteinMiller WL & Auchus RJ. Endocr Rev. 2011;32(1):81–151;Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.Testosterone biosynthesisTestosterone is synthesized from free cholesterol in a process involving 5 enzymatic stages Leydig cell LH ATP cAMP AC Mitochondria Smooth ER Free cholesterol CE StAR , PBR Pregnenolone Progesterone 17  -Hydroxy- progesterone Androstene- dione Testosterone 17  -Hydroxy- pregnenolone Dehydroepi- androsterone Androstenediol 3 β -HSD 17 β -HSD 17 ,20 Lyase 17 α -Hydroxylase P450scc

Cholesterol is the key substrate for testosterone biosynthesis Conversion of cholesterol to pregnenolone is the first and rate-limiting step in testosterone biosynthesis 1The cholesterol utilized for steroidogenesis is derived from multiple sources, including:2De novo synthesis in the ERMobilization of CEs stored in lipid droplets via the action ofCE hydrolase, which is mediated by LH-induced formation of cAMP and stimulation of protein kinase APlasma lipoprotein-derived CEs obtained by eitherLDLR-mediated endocytosis and/or SR-BI-mediatedselective uptakecAMP, cyclic adenosine monophosphate; CE, cholesteryl ester; ER, endoplasmic reticulum;LDLR, low-density lipoprotein receptor; LH, luteinizing hormone; SR-BI, scavenger receptor class B type 11. Haider SG. Endocrinology. 2007;148(6):2581–2. 2. Hu J et al. Nutr Metab (Lond). 2010;7:47.

The hypothalamus regulates testicular function and testosterone production Regular, intermittent GnRH secretion from hypothalamic neurons stimulates biosynthesis of LH and FSH by the pituitary gland 1,2 LH and FSH travel via the bloodstream to the testes, where they initiate testosterone production and spermatogenesis, as well as systemic testosterone secretion and virilization 3 This episodic GnRH release results in pulsatile LH secretion every ~60–120 min on average, although considerable variability in LH pulse patterns can exist 2,3 GnRH pulse amplitude and frequency influence the amountof LH and FSH secreted by the pituitary gland4FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone1. O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); 2017. 2. Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); 2016.3. Hayes F et al. Hypogonadotropic hypogonadism (Hh) and gonadotropin therapy. Endotext. South Dartmouth (MA); 2013. 4. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.

Role of LH in testosterone biosynthesis LH promotes Leydig cell steroidogenesis by increasing cholesterol availability and activating rate-limiting steroidogenic enzyme and cholesterol-transport proteins1LH thus regulates testosterone biosynthesis in two ways:Acutely at the level of StAR2Chronically by enhancing the transcription of genes thatencode a variety of enzymes in the steroidogenic pathway3The pulsatile pattern of trophic hormone exposure maintains Leydig cell sensitivity to LH, sustaining mature male patterns of testicular testosterone secretion1cAMP, cyclic adenosine monophosphate; LH, luteinizing hormone; StAR, steroidogenic acute regulatory protein1. Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); 2016.2. Haider SG. Endocrinology. 2007;148(6):2581–2. 3. O’Donnell L et al. Endocrinology of the male reproductive systemand spermatogenesis. Endotext. South Dartmouth (MA); 2017.

Control of testosterone biosynthesis in the hypothalamic-pituitary-testis axis Testosterone is key to the testicular negative feedback cycle on the hypothalamus and pituitary gland 1 It acts directly on androgen receptors and indirectly, by aromatization (oxidation) in the hypothalamus to estradiol1This leads to reductions in GnRH pulse frequency and decreases in the amplitude of LH pulses, caused by reduced GnRH secretion and gonadotropin responseto GnRH stimulation1Pituitary LH and FSH secretion is also suppressed by feedback inhibition via testosterone and estradiol2FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone1. Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); 2016. 2. O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); 2017.

Control of testosterone biosynthesis in the hypothalamic-pituitary-testis axis (2) Other regulatory molecules include: Inhibins , hormone inhibitors of FSH production secreted by Sertoli cells in the testes Follistatin , a pituitary autocrine glycoprotein, which inhibitsFSH secretion by binding to and blocking the action of activins, paracrine hormones that enhance FSH biosynthesis and secretionKisspeptins, a family of neuropeptides localized to thearcuate nucleus in the brain, which are upstream stimulatorsof GnRH secretionGnIH, which acts upstream of GnRH and may also operateat the level of the testes as an autocrine/paracrine regulatorof steroidogenesisFSH, follicle-stimulating hormone; GnIH, gonadotropin-inhibitory hormone; GnRH, gonadotropin-releasing hormoneO’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis.Endotext. South Dartmouth (MA); 2017.

Regulation of testosterone secretion FSH, follicle-stimulating hormone; GnIH , gonadotropin-inhibitory hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone Adapted from Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.GnRHInhibins Gonadotropins (LH, FSH) Testosterone ( Estradiol ) Pituitary gland Hypothalamus Testis + – – – Activins + Follistatin – + Kisspeptins – GnIH

Testosterone binding Approximately 98% of testosterone is bound to transport proteins and only 2% is free or ‘biologically active’SHBG is the carrier protein fortestosterone and estradiol, and hasa 3-fold higher affinity for the formerSHBG synthesis occurs mainly inthe liver and is regulated by theopposing actions of sex steroidsAndrogen inhibits SHBG synthesisEstrogen stimulates SHBG synthesisAn increase in SHBG plasmaconcentrations leads to an acute decreasein free testosterone and simultaneousstimulation of testosterone biosynthesisSHBG, sex hormone-binding globulinNieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010. Free2% Bound with higher affinity to SHBG54% Bound withlower affinityto albumin44%

Biological effects of testosterone and its metabolites Module 1: Testosterone backgrounder

DHT, dihydrotestosterone Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010. Testosterone is important in every phase of male life Testosterone is the most important endogenous sex hormone and androgen in the maleIt is vital for the proper development and functioning of the male phenotypeTestosterone and its main metabolites, DHT and estradiol, exert a number of anabolic and metabolic effects that can influence many physical and mental functions in menStage of male lifeEffect of testosterone In utero (embryo) Sex differentiation Puberty Virilization Adulthood Maintenance of the male phenotype Sexual function Anabolic effects

Testosterone acts on diverse target tissues to cause a variety of biological effects HbA 1c , glycated hemoglobin ; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SHBG, sex hormone-binding globulin1. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.2. Traish AM. Sex Med Rev. 2018;6(1):86–105. 3. Nebido® Product Monograph, Bayer AG, 2017.4. Cutolo M et al. Ann N Y Acad Sci. 2002;966:131–42.TargetBiological effect of testosterone and its metabolites Reproductive organs 1,2 Prenatal sex differentiation; pubertal virilization and development of testes, penis, epididymis, seminal vesicles and prostate; in adults, maintenance of reproductive organs, potency and sexual function, and initiation/maintenance of spermatogenesis Muscle 1 Increased volume, strength and lean body mass Skin and hair 1 Increased sebum production, hair growth and male hair pattern Liver 1,3 Increased synthesis of clotting factors, hepatic triglyceride lipase, sialic acid, α1-antitrypsin, haptoglobin; decreased production of SHBG and other hormone-binding proteins, transferrin, fibrinogenLipids1,2Improvements in dyslipidemia; decreased plasma total cholesterol, LDL-C and triglycerides, and increased plasma HDL-C concentrationsBlood glucose2 Improvements in hyperglycemia; decreased fasting blood glucose and HbA1cBone1Accelerated linear growth; closure of epiphyses; increased bone massBrain1Improvements in cognitive function, socialization, concentration, self-confidence, mood, libido; in adults, possible neuroprotective effect Hematopoietic system1,2Stimulation of erythropoietin biosynthesis in the kidney; direct effects on hematopoietic stem cells, leading to increased hemoglobin productionImmune cells4Suppression of humoral and cellular immune responses; anti-inflammatory effect

Conversion of testosterone to DHT and estradiol is a necessary process for its biological action Testosterone is metabolized to DHT and estradiol by 5α -reductase and aromatase, respectively1Testosterone and DHT bind to the same androgen receptor, while estradiol binds to the estrogen receptor, to regulate gene expressionin target tissues1,2Androgen and estrogen receptors are members of the steroid nuclear receptor superfamily and have a highly homologous structure, differing mostly in the binding domain2Compared with testosterone, DHT is a more potent androgen:Higher binding affinity for the androgen receptor23–10 times greater molar potency in transactivation2Slower dissociation from the androgen receptor3Greater ability to slow natural degradation of the androgen receptor3The complete spectrum of action of testosterone therefore incorporates effects that are indirectly induced by conversion to DHT and estradiolDHT, dihydrotestosterone 1. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010 .2. Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); 2016.3. Zhou ZX et al. Mol Endocrinol . 1995;9(2):208–18.

Direct and indirect biological effects of testosterone, DHT and estradiol DHT, dihydrotestosterone Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010; Rochira V et al. Estrogens and male reproduction. Endotext. South Dartmouth (MA); 2016. Testosterone DHT Estradiol 5 α-reductase Aromatase Sex differentiation Musculature Secondary hair growth Sebum production Prostate development Sex differentiation Spermatogenesis Musculature Bone mass Erythropoiesis Psychotropic action Potency/libidoLipid metabolismBone massEpiphyseal closurePsychotropic actionLipid metabolismProstate developmentAndrogen receptor effectsEstrogen receptor effects

Summary Testosterone is synthesized from cholesterol in the Leydig cells of the testis in 5 enzymatic stages As a necessary process for its biological action, testosterone is also converted to DHT by 5α-reductase and estradiol by aromataseTestosterone biosynthesis and secretion is regulated by the hypothalamic-pituitary-testis axisThis involves pulsatile GnRH secretion from the hypothalamus, LH and FSH biosynthesis by the pituitary gland, negative feedback from testosterone and estradiol, and the action of other regulatory molecules, including inhibins, follistatin, kisspeptins and GnIHApproximately 98% of testosterone is bound to the transport proteins, SHBG and albumin, and only 2% is free or ‘biologically active’Testosterone is the most important endogenous sex hormone and androgen in men, and is vital for proper male development and physical and mental functioningDHT, dihydrotestosterone; FSH, follicle-stimulating hormone; GnIH, gonadotropin-inhibitory hormone;GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; SHBG, sex hormone-binding globulin