Clinical Neuroendocrinology: An Introduction [1st ed.] 9781108149938

Table of contents :
Dedication; Preface; Acknowledgements; 1. Basic principles in clinical neuroendocrinology I: receptor mechanisms; 2. Basic principles in clinical neuroendocrinology II: assays, rhythms and pulses; 3. Neuroendocrinology of female reproduction; 4. Neuroendocrine regulation of appetite and body weight; 5. Hypothalamic-pituitary-adrenal cortex axis; 6. Hypothalamic regulation of thyroid function; 7. Hypothalamic regulation of prolactin secretion; 8. Regulation of growth hormone secretion; 9. Posterior pituitary; 10. An introduction to sellar masses; Appendix; Index.

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Preface The human body is primarily governed by two intricate communicating networks: the nervous system and the endocrine system. The study of the interrelationship of these two networks created the discipline of neuroendocrinology. Recent advances in this field have transformed our view of how human endocrine homeostasis is maintained. For example, the discovery of the adipokine leptin revolutionized our understanding of the neural mechanisms by which we regulate body weight. A further case is the discovery of the KISS1 gene, and its encoded neuropeptide kisspeptin, now recognized as obligatory for successful human reproductive function. Although several texts are currently available that cover the field of clinical neuroendocrinology, they are almost exclusively advanced, multi-author books written by experts and largely aimed at medical specialists. While these texts provide a comprehensive clinical and basic science review of the subject, there is a compelling need for an introductory description of the human neuroendocrine system in health and disease. Our book is therefore designed to emphasize the key physiological principles necessary for an understanding of various clinical neuroendocrine disorders. Introductory chapters discuss the fundamentals that govern how the hypothalamic–pituitary system interacts with various endocrine target tissues. Topics include cellular communication, hormone receptor systems, hormone assays and a description of the importance of hormonal secretory rhythms. Subsequent chapters outline the essentials of human female reproduction, the regulation of body weight and metabolism with a focus on obesity, the control of prolactin secretion and the principles of adrenal, thyroid and growth hormone physiology. Finally, in

a separate chapter on sellar masses, key elements of clinical history, biochemical and radiological assessment as well as epidemiology of sellar masses are discussed. The clinical implications of various physiological principles as well as cases from our clinics are included in the text. The text itself is liberally illustrated with full-color, high-resolution images to provide concise summaries of information. Extensive lists of references emphasize original papers based on human data. We intend the book to be useful at multiple levels, though it is especially aimed at those students and clinicians not previously exposed to a specific course in neuroendocrinology. For example, senior medical students making decisions to pursue a specialty will find it helpful, as will those residents and clinical fellows who are embarking on their chosen fields. In addition, this book should provide a crucial clinical context for biomedical science graduate students who may already be familiar with basic science research principles. We include an extensive and up-to-date reference list for each chapter, and additional material is provided under a further reading list. The latter tend to be clinical reviews that may be particularly useful to the more advanced reader and will provide a convenient link to the available specialized texts. A selection of review questions is provided at the end of each chapter. The field of neuroendocrinology is a rapidly evolving area of health sciences. This introduction to clinical neuroendocrinology should serve as a guide to medical students, clinicians and biomedical science students, as well as their teachers, in negotiating a fascinating and essential clinical field of study.

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Chapter

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Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

The homeostatic functions of the body are primarily controlled by neuronal cells communicating through electrical impulses and endocrine cells communicating through chemicals. Neuroendocrinology is the branch of endocrinology that is concerned with how the brain regulates the endocrine milieu. An essential and critical characteristic of this neural control is that endocrine hormones have profound effects on brain function through homeostatic feedback systems. An understanding of the chemical mechanisms that underpin neuroendocrine regulation is critical when dealing with clinical disorders of the neuroendocrine system. For example, the clinical complications of abnormal growth, thyroid disorders, obesity and Cushing’s syndrome can be

confronted through addressing the underlying neuroendocrine principles. For the most part, the brain influences endocrine targets in tandem with the pituitary gland; that is, pituitary hormone secretion is directed by various stimuli secreted from hypothalamic neurons. Thus, luteinizing hormone (LH) is released from pituitary gonadotropes following stimulation by gonadotropin releasing hormone (GnRH), a neuropeptide produced by hypothalamic neurons. Figure 1.1 illustrates the hypothalamic releasing hormones that regulate anterior pituitary hormone secretion. Note that dopamine – a neurotransmitter – controls prolactin (PRL) secretion. In contrast, posterior pituitary hormones, such as oxytocin, are not under the influence

HYPOTHALAMUS Thyrotropin releasing hormone Growth hormone releasing hormone Dopamine Gonadotropin releasing hormone Somatostatin Corticotropin releasing hormone

POSTERIOR PITUITARY

ANTERIOR PITUITARY Growth hormone Luteinizing hormone Follicle stimulating hormone Adrenocorticotropic hormone Thyroid stimulating hormone Prolactin

Vasopressin Oxytocin

Figure 1.1 Hypothalamic and pituitary hormones of the neuroendocrine system. Public domain images from Ladyofhats: http://en.wikipedia .org/wiki/File:Endocrine_central_nervous_en.svg.

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Regulatory neurons + –

–

±

GnRH neurons

–

Infundibular nucleus GnRH

Portal vasculature ±

–

G protein-coupled receptor (GPCR) superfamily. Other hormones, such as leptin, bind to so-called tyrosine kinase-dependent receptors. An additional, intracellular, superfamily of steroid hormone receptors mediates the feedback effects of steroid hormones such as estradiol and cortisol at the level of the anterior pituitary and hypothalamus. Thus, an interplay of fluctuating hormonal levels with receptor sensitivity dictates homeostatic neuroendocrine regulation. The following sections will summarize the receptorsignaling systems that: (a) regulate target organ activity, for example, stimulation of gonadotrophs by GnRH acting through cell surface GPCRs; and (b) control hormonal feedback in the hypothalamus and pituitary, for example, intracellular estradiol receptors in brain and pituitary.

Anterior pituitary

1.1 Cell Membrane Receptors LH/FSH

Estradiol

Testosterone

Progesterone Ovary

Testis

Figure 1.2 Homeostatic control of GnRH secretion and the reproductive system. Schematic diagram highlighting the role of GnRH neurons in the control of human reproduction. Red arrows indicate the positive and negative feedback effects of serum estradiol and progesterone exerted on GnRH secretion. This control is imposed at several levels: directly on GnRH neurons; at the level of gonadotrophs; and on neurons (stimulatory and inhibitory) that regulate GnRH neurons. Blue arrows illustrate the negative feedback effects of testosterone on GnRH release in the male. Abbreviations: GnRH, gonadotropin releasing hormone; LH, Luteinizing hormone; FSH, Follicle stimulating hormone.

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of releasing hormones but instead are secreted directly from hypothalamic neuron terminals in the posterior pituitary. A typical neuroendocrine feedback network is exemplified by the reproductive system in which the target organs (ovary and testis) respond to gonadotropin stimulation by releasing sex hormones (estradiol, progesterone or testosterone) that, in turn, exert negative feedback on the hypothalamus to influence secretion of GnRH and LH/follicle stimulating hormone (FSH) (see Figure 1.2 and Chapter 3). Other examples will be described in subsequent chapters. The target organ sensitivity to stimulation, and the neuronal response to hormonal feedback, is dependent upon a variety of receptor mechanisms. For example, in Figure 1.2, the response of pituitary gonadotrophs to stimulation with the peptide GnRH is governed by specific membrane receptors of the

Peptide hormones, neuropeptides and neurotransmitters are generally water soluble and cannot easily enter their target cells. They regulate cellular activity by binding to specific receptors located in the plasma membranes of their target cells. In order to induce biochemical changes within the target cell, they act as first messengers to activate an intracellular second messenger, such as cyclic adenosine monophosphate (cAMP). The transduction of information from the first to the second messenger is accomplished through the activation of membrane proteins (e.g., G proteins) and enzymes, such as adenylate cyclase. This section illustrates the role of membrane receptors for peptide hormone and neurotransmitter action, the mechanisms by which signal transduction across the cell membrane occurs, the role of G proteins and receptor tyrosine kinases in this signal transduction, and the second messenger systems activated.

1.2 G Protein-Coupled Receptors GPCRs are characterized by their seven transmembrane domain structures attached to trimeric G proteins (Figure 1.3). They bind multiple neurotransmitters, hormones and peptides, and control almost all known physiological processes, including neuroendocrine regulation, cardiovascular function, behavior and immune function. A variety of G proteins together with receptors, effectors and various regulatory intracellular proteins are the components of a complex and versatile signal transduction system (for a detailed review see Wettschureck and

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

GnRH CRH TSH dopamine

GHRH GPCR

6

7

GH

5 4

Cytoplasm

12 3

Gα GDP

Gα GTP Gαs AC

Gαs

cAMP

Gαq

PLC

Gαi

cAMP

+

γ

γ β

M SO

RE LIN

Activated G protein Extracellular

+

β

–

Gαq PLC

cAMP/Ca2+ PLC/IP3/Ca2+

Inhibition of cAMP

cAMP PKA

Biological response

PKC

–

GH gene Inhibition expression; of GH release GH secretion

Gene expression

Nucleus

+

DNA

Figure 1.3 Schematic diagram of GPCR signaling via G proteins and second messengers. GPCRs, in the inactive state, possess seven transmembrane domains coupled to a G protein complex consisting of α-, β- and γ-subunits, plus a molecule of GDP. Binding of a specific ligand activates the Gα-subunit by replacing GDP with GTP, followed by dissociation of the βγ-subunit. This step is reversible following dissociation or degradation of the receptor stimulus. The Gα-subunit exists as three forms: (a) a stimulatory Gαs that increases cAMP production and the activation of protein kinase A; (b) a stimulatory Gαq that selectively stimulates the PLC pathway to activate PKC; and (c) an inhibitory G03B1i pathway that inhibits cAMP production. The protein kinases may regulate enzyme activity or gene expression via transcription factors (dotted arrows) that bind to target genes. Examples of ligands that bind to GPCRs are GnRH, CRH, TSH and dopamine. Image reproduced with permission (Neumann et al., 2014). Abbreviations: AC, adenylate cyclase; GDP, guanosine diphosphate; GnRH, gonadotropin releasing hormone; GPCR, G protein-coupled receptor; GTP, guanosine triphosphate; PIP2, phosphatidylinositol 4,5 bisphosphate; PKC, protein kinase C; PLC, phospholipase C.

Offermanns, 2005). For the purposes of this chapter, Figure 1.3 outlines, in general terms, the processes by which extracellular stimuli are rapidly transduced to intracellular signals that ultimately control gene expression and biological response. The figure includes typical neuroendocrine stimuli (GnRH, corticotropin releasing hormone [CRH], thyroid stimulating hormone [TSH] and dopamine) that act via GPCRs. Other examples will be covered in subsequent chapters. By way of illustration, the pituitary somatotroph is a cell type that utilizes all three of the GPCRs illustrated in Figure 1.3; that is, ghrelin and growth hormone releasing hormone (GHRH) synergize to stimulate growth hormone (GH) secretion, whereas somatostatin serves to inhibit GH secretion

GH SECRETION

SOMATOTROPH

Figure 1.4 GPCR-dependent mechanisms regulating GH secretion from pituitary somatotrophs. Receptors for GHRH and ghrelin are stimulatory G protein coupled, linked to cAMP/PKA and PLC/PKC signals, respectively. GHRH and ghrelin act synergistically to induce intracellular calcium ion mobilization that, in turn, controls GH secretion. These stimuli also induce GH gene expression and GH synthesis in somatotrophs. In contrast, SOM binds to an inhibitory GPCR that reduces the accumulation of cAMP, decreasing the release of GH(Ben-Shlomo and Melmed, 2010). Abbreviations: GPCR, G protein-coupled receptor; IP3, inositol triphosphate; PKA, protein kinase A; PKC, protein kinase C; SOM, somatostatin.

(Figure 1.4). This system is covered in more detail in Chapter 8. A further example is the synergistic stimulation of adrenocorticotropin (ACTH) from pituitary corticotrophs by CRH and vasopressin (see Chapter 5; Figure 5.5)

Clinical Significance of GPCRs G protein receptors are firmly implicated in all the hormonal systems described in this book. For example, Figure 1.3 illustrates that important neuroendocrine molecules such as GnRH (reproductive system), CRH (regulation of the hypothalamic–pituitary–adrenal system), TRH (hypothalamic–pituitary–thyroid regulation) and dopamine (PRL secretion) all function through specific GPCRs. Other examples will be covered in later chapters. These receptors represent

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

a superfamily of human membrane proteins. Drugs that target them account for approximately 30% of the global market share of therapeutic drugs, with estimated sales for 2011–2015 of approximately US$890 billion (Hauser et al., 2017). The responsiveness of GPCRs to stimulation may be compromised in some neuroendocrine disorders. Receptor function is affected in two principal ways: (a) when GPCRs are subjected to chronic stimulation to produce tachyphylaxis, and (b) when mutations occur in the genes for receptor proteins or G proteins.

Chronic Stimulation and GPCR Downregulation For example, continuous stimulation of the human female pituitary with the releasing hormone GnRH downregulates (desensitizes) the normal response such that LH secretion and ovulation is inhibited (Southworth et al., 1991). A major consequence of continuous stimulation of gonadotrophs is inhibition of the normal response through the loss of cell surface GnRH receptors (Engel and Schally, 2007).

Desensitization is reversible by replacing continuous GnRH treatment with an episodic, pulsatile stimulation. This will be described in more detail in Chapter 3. A similar phenomenon is reported with stimulation of LH secretion with the neuropeptide kisspeptin, which also binds to a GPCR (Jayasena et al., 2009; see Chapter 3).

Mutations in GPCR Genes The clinical significance of GPCRs in the neuroendocrine system is reinforced when taking into account endocrine/neuroendocrine disorders that result from loss-of-function or gain-of-function mutations in the genes for receptor proteins or G proteins (Vassart and Costagliola, 2011). Table 1.1 lists a group of mutations of GPCRs relevant to endocrine diseases, together with the appropriate chapter where these receptors are discussed (based on data from Lania et al., 2006; Vassart and Costagliola, 2011). A detailed discussion of gain-of-function GPCR mutations associated with endocrine disorders is covered elsewhere (Fukami et al., 2018).

Table 1.1 Clinical and biochemical features of endocrine disorders caused by GPCR mutations

Biochemical feature

GPCR affected (type of mutation)

Isolated central hypothyroidism with normal pituitary MRI

Normal or low TSH, low fT4 absent TSH and prolactin responses to TRH test

TRH receptor (inactivating)

Complete congenital hypothyroidism

High TSH, low fT4, no goiter, no antibodies to thyroglobulin or thyroperoxidase

TRH receptor (inactivating)

Juvenile hyperthyroidism with goiter

Low TSH, high fT4, no antibodies to thyroglobulin or TSH receptor

TSH receptor (activating)

Low sex steroids and low–normal LH and FSH responsive to GnRH test

Kisspeptin receptor (inactivating)

Low sex steroids and low–normal LH and FSH poorly responsive to GnRH test

GnRH receptor (inactivating)

Primary amenorrhea with normal development of primary and secondary sexual characteristics

High LH, normal or high FSH, and low estradiol and progesterone

LH receptor (inactivating)

Male precocious puberty with normal pituitary MRI

High testosterone and low LH and FSH with prepubertal response to GnRH test

LH receptor (activating)

Primary or early-onset secondary amenorrhea, variable development of secondary sex characteristics and premature arrest of follicular maturation

High LH, high FSH

FSH receptor (inactivating)

Ovarian hyperstimulation syndrome during in vitro fertilization

None

FSH receptor (activating)

Clinical features Thyroid disorders (Chapter 6)

Reproductive disorders (Chapter 3) Delayed puberty

4

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Table 1.1 (cont.)

Biochemical feature

GPCR affected (type of mutation)

Hyperinsulinemia

Melanocortin 4 receptor (inactivating)

Low GH and insulin-like growth factor 1, unresponsive to GHRH test

GHRH receptor (inactivating)

Nephrogenic diabetes insipidus

Hypernatremia, low urine osmolality, normal or high VP

V2R (inactivating)

Nephrogenic syndrome of inappropriate antidiuresis

Hyponatremia, low serum osmolality, inappropriately high urine osmolality, undetectable VP levels

V2R (activating)

Clinical features Obesity (Chapter 4) Early-onset or severe adult obesity, associated with hyperphagia Growth disorders (Chapter 8) Dwarfism associated with abdominal adiposity Water balance disorders (Chapter 9)

Abbreviations: FSH, follicle stimulating hormone; fT4, free thyroxine; GH, growth hormone; GHRH, growth hormone releasing hormone; GnRH, gonadotropin releasing hormone; GPCR, G protein-coupled receptor; LH, luteinizing hormone; MRI, magnetic resonance imaging; TRH, thyrotropin releasing hormone; TSH, thyroid stimulating hormone; VP, vasopressin; V2R, vasopressin receptor 2.

1.3 Tyrosine Kinase-Dependent Receptors Peptide hormones such as leptin, PRL and GH (see Chapters 4, 7 and 8, respectively) do not bind to GPCRs. As shown in Figure 1.5, the receptor structures – in this case the leptin receptor dimer – have only two transmembrane domains. Leptin binding to a single transmembrane protein induces receptor dimerization, which then stimulates a tyrosine kinase pathway rather than the G protein signaling shown in Figure 1.3. For leptin, PRL and GH, this is the second messenger JAK/STAT pathway (for details of the GH system see Chapter 8; Figure 8.12). For example, binding of leptin to its receptor activates (phosphorylates) Janus kinase (JAK) proteins that are docked on the intracellular domain. Activated JAKs then phosphorylate the signal transducer and activator of transcription (STAT) family of transcription factors. Dimerization of STAT proteins precedes translocation to the nucleus where binding to response elements on DNA modulates transcription of target genes. Unlike the leptin receptor, the PRL and GH receptors exist as preformed dimers (see Figure 8.12 for GH, and Brooks and Waters, 2010) but also employ the JAK/STAT pathway following binding of the ligand (Bernard et al., 2015).

Leptin receptor dimer

LEPTIN

Plasma membrane JAK Cytoplasm

JAK

P

P P

P P

P STAT

STAT

–

STAT

P STAT

SOCS3

P

Nucleus P STAT

STAT P

Gene expression

Figure 1.5 Leptin receptor and the JAK/STAT pathway. Binding of leptin to its receptor dimer activates (phosphorylates; P) JAK proteins. Activated JAKs then phosphorylate the STAT family of transcription factors. Dimerization of STAT proteins precedes translocation to the nucleus where binding of the transcription factor to response elements on DNA modulates transcription of target genes. Also included is a negative feedback system where gene expression of SOCS3 modulates leptin stimulation. Image reproduced with permission (Dodington et al., 2018). Abbreviations: STAT, signal transducer and activator of transcription; SOCS3, suppressor of cytokine signaling 3.

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Clinical Significance of Tyrosine Kinase-Dependent Receptors The clinical importance of leptin, PRL and GH is described in the appropriate chapters. The clinical consequences of abnormal receptor signaling are profound, and defects, either at the receptor or in the intracellular pathway, appear as a hormone deficiency. For example, as described in Chapter 4, obese individuals are insensitive to their own high levels of leptin. By analogy with the insulin resistance seen in type 2 diabetes, the common forms of diet-related obesity are thought to be attributable to “leptin resistance,” a state in which multiple cellular processes block leptin receptor signaling (Myers et al., 2010). Thus, high levels of endogenous leptin, derived from the increased fat mass, fail to reduce food intake or body weight. Also, patients with mutations in the leptin receptor are severely obese and effectively leptin-free, even though leptin levels are high (Farooqi et al., 2007). In these cases, leptin is unable to signal to downstream pathways. Due to loss-of-function mutations in the GH receptor (Rosenfeld et al., 2007; Brooks and Waters, 2010), patients insensitive to GH (Laron syndrome) show severe growth failure and insulin-like growth factor 1 (IGF-1) deficiency (see Chapter 8). Over sixty loss-of-function mutations in the GH receptor have been reported, although a new cause of GH

insensitivity – mutations in the intracellular STAT5 gene (see Figure 8.12) – is now described; that is, the intracellular signaling pathway is defective (Hwa, 2016). Loss-of-function mutations in the PRL receptor have also been described (Bernard et al., 2015). Such patients present with hyperprolactinemia, possibly due to the inability of PRL to exert negative feedback on pituitary lactotrophs.

1.4 Intracellular Receptors for Steroid Hormones Steroid hormones (e.g., testosterone, estradiol and cortisol; Figure 1.6) and thyroid hormones (see Figure 6.5) are fat-soluble molecules transported in the blood bound to carrier proteins. Steroid hormones readily diffuse through cell membranes into any cell in the body, but only their target cells, in brain and pituitary for example, possess specific intracellular receptors. These receptors have common structural elements and belong to a superfamily of receptor proteins (Figure 1.7). Each receptor protein contains a hormone binding domain (HBD) that is specific for each hormone. Thus, for example, the human glucocorticoid receptor (hGR) binds cortisol in the HBD, whereas the progesterone receptor (hPR) recognizes only progesterone. Binding of the hormone to its specific receptor Figure 1.6 Chemical structures of the major steroid hormones. Four principal families of steroid hormones are: androgens (e.g., testosterone), estrogens (e.g., estradiol), glucocorticoids (e.g., cortisol) and mineralocorticoids (e.g., aldosterone). They are all derived from cholesterol. An important family not shown is the progestogens (e.g., progesterone). Note that the female sex hormone estradiol is formed by aromatization from the male hormone testosterone. Copyright A. Pincock.

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

hAR

NTD

100

hERα

hERβ

hGR

hPR

15

NTD

15

15

NTD

15

NTD

NTD

DBD 100

H

DBD

H

DBD 56

H

DBD

H

51

77

80

DBD

HORMONE BINDING DOMAIN

100

HORMONE BINDING DOMAIN

20

22

50

H

53

HORMONE BINDING DOMAIN

HORMONE BINDING DOMAIN

HORMONE BINDING DOMAIN

Figure 1.7 Structures of members of the human steroid receptor superfamily. These intracellular receptors consist of a single polypeptide chain and all of the family members contain a hormone binding domain that is specific for each hormone. For example, hGR binds cortisol in this region, whereas the hPR will only recognize progesterone. The DBD enables the hormone-receptor complex to bind to the HRE (see Figure 1.8) on a target gene. The hinge region (H) is important, along with the DBD, for nuclear localization of the receptor, and the NTD is crucial for the activation of gene transcription once the hormone-receptor complex reaches the nucleus and binds with the HRE. Numbers indicate the degree of structural homology in each domain, compared with the hAR (McEwan and Brinkmann, 2016). Abbreviations: DBD, DNA binding domain; hAR, androgen receptor; hERα, estrogen receptor α; hERβ, estrogen receptor β; hGR, glucocorticoid receptor; hPR, progesterone receptor; HRE, hormone response element; NTD, amino terminal domain.

enables the DNA binding domain (DBD) to interact with specific sites on target genes called hormone response elements (HRE; see Figure 1.8). The hinge region (H) appears to be important, along with the DBD, for nuclear localization of the receptor, and the amino terminal domain (NTD) is crucial for the activation of gene transcription once the hormonereceptor complex reaches the nucleus and binds with the HRE (McEwen and Brinkmann, 2016). Note that the receptor proteins have no biological activity until they bind to a hormone; that is, the hormone-receptor complex acts as a transcription factor at specific sites (HRE) on target genes. Figure 1.8 illustrates the sequence of events that occur when a steroid hormone diffuses into target cells, such as pituitary or hypothalamus. The hormone is released from the binding globulin (BG), enters the cell and binds to a specific receptor (R) in the cell cytosol. The unoccupied R is coupled to a so-called molecular chaperone (heat shock protein 90; HSP90) that ensures the receptor is stabilized in the correct shape. Following hormone binding to the receptor-HSP90 complex, the HSP90 dissociates and the remaining hormone-receptor complex dimerizes. The dimer then enters the cell nucleus where it attaches to an HRE to modify gene expression and the export of mRNA into the cell cytosol where it is translated into protein.

Estrogen Receptors It is beyond the scope of this text to provide a description of the clinical role of all of the nuclear receptors illustrated in Figure 1.7, and the estrogen receptor (ER) will be used here as an example. ERs exist in three forms, two of which are nuclear (ERα and ERβ, also termed ESR1 and ESR2; Figure 1.7) and the third, located in cell membranes, is a GPCR. The latter – termed the G protein-coupled ER-1 (GPER-1) – is a relative newcomer to ER physiology, and these receptors permit rapid, non-genomic (non-nuclear), cellular responses to estrogen treatment. There is evidence of a physiological role for GPER-1 in the reproductive, nervous, endocrine, immune and cardiovascular systems (Prossnitz and Barton, 2011), and GPER-1 appears to be a promising and novel therapeutic target and prognostic indicator (Barton, 2016). Membrane receptors for androgens, glucocorticoids (GCs), aldosterone and thyroid hormone have also been described. ERα and ERβ, the products of two distinct genes, are localized in many tissues (Figure 1.9), with ERα especially concentrated within the reproductive system (hypothalamus, pituitary, breast and uterus). Details of their functional role in estrogen feedback and the regulation of anterior pituitary secretion of gonadotropins through the menstrual cycle is outlined in Chapter 3.

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Figure 1.8 Schematic view of a steroid hormone interacting with a target cell. The hormone is reversibly bound to a binding globulin (BG) before the free hormone freely diffuses through the cell membrane. The unoccupied steroid receptor (R) is coupled to a molecular chaperone (HSP90) that stabilizes R in the correct shape. When the hormone binds to the receptor-HSP complex, the HSP dissociates and the remaining hormone-receptor complex dimerizes before it enters the cell nucleus. The hormone-receptor dimer then binds to target genes via a specific HRE. Various factors such as GTFs and RNA POL II assist in inducing gene transcription and the export of mRNA into the cell cytosol where it is translated into protein. Abbreviations: GTF, general transcription factor; HSP, heat shock protein; HSP90, heat shock protein 90; mRNA, messenger RNA; POL II, polymerase II; RNA, ribonucleic acid.

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This figure reveals that ERs are implicated in regulating multiple complex physiological processes in humans, and abnormal ER function leads to a variety of diseases, such as cancer, metabolic and cardiovascular disease, neurodegeneration, inflammation and osteoporosis (Jia et al., 2015). Clinical significance: Using the menopause as a specific example, estrogen deprivation has profound effects particularly in the central nervous system; for example, vasomotor symptoms such as hot flashes and night sweats, development of anxiety, depression, poor quality of sleep and migraine. Figure 1.10 illustrates the widespread influence of estrogen depletion (Monteleone et al., 2018). Estrogen replacement at the menopause inevitably influences many systems, some not without risk, as in the possibility of breast cancer (Warner et al., 2017). Selective targeting of ERα and ERβ appears to be a promising way to achieve beneficial estrogenic effects while avoiding unwanted side effects. ERβ selective agonists are now available that have no effect in breast tissue but may be therapeutic agents considered for prevention and treatment of cancer, metabolic and cardiovascular diseases, neurodegeneration,

inflammation and osteoporosis (Paterni et al., 2014; Warner et al., 2017).

Hormone Resistance This section uses the estrogen and GC receptors as examples of the clinical consequences of hormone resistance. Other well-described examples include androgen receptor insensitivity (testicular feminization syndrome; Hiort, 2013) and thyroid hormonereceptor insensitivity (Ortiga-Carvalho et al., 2014; see also Chapter 6).

Estrogens ER resistance appears to be unknown within the neuroendocrine system. However, mutations in ERα in metastatic breast cancer are well-described and result from long-term treatment with antiestrogens, such as tamoxifen, and drugs such as fulvestrant that degrades ERα (Huang et al., 2017). Drug-induced estrogen resistance could take the form of changes in downstream signaling or as intrinsic activation of ERα in the absence of estradiol. It is possible that tamoxifen or fulvestrant could affect hypothalamic or pituitary ERα receptors.

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Figure 1.9 Distribution of hERα and hERβ throughout the body. Localization of hERα and hERβ gene expression in normal human pituitary (Chaidarun et al., 1998) and pituitary adenomas (Manoranjan et al., 2010) has been added. Not included in the figure is the localization of both receptors in the human uterus (Simmen and Kelley, 2016). Image reproduced with permission (Warner et al., 2017). Abbreviations: hERα, estrogen receptor α; hERβ, estrogen receptor β.

Glucocorticoids GCs influence multiple physiological processes (see Chapter 5), and are routinely used to treat disorders of inflammation, autoimmune diseases and cancer. The physiological effects of GCs can, in rare cases, be compromised because of mutations in the GC receptor that impair GC action. This reduces tissue sensitivity to GCs, compromises negative feedback and induces hypersecretion of ACTH, cortisol and androgens (Figure 1.11; Charmandari et al., 2008; Charmandari et al., 2013). GC receptor sensitivity is also reduced when synthetic GCs are used to suppress allergic, inflammatory and immune disorders. Patients receiving chronic treatment often develop GC insensitivity and

resistance, increasing patient vulnerability to exaggerated inflammatory responses. GC usage is increasing as a result of disease prevalence in an aging population. For example, GCs are used in the treatment of asthma, allergic rhinitis, hematologic malignancies, ulcerative colitis, rheumatoid arthritis, eczema and psychological disorders. GC resistance can also occur as a result of chronic stress as well as in major depression (Rodriguez et al., 2016). Efforts to generate new, clinically useful, GC receptor ligands that can overcome GC resistance are in progress (Vandewalle et al., 2018).

1.5 Chapter Summary The neuroendocrine hypothalamus influences endocrine targets in tandem with the pituitary gland; that

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Central nervous system • Vasomotor symptoms • Sleep disruption • Depression and anxiety • Cognitive changes • Migraine

Sexual function • Decreased sexual desire • Dyspareunia

Urogenital system • Vaginal dryness • Vulvar itching and burning • Dysuria • Urinary frequency • Urgency • Recurrent lower urinary tract infections

Skin, mucosal and hair changes • Reduced skin thickness • Reduced elasticity • Reduced hydration • Increased wrinkling • Hair loss

Figure 1.10 Effects of estrogen depletion in menopause. Symptoms of menopause include CNS-related disorders, weight gain, bodily alterations related to cardiometabolic changes, musculoskeletal alterations, urogenital and skin atrophy and sexual dysfunction. Perimenopause is associated with the worst menopausal symptom burden, arising from neurochemical changes within the CNS leading to severe vasomotor symptoms, sleep disorders and depression, which might affect cognitive function. Reproduced with permission (Monteleone et al., 2018). Abbreviation: CNS, central nervous system.

Weight and metabolic changes • Weight gain • Increased visceral adiposity • Increased waist circumference

Musculoskeletal system • Joint pain • Sarcopenia

Figure 1.11 Changes in the HPA axis due to glucocorticoid resistance. Glucocorticoid negative feedback at the hypothalamic and anterior pituitary levels is compromised because of glucocorticoid resistance. This results in increased secretion of CRH and ACTH, followed by adrenal hyperplasia, and increased secretion of adrenal cortisol and androgens. Abbreviations: HPA, hypothalamic–pituitary–adrenal. Figure derived from Charmandari et al. (2008).

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

is, pituitary hormone secretion is directed by stimuli secreted from hypothalamic neurons. The target organ sensitivity to stimulation, and the neuronal response to hormonal feedback, is dependent upon a variety of receptor mechanisms. Peptide hormones (e.g., oxytocin), neuropeptides (e.g., thyrotropin releasing hormone) and neurotransmitters (e.g., dopamine) regulate cellular activity by binding to specific receptors located in target cell membranes. Biochemical changes within the target cell are induced via intracellular second messengers, such as cAMP and protein kinase C. The transduction of information from the membrane to the second messenger is accomplished through the activation of membrane proteins (e.g., G proteins) and enzymes, such as adenylate cyclase. GPCRs are characterized by their seven transmembrane domain structures and are attached to trimeric G proteins. There are stimulatory (Gs) and inhibitory (Gi) proteins. For example, GH secretion is inhibited by somatostatin (Gi) and stimulated by GHRH (Gs). The responsiveness of GPCRs is compromised in some neuroendocrine disorders. For example: (a) when GPCRs are subjected to chronic stimulation to produce tachyphylaxis, and (b) when mutations occur in the genes for receptor proteins or G proteins; that is, loss-of-function or gain-offunction mutations. A second type of membrane stimulation is via tyrosine kinase-dependent receptors. Peptide hormones such as leptin, PRL and GH bind to receptors having only two transmembrane domains and which activate tyrosine kinase signaling, employing the second messenger JAK/STAT pathway. The clinical consequences of abnormal receptor signaling are profound. For example, obese individuals are insensitive to their own leptin (“leptin resistance”) and high levels of endogenous leptin, derived from the increased fat mass, fail to reduce food intake or body weight. Patients with mutations in the leptin receptor are also severely obese. Loss-of-function mutations in the GH receptor result in severe growth failure and IGF-1 deficiency. GH insensitivity is also caused by mutations in the intracellular STAT5 gene. Loss-offunction mutations in the PRL receptor present with hyperprolactinemia, possibly due to the inability of PRL to exert negative feedback on pituitary lactotrophs. In contrast, steroid hormones – such as estradiol and cortisol – exert their effects via intracellular receptors that have common structural elements

and belong to a superfamily of receptor proteins. The receptor proteins have no biological activity until they bind to a hormone; that is, the hormonereceptor complex acts as a transcription factor at specific sites (HRE) on target genes. Two ERs (ERα and ERβ) are encoded by two distinct genes and are localized in many tissues, including the hypothalamus and anterior pituitary. These sites are critical for regulation of the menstrual cycle. GC receptors are also localized to the hypothalamic–pituitary system where they control ACTH secretion. The physiological effects of GC can be compromised because of mutations in the GC receptor that impair GC action. This reduces tissue sensitivity to GCs, compromises negative feedback and induces hypersecretion of ACTH, cortisol and androgens. GC resistance can also occur as a result of chronic stress. Other welldescribed examples of hormone resistance include androgen-receptor insensitivity (testicular feminization syndrome) and thyroid hormone-receptor insensitivity.

1.6 Review Questions 1. Cyclic adenosine monophosphate is a welldescribed second messenger involved in the activation of many cell types. Name two other second messenger systems. 2. Neuropeptides and neurotransmitters bind to receptors located in the cell membrane. Based on structure alone, there are two major membrane receptor types. What are they? 3. Name five hormones that act through membrane receptors. 4. Which of the following signaling molecules act via a cytosolic receptor, rather than a membrane protein? a. b. c. d. e.

Insulin-like growth factor 1 Cortisol Growth hormone (GH) Thyroxine (T4) Somatostatin

5. The target cells of a hormone such as cortisol respond to stimulation because: a. the genome contains hormone response elements b. cortisol receptors are present in the cell membrane c. only target cells contain cortisol receptors

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Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

d. cortisol receptor dimers are formed e. cortisol stimulates G protein-coupled receptors 6. “Intracellular steroid hormone receptors have no intrinsic biological activity.” Is this statement true or false? 7. The somatotroph contains three distinct signaling mechanisms that regulate GH secretion; that is, GH releasing hormone (GHRH), somatostatin and ghrelin combine to control GH release. Which G proteins are involved? 8. Loss-of-function mutations in the genes for receptor proteins reveal their importance in endocrine/neuroendocrine disorders. Which disorders are associated with mutations in the following receptors? a. b. c. d. e.

Kisspeptin receptor Thyrotropin releasing hormone receptor Melanocortin 4 receptor GHRH receptor Vasopressin receptor

9. Which of the following hormonal patterns is associated with male precocious puberty due to an activating luteinizing hormone (LH) receptor mutation? a. Low testosterone, low LH and follicle stimulating hormone (FSH) b. Normal testosterone, high LH and FSH c. High testosterone, low LH and FSH d. Low testosterone, high LH and FSH e. Normal testosterone and LH and FSH 10. An activating mutation of the thyroid stimulating hormone (TSH) receptor is associated with which of the following? a. b. c. d. e.

High TSH High T4 Goiter Features of hyperthyroidism Congenital hypothyroidism

References Barton M. (2016). Not lost in translation: emerging clinical importance of the G protein-coupled estrogen receptor GPER. Steroids 111, 37–45. Ben-Shlomo A & Melmed S. (2010). Pituitary somatostatin receptor signaling. Trends Endocr Metab 21, 123–133.

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Bernard V, Young J, Chanson P & Binart N. (2015). New insights in prolactin: pathological implications. Nat Rev Endocr 11, 265–275.

Brooks A J & Waters M J. (2010). The growth hormone receptor: mechanism of activation and clinical implications. Nat Rev Endocr 6, 515–525. Chaidarun S S, Swearingen B & Alexander JM. (1998). Differential expression of estrogen receptor-β (ERβ) in human pituitary tumors: functional interactions with ERα and a tumor-specific splice variant. J Clin Endocr Metab 83, 3308–3315. Charmandari E, Kino T, Ichijo T & Chrousos G P. (2008). Generalized glucocorticoid resistance: clinical aspects, molecular mechanisms, and implications of a rare genetic disorder. J Clin Endocr Metab 93, 1563–1572. Charmandari E, Kino T & Chrousos G P. (2013). Primary generalized familial and sporadic glucocorticoid resistance (Chrousos syndrome) and hypersensitivity. Endocr Dev 24, 67–85. Dodington D W, Desai H R & Woo M. (2018). JAK/STAT – Emerging players in metabolism. Trends Endocr Metab 29, 55–65. Engel J B & Schally A V. (2007). Drug insight: clinical use of agonists and antagonists of luteinizing-hormone-releasing hormone. Nat Clin Practice Endocr Metab 3, 157–167. Farooqi IS, Wangensteen T, Collins S et al. (2007). Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor. New Engl J Med 356, 237–247. Fukami M, Suzuki E, Igarashi M, Miyado M & Ogata T. (2018). Gain-of-function mutations in G-protein–coupled receptor genes associated with human endocrine disorders. Clin Endocr 88, 351–359. Hauser A S, Attwood M M, Rask-Andersen M, Schiöth H B & Gloriam D E. (2017). Trends in GPCR drug discovery: new agents, targets and indications. Nat Revs Drug Discovery 16, 829–842. Hiort O. (2013). Clinical and molecular aspects of androgen insensitivity. Endocr Develop 24, 33–40. Huang D, Yang F, Wang Y & Guan X. (2017). Mechanisms of resistance to selective estrogen receptor down-regulator in metastatic breast cancer. Biochim Biophys Acta 1868, 148–156. Hwa V. (2016). STAT5B deficiency: impacts on human growth and immunity. Growth Horm IGF Res 28, 16–20. Jayasena C N, Nijher G M K, Chaudhri O B et al. (2009). Subcutaneous injection of kisspeptin-54 acutely stimulates gonadotropin secretion in women with hypothalamic amenorrhea, but chronic administration causes tachyphylaxis. J Clin Endocr Metab 94, 4315–4323. Jia M, Dahlman-Wright K & Gustafsson J A. (2015). Estrogen receptor alpha and beta in health and disease. Best Prac Res Clin Endocr Metab 29, 557–568. Lania A G, Mantovani G & Spada A. (2006). Mechanisms of disease: mutations of G proteins and G-protein-coupled receptors in endocrine diseases. Nat Clin Practice Endocr Metab 2, 681–693.

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Manoranjan B, Salehi F, Scheithauer B W, Rotondo F, Kovacs K & Cusimano M D. (2010). Estrogen receptors alpha and beta immunohistochemical expression: clinicopathological correlations in pituitary adenomas. Anticancer Res 30, 2897–2904. McEwan I J & Brinkmann A O. (2016). Androgen physiology: Receptor and metabolic disorders. In: Endocrinology of Male Reproduction; Simoni M & Huhtaniemi I T, Eds. Berlin: Springer. www.Endotext.org. Monteleone P, Mascagni G, Giannini A, Genazzani A R & Simoncini T. (2018). Symptoms of menopause – global prevalence, physiology and implications. Nat Rev Endocr 14, 199–215. Myers Jr. M G, Leibel R L, Seeley R J & Schwartz M W. (2010). Obesity and leptin resistance: distinguishing cause from effect. Trends Endocr Metab 21, 643–651. Neumann E, Khawaja K & Müller-Ladner U. (2014). G protein-coupled receptors in rheumatology. Nat Rev Rheumatol 10, 429–436. Ortiga-Carvalho T M, Sidhaye A R & Wondisford F E. (2014). Thyroid hormone receptors and resistance to thyroid hormone disorders. Nat Rev Endocr 10, 582–591.

Rodriguez J M, Monsalves-Alvarez M, Henriquez S, Llanos M N & Troncoso R. (2016). Glucocorticoid resistance in chronic diseases. Steroids 115, 182–192. Rosenfeld R G, Belgorosky A, Camacho-Hubner C, Savage M O, Wit J M & Hwa V. (2007). Defects in growth hormone receptor signaling. Trends Endocr Metab 18, 134–140. Simmen R C M & Kelley A S. (2016). Reversal of fortune: estrogen receptor-beta in endometriosis. J Mol Endocr 57, F23–F27. Southworth M B, Matsumoto A M, Gross K M, Soules M R & Bremner W J. (1991). The importance of signal pattern in the transmission of endocrine information: pituitary gonadotropin responses to continuous and pulsatile gonadotropin-releasing hormone. J Clin Endocr Metab 72, 1286–1289. Vandewalle J, Luypaert A, De Bosscher K & Libert C. (2018). Therapeutic mechanisms of glucocorticoids. Trends Endocr Metab 29, 42–54. Vassart G & Costagliola S. (2011). G protein-coupled receptors: mutations and endocrine diseases. Nat Rev Endocr 7, 362–372.

Paterni I, Granchi C, Katzenellenbogen J A & Minutolo F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids 90, 13–29.

Warner M, Huang B & Gustafsson J-A. (2017). Estrogen receptor β as a pharmaceutical target. Trends Pharmacol Sci 38, 92–99.

Prossnitz ER & Barton M. (2011). The G-protein-coupled estrogen receptor GPER in health and disease. Nat Rev Endocr 7, 715–726.

Wettschureck N & and Offermanns S. (2005). Mammalian G proteins and their cell type specific functions. Physiol Rev 85, 1159–1204.

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Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

The clinical investigation of neuroendocrine problems is dependent on the quantitative assay of hormone levels, for example, by immunoassay or mass spectrometry. Blood samples are the primary source for quantification of hormone concentrations, although saliva and hair samples can also provide valuable information. Automated blood collection and automated assays permit the determination of circadian and rapid, pulsatile changes in, for example, pituitary hormone secretion. This chapter will outline the methodology and application of these assay techniques.

2.1 Determination of Hormone Levels The levels of circulating hormones can be determined directly in blood samples or, in the case of steroid hormones such as cortisol, in the saliva or hair. The determination of glucocorticoid (GC) levels in hair, for example, is able to detect prolonged exposure to stress (Wester and van Rossum, 2015). This section will include two routine techniques for the determination of hormone levels: (a) immunoassay and (b) mass spectrometry.

Immunoassays

14

Since the 1960s the benchmark in determination of hormone levels was the radioimmunoassay. However, this method, employing antibodies specific to each hormone, and radioactively labeled hormones, is slow, labor intensive and raises safety problems in the use and disposal of radioactive materials. The ready availability of highly specific monoclonal antibodies for steroid and peptide hormones aided the development of rapid and automated chemiluminescent or immunometric assays that produce data in a matter of hours, rather than days. A widely used assay is the ELISA. One form of this technique (there are several variations; Aydin, 2015) for the quantification of cortisol levels in blood is illustrated in

Figure 2.1. To perform this assay, a specific cortisol monoclonal antibody (the “capture” antibody) is permanently attached to the bottom of a plastic microwell plate well (Step A). Cortisol samples are then added and allowed to complex with the immobilized antibody (Step B). Unbound products are then removed with a wash step, leaving cortisol bound to the antibodies. A biotin-labeled second antibody, also specific for cortisol (the “detection” antibody), is then added to the wells, binding to the captured cortisol (Step C). After a further wash step, the biotinylated detection antibody is itself now labeled with an enzyme conjugate capable of generating light or fluorescence in response to a particular substrate (Step D). The quantification step measures the intensity of light produced after the addition of the substrate (Step E). Standard curves are generated by adding known quantities of cortisol, and these curves then permit determination of unknowns in the blood samples. The availability of monoclonal antibodies for many hormones and peptides is a major advantage of this method. In general, the samples do not need to be purified prior to use, and the whole process can be automated to handle large numbers of samples. Typical uses of this technique are: (a) estimation of pulsatile secretion of human adrenocorticotropin (ACTH) and cortisol (Henley et al., 2009; cf. Chapter 5, Figure 5.3), and (b) the determination of salivary cortisol (Langelaan et al., 2018). A disadvantage of this technique is the inability to assay more than one hormone in a single clinical patient sample. Complex pathologies may require determination of a panel of hormones for diagnosis or prognosis. Automated multiplex immunoassay technologies are increasingly available, and Figure 2.2 outlines just such a method for the simultaneous assay of insulin, growth hormone (GH), leptin, thyroid stimulating hormone (TSH) and glucagon-like peptide-1 (GLP-1) (Stephen and Guest, 2017).

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Addition of enzyme complex Biotin-labeled detection antibody

Cortisol-specific monoclonal antibody

A

Addition of chemiluminescent substrate Chemiluminescent substrate

HRP conjugate

Light

B

B

B

C

D

E

Cortisol binding

B

Figure 2.1 Assay of cortisol using ELISA. Each well of a microwell plate has been pre-coated with a cortisol-specific monoclonal antibody (capture antibody) (A). Standard amounts of cortisol, or samples, are added to the wells and cortisol binds to the capture antibody (B). Unbound standard or sample is washed away. A biotin-conjugated, cortisol-specific, detection antibody is then added, which binds to the captured cortisol (C). Unbound detection antibody is washed away. An avidin–HRP conjugate is then added, which labels the biotin (D). Unbound avidin–HRP conjugate is washed away. Addition of a chemiluminescent substrate causes the HRP enzyme to induce light emission (E). The light units of each well are automatically measured. Light units from unknown samples can then be compared with a light unit standard curve generated using known cortisol concentrations in order to determine serum cortisol concentrations. See www.lsbio.com/pr oducts/elisakits/clia as a typical example of this technique. Abbreviations: HRP, horseradish peroxidase.

Specific antibodies are covalently attached to distinct, dye-coded microspheres and added to the hormone sample. Each hormone binds to the appropriate antibody (simplified here by only showing the sequence for GH). As in Figure 2.1, the hormonebound antibody is further decorated with a fluorescent biotin conjugate. The complete mixture – microspheres, hormones and decorated antibodies – is separated into antibody-specific groups by flow cytometry and then analyzed by lasers able to quantify each dyecoded microsphere as well as the fluorescence of the bound hormone. This fully automated technique enables much information to be derived from a single sample, with minimal reagent usage, at lower cost compared with the assay shown in Figure 2.1.

Mass Spectrometry A second powerful technique to quantify multiple hormones in single samples of biological fluids is mass spectrometry. The biggest advantage it possesses over the immunoassays is that it does not require antibodies to assay the hormones of interest. This is not to say that the ELISA is not useful; there are many hormones for which highly specific antibodies are available and therefore can be quantified with ELISAs. However, even the most specific antibodies

may still cross-react with other hormones and this lack of specificity produces misleading results. In contrast, the mass spectrometer can determine absolute values of many substances in blood, with high specificity, based on differences in molecular weight. High throughput testing is possible, using small sample volumes with minimal sample preparation, and eliminating the requirement for expensive immunoassay-specific reagents such as antibodies. For example, when coupled with liquid chromatography to separate sample components, mass spectrometry is an efficient and rapid way to assay steroid hormones in a clinical setting (Matysik and Liebisch, 2017). This technique may also be the only way to determine very low levels of hormones in blood; for example, estradiol levels in prepubertal children (Stanczyk and Clarke, 2014) or low levels of cortisol in saliva (Sturmer et al., 2018). The collection of biological samples such as saliva, hair and urine for the analysis of hormone levels is simple, non-invasive and stress-free compared with taking blood samples. Hormone levels can therefore be determined in newborn children, as well as adults, outside the laboratory or hospital setting, and mass spectrometry permits the analysis of multiple hormones simultaneously in the same sample. Also,

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Figure 2.2 Overview of the multiplex immunoassay protocol. Samples are added to dye-coded microsphere–antibody complexes that capture specific hormone targets. The capture of GH is shown in this sequence. Following incubation with a fluorescent biotinylated label, the mixtures are streamed through the detector – via flow cytometry to separate the individual groups of microspheres – which uses lasers for identification of the antibody–microsphere conjugates and quantitation of the fluorescent bound hormones. The example shows a 5-plex assay capable of binding the targets GLP-1, GH, insulin, leptin and TSH. This figure is based on the illustration from Stephen et al. (2017).

since cortisol is deposited in the growing hair shaft, an estimate of cortisol concentrations in hair serves as a measure of chronic exposure over weeks and months (Meyer and Novak, 2012; Wester and van Rossum, 2015). Nonetheless, it is important to remember that hair, saliva and urine analysis only provides a measure of the average hormone secretion over a number of hours or weeks. In the case of cortisol, for example, blood samples taken every few minutes is the only way to demonstrate pulsatile secretion and its circadian variation (see Figure 5.3 showing pulsatile secretion of ACTH and cortisol). Table 2.1 summarizes the variations in hair cortisol levels associated with some clinical situations (Wester and van Rossum, 2015).

2.2 Patterns of Hormone Secretion 16

The neuroendocrine system is defined by rhythms that orchestrate the dialogue between the brain and

critical systems such as regulation of growth, reproduction and stress. Some of these rhythms have periods longer than 24 hours, typified by the female menstrual cycle. Circadian or 24-hour rhythms include the sleep–wake cycle and the increase, for example, in GH secretion seen at night. There are also cycles of less than 24 hours – the ultradian cycles – such as the pulsatile release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and GH. Section 2.1 outlined the analytical approaches used to assess hormone levels in biological fluids. Since hormone levels often vary dramatically through 24 hours, it is critical to determine hormone secretory patterns in normal individuals to inform when hormone samples should be obtained in clinical practice. The following sections outline the clinical importance of normal hormonal rhythms.

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Table 2.1 Clinical and situational factors associated with variations in hair cortisol levels

Increased hair cortisol

Decreased hair cortisol

Cushing’s syndrome

Childhood asthma with inhalation glucocorticoids

Somatic health factors Hydrocortisone use Obesity Metabolic syndrome Diabetes mellitus Cardiovascular disease Heart failure severity Recent myocardial infarction Chronic and acute stressors Intensive aerobic exercise

Traumatic experience

Trauma Life events Unemployment Shift work Severe chronic pain Psychopathology

a

PTSDa

PTSDa

Major depressive disorder

Generalized anxiety disorder

Bipolar disorder, late onset

Panic disorder

Post-traumatic stress disorder (PTSD) has been associated with both increased and decreased hair cortisol concentrations (depending on the type of traumatic event, characteristics of the patient sample examined, and the timespan between the trauma and assessment) compared with controls (Wester and van Rossum, 2015).

Daily Rhythms As an example of a well-described daily rhythm, Figure 2.3 illustrates that pulsatile cortisol levels in healthy individuals vary with a 24-hour period, with blood levels beginning to rise late in the night to peak at the sleep–wake transition (cf. Chapter 5; Figure 5.3). The figure also reveals that in patients with pituitarydependent Cushing’s disease, although the frequency of cortisol pulses is increased, producing significant cortisol excess, the diurnal pattern of cortisol output is absent (van den Berg et al., 1995). These data indicate dysfunction of the hypothalamic–pituitary system in Cushing’s disease. These rhythms may also be disrupted in other disease states. For example, psychotic disorders such as major depression are associated with changes in circadian rhythms similar to Cushing’s syndrome (Keller et al., 2006). Levels of several other circulating endocrine factors are known to oscillate through 24 hours,

and peak secretion for these is illustrated in Figure 2.4. The secretory rhythms of some hormones (e.g., cortisol and TSH) occur independently of sleepwake cycles. In a study on healthy young men the cortisol profile was minimally affected by the absence of sleep or when they slept at an abnormal time of day. Over a period of 28 hours of continuous wakefulness, constant light and constant caloric intake, the normal wave shape of the rhythm of cortisol release was maintained (Oster et al., 2017). This finding indicates that fluctuations in the levels of some hormones, over the course of the day, are driven, at least in part, by an endogenous circadian mechanism (i.e., independent of behavioral rhythms). In contrast, 24-hour oscillations in the levels of other endocrine factors (e.g., GH and prolactin) are dependent on sleep–wake cycles. For example, sleep deprivation abolishes GH secretion, but daytime sleep, several hours later, restores the sleep-related surge of GH

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Cortisol (nmol/L)

1500

Figure 2.3 Diurnal rhythm of human cortisol secretion: control vs. Cushing’s disease. Circulating cortisol concentrations in a control male (lower profile) and a male patient with Cushing’s disease (upper profile). Note that both profiles demonstrate the presence of pulsatile secretion of cortisol, but the 24-hour variation is absent from the Cushing’s patient. Data obtained from van den Berg et al. (1995).

Cushing’s disease

1000 800 Lights off

600 400 200

Control

0 0900 1200 1500 1800 2100 2400 0300 0600 0900 Clock time

Figure 2.4 Peak variations in circulating endocrine factors. The time of day at which circulating levels of key endocrine factors peak in humans. Abbreviations: FGF 21, fibroblast growth factor 21; RAAS, renin–angiotensin– aldosterone system; T3, triiodothyronine. Image reproduced with permission (Gamble et al., 2014).

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(van Cauter et al., 1998; cf. Figure 8.2). Seminal studies in experimental animals revealed that a specific structure in the hypothalamus – the suprachiasmatic nucleus (SCN) – is the site of the clock mechanism that governs circadian variations in hormone release, as well as a variety of physiological functions. Most peripheral tissues, throughout the body – including

adrenal, liver and adipose tissue – also contain secondary clock mechanisms that synchronize with the SCN master oscillator (Figure 2.5; reviewed in Oster et al., 2017). The molecular circuitry that governs the production of rhythms, perhaps common to all these oscillators, is represented schematically in Figure 2.6. In its

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Pineal gland

Plasma melatonin

SCN

Plasma GCs

sleep

0 12 24 Time of day (h) sleep

0 12 24 Time of day (h)

Figure 2.5 Schematic representation of synchronization of SCN clock with peripheral oscillators. Both neural signals (transmitted by the autonomic nervous system; black arrows) and hormonal signals are involved. The 24-hour rhythms of circulating melatonin (released by the pineal gland) and cortisol (from the adrenal cortex) are considered as primarily controlled by the central SCN clock. The blue and purple arrows symbolize, respectively, the synchronizing effects of the GC and melatonin rhythms. Due to the ubiquity of glucocorticoid receptors in the entire organism, the 24-hour rhythm of circulating GCs plays a major role in synchronizing central and peripheral clocks. Reproduced through open access (Oster et al., 2017).

Adrenal gland

RHYTHMIC SIGNALS

BMAL1 CLOCK

+

clock

bmal1

bmal1 mRNA clock mRNA

per

cry

cry mRNA

NUCLEUS

per mRNA

– CRY SLEEP, CIRCADIAN INFORMATION HORMONES, STRESS, etc.

Figure 2.6 Schematic outline of how clock gene expression in the SCN regulates circadian rhythms. Positive and negative gene expression loops produce output signals responsible for rhythmic cell function over 24 hours. The clock genes bmal1 and clock encode the proteins BMAL1 and CLOCK that form a heterodimer transcription factor that stimulates the per and cry genes. In turn, their protein products, CRY and PER, reach a critical level before negatively regulating the activity of bmal1 and clock, thereby reducing the amount of CLOCK and BMAL1. Image of DNA structure reproduced with permission: https://com mons.wikimedia.org/w/index .php?title=File:DNA_structur e_and_bases_color_FR.svg &oldid=124826534.

PER CYTOPLASM

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

simplest form, neurons in the SCN begin to produce two proteins at the start of the circadian day. These proteins – BMAL1 and CLOCK – form a heterodimer that acts as a transcription factor able to stimulate expression of two further genes – per and cry – that encode the proteins PER and CRY. The buildup of PER and CRY over the course of the day establishes an autoregulatory feedback loop that represses the clock and bmal1 genes, reducing the levels of CLOCK and BMAL1. This feedback cycle takes approximately 24 hours, driving the circadian rhythm. For example, the circadian signal necessary for the production of nighttime cortisol (Figure 2.3) would be an increase in corticotropin releasing hormone (CRH) neuron activity, increasing CRH secretion into the pituitary portal system (see Figure 5.2). Current evidence suggests that the human circadian system also uses clock genes. The human SCN expresses the clock gene (Steeves et al., 1999), and circadian variations in clock mRNA and bmal1 mRNA have been detected in human placenta (Pérez et al., 2015). A clock/bmal1 mechanism may therefore be responsible for human circadian rhythms, including variable adrenal sensitivity to stimulation by ACTH (Angelousi et al., 2018). In addition, polymorphisms in clock genes are implicated in the susceptibility to obesity (Gómez-Abellán et al., 2008; Sookoian et al., 2008). For example, per2 expression in adipose tissue was negatively correlated with waist

circumference in morbidly obese patients (GómezAbellán et al., 2008), and a clock polymorphism (in white blood samples) was associated with a 1.8-fold risk of obesity (Sookoian et al., 2008).

Neuroendocrine Consequences of Circadian Disruption Human circadian rhythms are especially sensitive to multiple adverse influences; for example, shift work, artificial lighting at night and long-distance flights across time zones (jet lag). The evidence is clear that chronic perturbation of circadian function provokes metabolic, reproductive, sleep and mood disorders (Sheikh-Ali and Maharaj, 2014; Bedrosian et al., 2016; Nicolaides et al., 2017). Some of these issues are summarized in Table 2.2 (Potter et al., 2016). The light-dependent rhythm of melatonin secretion from the pineal gland (see Figure 2.5) is a sensitive indicator of circadian disruption. For example, low light levels in the early night phase are sufficient to delay the melatonin rhythm, and the use of a computer or e-book before bed depresses melatonin secretion and may disrupt sleep (Bedrosian et al., 2016; Potter et al., 2016). Night-shift workers are exposed to light levels that far exceed those that affect the circadian system. Several studies of shift workers indicate that exposure to nighttime light leads to metabolic problems. For example, healthcare

Table 2.2 Human circadian rhythm disruption

Mechanism

Source Behavioral

20

Biological

Light cycle

Meal/fasting cycle disruption

Rest/activity disruption

Genetic disruption (e.g., clock gene mutations)

Physiological disruption (e.g., retinal dysfunction)

Work schedules (e.g., shift work)

✓

✓

✓

✗

✗

Jet lag

✓

✓

✓

✗

✗

Unusual photoperiods (e.g., polar regions)

✓

✗

✗

✗

✗

Circadian rhythm sleep/wake disorders (e.g., non-24-hour sleep/wake disorder)

✓

✗

✗

✓

✓

Aging

✗

✗

✗

✗

✓

Disease states (e.g., Alzheimer’s)

✗

✗

✗

✓

✓

Data derived from Potter et al. (2016)

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Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

US light pollution

US obesity trends

mid-1970s

Figure 2.7 Exposure to nighttime light parallels incidence of obesity. The left panel shows light pollution trends in the United States from the mid1970s through projected levels in 2025. The right panel shows obesity trends in the United States from 1990, 2000 and 2010. Reproduced with permission (Fonken and Nelson, 2014). Abbreviation: BMI, body mass index.

1990

1977 2000

2025 35% rise in ACTH after CRH or VP. is suggestive of an ACTH-producing pituitary adenoma (Cushing’s disease)

• Dopamine agonists (e.g., cabergoline) or somatostatin analogs, such as paseriotide, are used for the management of Cushing’s disease • Acute morphine

Thyroid stimulating hormone (TSH)

• Acute morphine

• Somatostatin analogs bind to somatostatin receptors in pituitary adenomas; therapy is used for management of TSH-producing pituitary adenoma

and magnitude of GnRH pulses, a product of the socalled neural pulse generator (Krsmanovic et al., 2009). As an example, Figure 3.4 illustrates a normal pattern of episodic LH release in a healthy male, compared to the apulsatile pattern seen in a typical Kallmann syndrome (KS) patient who is infertile, lacking GnRH/LH secretion and testosterone (see later). LH is also secreted in a pulsatile fashion throughout the normal menstrual cycle (see Figure 3.1). The frequency and magnitude of the pulses varies according to feedback by E2 and progesterone (Filicori et al., 1986). For example, high frequency, high magnitude pulses drive the ovulatory surge of LH at midcycle (Figure 3.5A). Contrast this pattern with the low-amplitude and lower-frequency pulses seen during menstruation, following the LH surge, when the peak values of LH have declined into the

normal range (Figures 3.5A and 3.5B). Consequently, serum levels can vary widely during different parts of the menstrual cycle. The critical importance of LH pulses for fertility is illustrated in patients suffering significant weight loss. For example, when LH pulsatile secretion is inhibited by loss of body weight, as in patients with anorexia nervosa or in those engaged in strenuous exercise, menstrual cyclicity is abolished (Figure 3.6). This figure illustrates that the process of regaining body weight is sufficient to restore pulsatile secretion of LH and menstruation.

Development of Pulsatile Secretion GnRH neurons are functional in the human fetus and in early infancy, producing gonadotropin secretion that stimulates the ovary and testis to release E2 and

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Blood levels of LH (mIU/ml)

5

Figure 3.4 Pulsatile secretion of LH in a normal male. Blood levels of LH (mIU per ml; upper trace) measured during a 24 hr period in a 36-year-old adult male with normal levels of testosterone. * Indicates significant pulses above the background. Pulses occur approximately every 2–3 hr. The lower trace illustrates the attenuated, non-pulsatile, secretion of LH in a KS patient. Reproduced with permission and redrawn (Besser and Thorner, 2002).

*

4

*

Normal male

*

*

*

3

2

* *

*

* *

*

1 Kallmann syndrome patient

0 0

6

12

18

24

Hours

* 150

A

* **

* LH (mIU/ml)

100

*

* *

* *

* *

**

**

*

* *

50

0

LH (mIU/ml)

1725 40 30 20

2125

0125 0525 0925 Clock time

1325

*

B

*

*

*

1725

* *

*

* *

*

10 0 1725

2125

0125 0525 0925 1325 Clock time

1725

Figure 3.5 Pulsatile secretion of LH in the menstrual cycle. The frequency and magnitude of LH pulses varies according to feedback by E2 and progesterone. In A, high-frequency, highmagnitude pulses drive the ovulatory surge of LH that induces ovulation at midcycle. In B, note the low-amplitude and lowerfrequency pulses seen during menstruation, following the LH surge, when the peak values of LH have declined into the normal range. * Indicate significant pulses of LH above the background. Redrawn from data in Filicori et al. (1986).

testosterone, respectively (Grumbach, 2002). This activity is gradually attenuated through childhood (approx. ages five to eleven years) until it is reinstated at the time of puberty. The onset of the pubertal process is signaled by a remarkable increase in sleeprelated pulsatile LH release (Figure 3.7). This occurs in boys and girls (Boyar et al. 1974; Kapen et al., 1974) with the pulses being correlated with slow wave sleep (Shaw et al., 2012). In post-pubertal adults, LH pulses occur throughout 24 hrs. The transition from childhood quiescence to sleepinduced LH secretion at puberty represents a reactivation of the pulse generator. This probably occurs as a result of two processes occurring simultaneously: (a) the lifting of a hypothalamic neurochemical inhibition and (b) the activation of an excitatory drive to release GnRH (McCarthy, 2013). The latter may involve kisspeptin (KP) neurons (see Section 3.4).

Why Are Pulses Necessary to Induce Puberty and Sustain Fertility? An answer to this question became clear from classical experiments in monkeys. Continuous infusion of GnRH was observed to induce a paradoxical suppression of LH and FSH secretion (Belchetz et al., 1978; Figure 3.8). Restoration of a pulsatile stimulation with GnRH restored LH and FSH levels. These data, and others, suggested that there is an optimal frequency of GnRH stimulation that maintains LH and FSH at levels consistent with fertility. Continuous stimulation of GnRH receptors induces a state of receptor downregulation, reversible by

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33

Clinical remission

LH (mIU/ml)

16 14 12 10 8 6 4 0

INCREASE IN BODY WEIGHT (+10.5 kg)

LH (mIU/ml)

Anorexia nervosa

LH (mIU/ml)

LH (mIU/ml)

Chapter 3: Neuroendocrinology of Female Reproduction

6 4 2 0 2200

0200

0600

1000

1400

1800

2200

Clock time

4 2 0 2200

18 16 14 12 10 8 6 4 2 0 2200

SLEEP 0200

0600

1000 1400 Clock time

1800

2200

EARLY PUBERTAL

SLEEP 0200

0600

41 37 LH (mIU/ml)

Figure 3.6 LH pulses in anorexia nervosa and following weight gain. The lower graph shows a typical non-pulsatile pattern of LH secretion over 24 hr in a female patient (22 years) with low body weight due to anorexia nervosa. Such a pattern of LH secretion causes amenorrhea. The upper graph shows the dramatic effect of restoration of normal body weight. LH pulses are now greatly increased in frequency and magnitude. Treatment with clomiphene restored menstruation. Graphs based on data from Boyar et al. (1974).

PREPUBERTAL

6

1000 1400 Clock time

1800

2200

LATE PUBERTAL

29 21 13

applying pulses of GnRH. A similar phenomenon is also observed in humans (Rabin and McNeil, 1980; Southworth et al., 1991). The clinical implications arising from these studies are two-fold: (a) pulsatile stimulation of pituitary secretion of LH and FSH is mandatory for restoration of gonadotropin secretion in the treatment of, for example, hypogonadotropic hypogonadism (see in Section 3.3.1, “A Case of Delayed Puberty”); (b) the ability of continuous stimulation with GnRH to suppress fertility is clinically advantageous in various ways. For example, synthetic, long-acting GnRH agonists are very effective in reducing gonadotropin and sex hormone levels in advanced prostate cancer, treatment of estrogensensitive breast cancer, precocious puberty and in in vitro fertilization (IVF) protocols (Engel and Schally, 2007).

Absence of Pulsatile LH Secretion: Kallmann Syndrome

34

Hypothalamic amenorrhea (HA) is characterized by the absence of menstrual cyclicity and the loss of ovarian function due in large part to the lack of pulsatile GnRH secretion. Several extraneous factors, such as excessive exercise, loss of body weight and psychological stress, contribute to this reversible condition (Liu et al., 2016).

5 2200

SLEEP 0200

0600

1000 1400 Clock time

1800

2200

Figure 3.7 Appearance of sleep-induced pulsatile secretion of LH at puberty. The onset of the pubertal process is signaled by a remarkable increase in sleep-related pulsatile LH release. This occurs in boys and girls and the pulses are correlated with slow wave sleep. Data from three examples are shown here: a prepubertal girl, a normal pubertal girl and a girl in late puberty. Note the emergence of sleep-related LH pulses during puberty and the appearance of additional LH pulses during the day in late puberty. Graphs are based on data from Boyar et al. (1974).

Although certain genetic and developmental defects can lead to absence of GnRH/LH secretion, there is generally no obvious neuroanatomic abnormality – other than atypical GnRH/LH secretion – in most cases of HA. Isolated GnRH deficiency (IGD) typically induces hypogonadotropic hypogonadism (Stamou et al., 2015), which is characterized by low LH and FSH levels leading to low E2 or testosterone production. Consequently, congenital or very early-onset GnRH deficiency, typically leads to incomplete sexual maturation. Similarly, lack of pulsatile GnRH secretion, which is crucial for the appropriate release of LH and FSH (see e.g., Figure 3.4), impairs gonadal sex steroid synthesis and

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Chapter 3: Neuroendocrinology of Female Reproduction

25

200 PULSATILE GnRH

CONTINUOUS GnRH

PULSATILE GnRH

20

150

15 100 10

FSH

FSH (ng/ml)

LH (ng/ml)

LH

50 5

0

0 –15 –10 –5

0

5 10 15 20 25 30 35 Time (days)

Figure 3.8 Non-pulsatile, continuous stimulation with GnRH suppresses LH and FSH secretion from the anterior pituitary. The figure compares the pituitary response to continuous vs. pulsatile stimulation with GnRH. The switch from pulsatile to continuous infusion of GnRH markedly reduces LH and FSH secretion. This occurs because the GnRH receptors become overstimulated, causing desensitization to further treatment with GnRH. However, restoration of a pulsatile pattern gradually, over several days, restores normal secretion. Reproduced with permission (Besser and Thorner, 2002).

gametogenesis, culminating in delayed puberty and infertility. IGD has an incidence of around 1 in 4,000 males, and occurs three to five times more commonly in men than in women (Boehm et al., 2015). KS accounts for approximately 50–60% of all cases and will be used here as an exemplar of IGD (Stamou and Georgopoulos, 2017). KS patients present with associated anosmia, a partial or total lack of the sense of smell. Investigations into the association of anosmia with the failure of GnRH neurons to support fertility led to our current understanding of the embryonic origin of GnRH neurons; that is, GnRH neuronal precursor cells originate embryonically in the nasal placode and migrate through the cribriform plate into the brain, and thence to the hypothalamus, via guidance from olfactory axons (Figure 3.9A; Stamou et al., 2015). Several genetic mutations result in KS by preventing the migration of GnRH neurons to the hypothalamus (Figure 3.9B). These mutations may also cause failure of development of the olfactory bulb, giving rise to anosmia (Bianco and Kaiser, 2009).

Although several mutations have been associated with KS (Balasubramanian et al., 2010; Valdes-Socin et al., 2014), genetic abnormalities in three genes – KAL1, FGFR1 and PROKR2 – are particularly well described. KAL1 encodes a protein, anosmin 1, which acts as a cell-adhesion molecule essential for migration of GnRH and olfactory neurons. FGFR1 encodes a receptor, FGFR1, which binds a fibroblast growth factor (possibly FGF8) that is also responsible for neuron migration and development of the olfactory bulb. PROKR2 encodes a receptor protein that binds prokineticin-2, which is involved in the maturation of GnRH precursors and development of the olfactory system (shown in Figure 3.9A). Mutations in these genes impair the development and migration of GnRH neurons (Figure 3.9B) and loss of olfactory neurons, leading to impairment of the sense of smell. In all, mutations in the three genes mentioned here account for only approximately 25% of KS cases, suggesting the involvement of other genes (Bianco and Kaiser, 2009).

3.3.1 A Case of Delayed Puberty An 18-year-old Caucasian male was referred to the endocrinology clinic for delayed puberty. He was born through normal vaginal delivery and had been healthy in the past apart from a knee injury while skating that required an arthroscopy when he was 15. A lack of pubertal development was first noticed by the parents when his younger brother (who was 14) began growing facial hair. On examination, he appeared younger than his stated age and had a high-pitched voice. He was 174 cm tall (mid-parental height = 176 cm) and weighed 59.7 kg. There was no evidence of facial hair and he had sparse axillary and pubic hair (Tanner stage 2). The arm span was 178 cm with an increased limb to trunk span of >2 cm; he had prepubertal male genitalia with testicular volume of around 3 ml bilaterally and micropenis (penile length of 5 cm). He also had anosmia which was confirmed by using serial dilutions of multiple odorants. Lab results were: Total Testosterone = 2.7 nM (8.4–28.7 nM) Serum LH = 1.1 IU/L (1.5–9.3 IU/L) Serum FSH = 1.6 IU/L (1.4–18.1 IU/L) Based on the clinical and biochemical features, a diagnosis of KS was made, which was subsequently confirmed through a genetic test revealing a mutation in the KAL1 gene.

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Chapter 3: Neuroendocrinology of Female Reproduction

A

Olfactory bulb

FGFR1 KP neurons GnRH neurons

Median eminence

KAL1

GnRH

Cribriform plate

Pituitary gland

Olfactory axons PROKR2 LH/FSH Nasal placode

GnRH neuron precursor cells

B

Olfactory bulb

X

FGFR1 xons

tory a

Olfac

KP neurons

Median eminence

X

KAL1

PROKR2 X

X

Cribriform plate

Olfactory axons

GnRH absent

Pituitary gland

X

LH/FSH LH/FSH absent

GnRH neuron precursor cells Figure 3.9 Neurodevelopmental and neuroendocrine regulation of GnRH neurons. GnRH neuronal precursor cells originate embryonically, in the first weeks of fetal life, in the nasal placode and migrate through the cribriform plate and across the olfactory bulb (A). They reach the infundibular nucleus and preoptic area, via guidance from olfactory axons, and synchronize with other neurons, such as KP neurons, to regulate GnRH secretion (see also Figure 3.3). Mutations in several genes – including KAL1, PROKR2 and FGFR1 – prevent the migration of GnRH neurons and eliminate LH and FSH secretion (B). These mutations may also cause failure of olfactory bulb development, giving rise to anosmia. Reproduced and redrawn with the generous permission of Dr. F. Crowley, Jr. (Stamou et al., 2015). Abbreviations: GnRH, gonadotropin releasing hormone; KP, kisspeptin.

36

The primary goal of management of KS in male patients is to restore serum testosterone levels into the normal range, thereby establishing secondary sexual characteristics and sexual function. This can be achieved by initiating testosterone therapy, either as long-acting testosterone injection, such as testosterone enanthate and testosterone cypionate, or as

a topical preparation such as Androderm, Androgel and Testim. In order to avoid a vigorous rise in testosterone level in previously hypogonadal adolescent males, it is generally advisable to start testosterone therapy at a low dose and gradually increase to a full dose over several months to avoid a too-rapid induction of puberty with concomitant changes in mood

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Chapter 3: Neuroendocrinology of Female Reproduction

and sleep. However, testosterone therapy alone does not improve fertility, but rather suppresses LH and FSH through its negative feedback effect on GnRH. In order to induce fertility and stimulate spermatogenesis, patients with KS will typically require either a combination of human chorionic gonadotropin (hCG; which acts like LH to stimulate testosterone production by Leydig cells) and recombinant FSH (such as Gonal F to stimulate spermatogenesis) or pulsatile GnRH therapy via a subcutaneous pump. Female KS patients also lack gonadotropins, and therefore ovarian function, and require transdermal treatment with E2 to maximize breast development, and an E2/progestin regimen to induce menstrual bleeding. Such treatment does not induce ovulation and fertility and female patients seeking fertility can be treated with GnRH pump therapy to achieve pulsatile secretion of LH/FSH and ovulation (Bianco and Kaiser, 2009).

3.4 Neurochemical Regulation of GnRH Secretion GnRH neurons are sensitive to a variety of exogenous and endogenous neurochemical stimuli (see Section 3.2; Table 3.1). Detailed neuroanatomical studies on human hypothalamic tissue have revealed close apposition of various nerve terminals with GnRH neuron axons and dendrites, which further implicates endogenous neural control of GnRH release. These include investigation of classical neurotransmitters (e.g., norepinephrine; Dudas and Merchenthaler, 2001), the excitatory neurotransmitter glutamate and the inhibitory amino acid γamino butyric acid (Hrabovszky et al., 2012). Several neuropeptides have also been identified: the opioid peptides β-endorphin (Dudas and Merchenthaler, 2004) and leu-enkephalin (Dudas and Merchenthaler, 2006); orexin and melanin-concentrating hormone (Skrapits et al., 2015); substance P and neurokinin B (NKB; Borsay et al., 2014); neuropeptideY, galanin and corticotropin releasing hormone (Dudas and Merchenthaler, 2004) and the excitatory neuropeptide KP (Hrabovszky, 2014). The following sections will use two neuropeptides as examples of how GnRH secretion can be inhibited or stimulated. The first will outline the extensive clinical importance of opioids in inhibiting LH secretion and fertility. We will then describe the detailed studies on KP and its ability to stimulate the human reproductive system. KP is the most potent GnRH secretagogue known.

Inhibitory Effects of Opioids on the Human Hypothalamic–Pituitary–Gonadal System Opioid peptides (e.g., β-endorphin) and non-peptides (e.g., morphine) are potent inhibitors of gonadotropin secretion, consistent with the presence of opioid receptors in the human hypothalamus. Opioid receptors are G protein-coupled (see Chapter 1) and they exist as three major subtypes: μR, δR and κR. Gene expression studies revealed μR messenger ribonucleic acid (mRNA) and κR mRNA, but not δR mRNA, localized to large numbers of human hypothalamic neurons, indicating that these cells biosynthesize the μR and κR receptor proteins (Peckys and Landwehrmeyer, 1999). There is good evidence from animal studies that injections of morphine can inhibit GnRH and LH secretion via hypothalamic μ-opiate receptors (Ferin et al., 1982; Williams et al., 1990). It is assumed that drugs such as morphine and βendorphin bind to human hypothalamic μR to inhibit LH secretion (Reid et al., 1981; Rasmussen et al., 1989). The human reproductive system is also regulated by endogenous opioid peptides (EOPs); that is, peptides biosynthesized in hypothalamic neurons. One of these, β-endorphin, has been referred to previously but several families of EOP are known (Table 3.2; Wilkinson and Brown, 2015). Each precursor peptide (a propeptide) is derived from a different gene; that is, pro-opiomelanocortin is encoded by the pomc gene, proenkephalins from the enkA gene and prodynorphin is derived from the proenkephalin B gene (enkB; Figure 3.10). The large precursor peptides are cut into smaller fragments by proprotein convertases to produce the final peptide structure, such as β-endorphin. Some of the gene products have been linked to inhibition of LH secretion in humans: β-endorphin (Reid et al., 1981); Table 3.2 Endogenous opioid peptides

Precursor Proenkephalin A

Opioid peptide [Met]enkephalin [Leu]enkephalin

Pro-opiomelanocortin

β-endorphin

Proenkephalin B

Dynorphin A

Prodermorphin

Dermorphin

Prodeltorphin

Deltorphin Deltorphin I Deltorphin II

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Chapter 3: Neuroendocrinology of Female Reproduction

pomc

SIGNAL PEPTIDE

enKA

SIGNAL PEPTIDE

enKB

SIGNAL PEPTIDE

pre-pro-opiomelanocortin

γ–MSH

α–MSH

β–MSH β–ENDORPHIN

ACTH

pre-proenkephalin A

met-enk met-enk met-enk

met-enk

leu-enk

met-enk

pre-proenkephalin B

leu-enk

leu-enk leu-enk DYNORPHIN

Figure 3.10 Schematic illustration of three opioid pre-propeptides. Pro-opiomelanocortin (pomc), enkephalin (enkA) and DYN (enkB) genes encode large pre-propeptides that are proteolytically cleaved (scissors) to a series of smaller, biologically active neuropeptides (e.g., β-endorphin; metenkephalin), by enzymes called proprotein convertases. Abbreviation: MSH, melanocytestimulating hormone; met-enk, met-enkephalin.

metenkephalin (Giusti et al., 1992) and dermorphin (Petraglia et al., 1985). A role for dynorphin (DYN) in LH secretion will be described later in this chapter (see Figure 3.13). In summary, opioids, such as morphine, and endogenous opioids, such as β-endorphin, bind to hypothalamic opioid receptors to inhibit LH secretion in humans. The following sections provide clinical examples of these effects, keeping in mind that the influence of endogenous opioids can best be illustrated by blocking their action with antagonists such as naloxone or naltrexone.

Inhibitory Effects of Exogenous Opioids on LH Secretion

38

The neuroendocrine hypothalamus is highly sensitive to opioids (review: Vuong et al., 2010) and both therapeutic and recreational use of opioids can result in hypogonadism. This is of widespread concern since 243,000 new heroin abusers were admitted for treatment in the United States during the year 2000, of which 40% were women (Schmittner et al., 2005). In addition, the increasing therapeutic use of opioid analgesics for non-cancer pain – such as back injuries, sport and automobile injuries and headache – has led

to widespread opioid prescription abuse. In 2014, more than 10 million persons were recorded as using prescription opioid analgesics for nonmedical purposes in the United States (Compton et al. 2016). The full extent of the implications for the reproductive system is yet to be explored in detail (see reviews: Katz and Mazer, 2009; Brennan, 2013), and women have been found to be at greater risk for opioid abuse in all age groups (Koons et al., 2018). Opioids have both acute and chronic effects. In men, gonadotropins and testosterone secretion are inhibited within 4 hrs after acute opioid administration, regardless of whether it is administered by intravenous, oral, transdermal or intrathecal routes (reviewed in Daniell, 2008). Similarly, long-term intrathecal morphine or hydromorphone administration for nonmalignant pain, in women, causes hypogonadotropic hypogonadism (Abs et al., 2000). Of the 21 premenopausal women receiving opioid treatment, 14 became amenorrheic and seven developed irregular cycles, compared with controls who had normal cycles. Serum LH levels were lower in the opioid group. In the 18 postmenopausal women, serum LH concentrations were significantly lower than those in the six postmenopausal control women. Similar findings were obtained in women receiving long-term (>1 year) oral methadone (Rhodin et al., 2010). In summary, there is strong evidence that both acute and chronic opioid administration can cause suppression of sex hormones, leading to sexual dysfunction, infertility, loss of muscle mass and mood disorders. Additionally, opioid therapy can also cause loss of other hypothalamic pituitary hormones, resulting in hypothyroidism, hypocortisolism and growth hormone (GH) deficiency (Abs et al., 2000).

Inhibitory Effects of Endogenous Opioids on LH Secretion: Stimulatory Effects of Naloxone As noted, the human hypothalamus expresses several opioid genes encoding peptides such as β-endorphin and DYN. These endogenous opioids are implicated in the inhibition of LH secretion. A convenient experimental route to test whether EOPs regulate human LH secretion is to use opioid antagonists such as naloxone, naltrexone and nalmefene to reverse the inhibitory effects of the endogenous peptides (Tenhola et al., 2012). In men, for example, naloxone infusion or oral nalmefene rapidly and significantly elevated LH secretion (Graves et al., 1993). Increased LH release was also seen in women given

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Chapter 3: Neuroendocrinology of Female Reproduction

oral naltrexone (Teoh et al., 1988; Roche and King, 2015), with similar responses in the follicular and luteal phases of the cycle. In contrast, naloxoneinduced LH secretion occurred only in the late luteal phase of the cycle (Rossmanith et al., 1989), emphasizing that naloxone and naltrexone differ in their ability to inhibit endogenous opioids. Nevertheless, it is clear that EOPs exert inhibitory control of LH secretion. The literature on the ability of opioid antagonists to modify normal hypothalamic–pituitary control of LH secretion is extensive, and in subsequent chapters we will see that other pituitary hormones are similarly regulated. However, the clinical utility of opioid antagonists in treating anovulatory conditions by increasing blood LH levels remains limited. While initial studies with naltrexone reported significant increases in LH values, and resumption of menstrual cycles, in women with ovarian failure (Wildt et al., 1993), others were unable to confirm these results (e.g., Couzinet et al., 1995). Similarly, naloxone has also been shown to stimulate LH secretion in women with HA (Khoury et al., 1987; Figure 3.11). In addition, long-term naltrexone therapy (3–6 months) in patients with HA due to low body weight restored normal menstrual bleeding in 80% of the patients (Genazzani et al., 1995). In another study of clomiphene-resistant, normogonadotropic, anovulatory women treated with naltrexone, 19 out of 22 women

resumed normal menstrual cycles and 12 of the 19 became pregnant (Roozenburg et al., 1997). Naltrexone has also been used in women with polycystic ovary syndrome (PCOS). PCOS is the most common cause of female infertility and affects over 100 million women worldwide (Azziz et al., 2005; Padmanabhan, 2009). It is a heterogeneous disorder characterized by anovulation, hyperandrogenism, enlarged polycystic ovaries, insulin resistance and obesity. Both central and peripheral opioids may play a role in the pathogenesis of PCOS (Eyvazzadeh et al., 2009). One study looked at a group of anovulatory PCOS women treated with either naltrexone or naltrexone combined with clomiphene (Ahmed et al., 2008). Results showed that naltrexone not only restored sensitivity to clomiphene in most of the patients but an improvement in other metabolic parameters – including reduction in body mass index (BMI) and serum testosterone as well as increased insulin sensitivity – was also observed. Another study showed similar results, with restoration of menstrual activity in 80% of women, a reduction in BMI, testosterone and cortisol levels and an improvement in insulin sensitivity (Fruzzetti et al., 2002). In summary, these studies suggest that opioid systems are abnormal in PCOS, although it is not possible to conclude that central opioids are solely responsible. For example, the reduction in BMI could be an indirect contributor to improved menstrual cyclicity.

Figure 3.11 Naloxone-stimulated LH release in hypothalamic amenorrhea. The patient was amenorrheic due to weight loss (8 years). Despite recovery to 90% ideal body weight, she remained amenorrheic. Infusion of saline did not affect LH or FSH secretion but treatment with naloxone (1.0 mg/m2; 4 hrs) markedly increased LH pulses but not FSH pulses. Reproduced with permission (Besser and Thorner, 2002).

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KP Is a Stimulus for GnRH Secretion KP is a hypothalamic neuropeptide that functions as a neurotransmitter/neuromodulator to regulate the secretion of GnRH (Roa et al., 2008; Skorupskaite et al., 2014; Wilkinson and Brown, 2015). Subcutaneous infusion of the peptide KP is a powerful stimulator of LH (and to a lesser extent, FSH) secretion in women, an effect comparable with that of GnRH (Skorupskaite et al., 2014; Narayanaswamy et al., 2015). In keeping with many neuropeptides, KP is also expressed in several other tissues. It was originally isolated as a metastasis-inhibiting peptide (metastin) in several tumors, such as pancreas, ovary, thyroid and breast, and is also normally expressed in placenta, ovary, pancreatic islets, cardiovascular system, fat tissue and several brain areas outside of the hypothalamus (Roa et al., 2008; Brown et al., 2008; Oakley et al., 2009; Cockwell et al., 2013). The human KISS1 gene encodes a prepropeptide (pre-prokisspeptin) that contains the sequence for metastin (KP-54; Figure 3.12; Roa et al., 2008). The bioactive KP-54 is formed by cleavage, as shown, and three other smaller peptides have been isolated. However, only KP-10 appears to have significant biological activity in stimulating LH secretion in healthy men (Jayasena et al., 2015). Human brain autopsy samples, from infants and adults of both sexes, revealed KP-immunoreactive neurons in the infundibular region of the hypothalamus

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(Taziaux et al., 2016). Moreover, tissue from pre- and post-menopausal women showed that KP neurons were co-localized with GnRH neurons in the infundibular nucleus, with a second population in the preoptic area (Hrabovsky et al., 2010; Hrabovsky, 2014). KP neuron axons form a dense plexus that makes axosomatic, axo-dendritic and axo-axonal contacts with GnRH neurons. It seems likely that KP regulates the secretion of GnRH from human GnRH neurons via KP receptors (KISS1 R). However, unlike the situation in experimental animals (Messager et al., 2005), where KISS1 R are located on GnRH neurons, the precise location of the receptors in the human hypothalamus is presently unknown. The mechanism by which KP neurons regulate LH secretion, and specifically in generating the preovulatory surge in LH, was revealed following the discovery that KP is co-localized and co-released with two other neuropeptides: DYN, an endogenous opioid, and NKB (a tachykinin peptide). These cells have been labeled as KNDy neurons and they play a critical role in controlling reproductive function (Lehman et al., 2010; Pinilla et al., 2012). A possible cellular arrangement is shown in Figure 3.13, where NKB (stimulatory) and DYN (inhibitory) are shown acting in an autocrine fashion to control KP release. Alternate, reciprocal signaling by NKB and DYN is then a possible locus of pulsatile LH secretion.

Figure 3.12 Structure and cleavage of human pre-prokisspeptin. Pre-prokisspeptin is a 145 amino acid peptide that contains a 54 amino acid region (KP-54), flanked by two cleavage sites (denoted by scissors). Proteolytic processing of pre-prokisspeptin by the enzyme furin generates KP-54 (also called metastin) (Harihar et al., 2015). Further cleavage of KP-54 produces KPs of lower molecular weight: KP-14, KP-13 and KP-10. Reproduced with permission (Roa et al., 2008).

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Figure 3.13 Tentative model for the role of KNDy neurons in the control of pulsatile GnRH secretion. A schematic model shows the putative roles of NKB (stimulatory) and DYN (inhibitory) as co-transmitters and putative regulators of the secretory activity of KP neurons as the major driving signal for GnRH pulsatile release. In this model, NKB would operate as a stimulatory autocrine signal for KNDy neurons, and DYN would inhibit KP secretion. Reciprocal signaling by NKB and DYN, at G protein-coupled receptors, could possibly produce pulsatile GnRH secretion. Abbreviations: KOR, kappa opioid receptor; NK3 R, NKB receptor. Figure is adapted and redrawn from Pinilla et al. (2012). The GnRH neuron image is reproduced with permission (Herbison, 2016).

The neuroanatomical and neurochemical details of such a system were established in experimental animals, including primates, but there is good evidence for colocalization of NKB and KP in human hypothalamic neurons (Hrabovszky et al., 2010; Hrabovszky, 2014). Supporting evidence exists for a stimulatory effect of NKB in humans; that is, lossof-function mutations in the NKB gene, or its receptor gene, produces isolated hypogonadotropic hypogonadism (Semple and Topaloglu, 2010). In addition, treatment of healthy women (regular menses) with an NKB antagonist reduced LH and E2 secretion (Skorupskaite et al., 2018a), demonstrating the importance of stimulatory NKB signaling in women. In contrast, there is little evidence that places DYN in the same neurons, although DYN gene expression is present in human hypothalamus (Rometo and Rance, 2008). Further, and as discussed already, blockade of endogenous opioids (such as DYN) with naloxone or naltrexone increases LH secretion. An additional key point is that KP neurons possess E2 receptors, strategically located to facilitate hormone feedback. E2 receptors, NKB and KP are all colocalized in human hypothalamic neurons (Rance,

2009), suggesting that E2 feedback, both positive and negative, may be mediated through steroid-sensitive KP neurons. A speculative arrangement is shown in Figure 3.14, where KP neurons mediate the positive feedback effect of E2 to increase GnRH secretion, and KNDy neurons respond to E2 by reducing KISS1 gene expression and limiting the release of GnRH (Skorupskaite et al., 2014). In summary, KP, along with NKB and DYN, is a key neuropeptide in the hypothalamic control of the human reproductive system. Acting as a potent stimulus to LH secretion in healthy men and women it is likely that KP could also be clinically invaluable in the treatment of human reproductive disorders (Skorupskaite et al., 2014; Prague and Dhillo, 2015). NKB antagonism also appears to be a novel treatment for menopausal hot flushes (Prague et al., 2017; Skorupskaite et al., 2018b).

Stimulatory Effects of KP in the Human Reproductive System Exogenous KP potently stimulates the secretion of LH and is particularly effective in the early follicular phase of the menstrual cycle (Dhillo et al., 2007; Skorupskaite et al., 2014). The results from several

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Figure 3.14 KP neurons mediate hypothalamic E2 feedback. This schematic representation suggests that E2 exerts hypothalamic feedback via KP neurons in two anatomical sites. E2 receptors, NKB and KP are all co-localized in human hypothalamic neurons (Rance, 2009). KP neurons could therefore mediate the positive feedback effect of E2 to increase GnRH secretion, and KNDy neurons respond to the negative feedback effects of E2 by reducing KISS1 gene expression and limiting the release of GnRH.

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studies on healthy men and women are summarized in Figure 3.15. For the most part, these data represent the influence of single injections, illustrated as peak concentrations or mean LH values. A subcutaneous infusion of KP over several hours was also highly effective in increasing LH secretion in healthy women (Narayanaswamy et al., 2015; Figure 3.16). This result is notable because of the absence of desensitization of the KP response (tachyphylaxis), suggesting that this may constitute a useful therapeutic approach to treating infertility. Of equal significance is the report that a single subcutaneous injection of KP (0.6 nmol/kg; see Figure 3.15), given to healthy women in the follicular phase of the menstrual cycle, significantly increased the frequency of LH pulses (Jayasena et al., 2013). These data emphasize that KP stimulation is effective, not only in elevating LH levels over several hours, but in increasing pulsatile secretion as well. This observation is in marked contrast to the effect on LH of

continuous stimulation with the secretagogue GnRH, or its long-acting analogs, that profoundly inhibits LH secretion (see Section 3.3 and Figure 3.8; Fraser, 1993). However, note that the effect of KP on LH pulsatility in women may be dose- and E2dependent. For example, chronic subcutaneous injections of high-dose KP (6.4 nmol/kg vs. 0.6 nmol/kg in their 2013 study referred to previously) induced tachyphylaxis in the LH response in women (Jayasena et al., 2009).

Potential Clinical Uses of KP Adult humans possessing inactivating mutations in the KISS1 gene or in its receptor gene, KISS1 R, show hypogonadotropic hypogonadism characterized by delayed puberty, low gonadotropins and sex steroids (Silveira et al., 2013; Silveira and Latronico, 2013; Prague and Dhillo, 2015). An activating mutation in KISS1 R, leading to precocious puberty, has also been described (Teles et al., 2008). These seminal studies led to the current acceptance of an obligatory role for

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Subgroup

lsoform

Protocol

LH (IU/L) Baseline Stimulated Reference

Healthy Men

Healthy Women Follicular Follicular Follicular Follicular Follicular Follicular Follicular Follicular Follicular Follicular Follicular Late follicular Late follicular Preovulatory Luteal Luteal Menopause Implanon COCP

KP-54 iv 90 min 4 pmol/kg/min

4.2

10.8

Dhillo 2005

KP-10 iv bolus 0.24 nmol/kg

2.8

7.8

Chan 2011

KP-10 iv bolus 0. 77 nmol/kg

4.1

12.4

George 2011

KP-10 iv bolus 2.3 nmol/kg

4.1

7.0

George 2011

KP-10 iv bolus 10 nmol/kg

2.9

6.5

Jayasena 2011

KP-10 iv 9 h 1.1 nmol/kg/h

5.2

14.1

George 2011

KP-10 iv 22.5 h 3.1 nmol/kg/h

5.4

20.8

George 2011

KP-54 KP-54 KP-54 KP-54 KP-54 KP-54 KP-10 KP-10 KP-10 KP-10 KP-10 KP-54 KP-10 KP-10 KP-54 KP-10 KP-10 KP-10 KP-10

4.2 3.4 5.7 9.4 6.0 3.8 3.8 6.3 2.9 3.8 3.8 14.5 34.7 6.8 3.6 3.4 35.3 4.6 2.3

4.3 5.7 14.3 17.7 18.7 12.1 3.8 9.4 3.8 3.8 3.8 35.1 61.3 26.8 5.8 7.1 44.7 7.5 3.7

Dhillo 2007 Jayasena 2013b Jayasena 2013a Jaysasena 2013a Jayasena 2013a Jayasena 2011 Jayasena 2011 George 2012 Chan 2012 Jayasena 2011 Jayasena 2011 Dhillo 2007 Chan 2012 Jayasena 2011 Dhillo 2007 Chan 2012 George 2012 George 2012 George 2012

sc bolus 0.4 nmol/kg sc bolus 0.6 nmol/kg sc bolus 6.4 nmol/kg sc bolus 6.4 nmol/kg BD5d sc bolus 6.4 nmol/kg BD8d iv bolus 1 nmol/kg sc bolus 2–32 nmol/kg iv bolus 0.23 nmol/kg iv bolus 0.24 nmol/kg iv bolus 1–10 nmol/kg iv 90 min 20–720 pmol/kg/min sc bolus 0.4 nmol/kg iv bolus 0.24 nmol/kg iv bolus 10 nmol/kg sc bolus 0.4 nmol/kg iv bolus 0.24 nmol/kg iv bolus 0.23 nmol/kg iv bolus 0.23 nmol/kg iv bolus 0.23 nmol/kg

Men

Women

0

50

100

150

200

250

350

% Change in LH secretion

Figure 3.15 KP stimulates LH secretion in healthy men and women. KP was administered in different isoforms (KP-54 and KP-10) and by different protocols (intravenous or subcutaneous, single boluses or continuous infusion). Note that stimulated LH values are either mean LH or peak LH concentrations depending on how the data were originally presented. Abbreviations: BD5d, twice daily for five days; BD8d, twice daily for eight days; COCP, combined oral contraceptive pill; Implanon, etonogestrel contraceptive implant; iv, intravenous; sc, subcutaneous. Reproduced with permission (Skorupskaite et al., 2014).

KP in regulating GnRH/LH release and the neuroendocrine regulation of human reproduction. As already described in the previous section, KP is a potent stimulus for LH secretion in healthy individuals, and a logical question is whether KP treatment might be capable of reversing some forms of infertility, such as HA. Two examples of KP as a therapeutic intervention will be described. Functional HA is a common cause of absent menstruation. The primary defect appears to be an inability of the hypothalamus to increase GnRH output in response to severe hypoestrogenism. The decrease in LH secretion in these women probably reflects an impairment of the GnRH pulse generator (Liu et al.,

2016). In practice, functional HA is the reversible absence of menstruation for more than 6 months, providing there is no indication of anatomical or organic abnormalities (Meczekalski et al., 2008). It is commonly associated, for example, with stress, weight loss or intense exercise. HA is observed in many female athletes with low body weight and who exercise excessively, as well as in anorexic women and girls. Restoration of fertility in HA requires the return of hypothalamic–pituitary function through normalization of pulsatile LH secretion, a state that is not provided by supplementation with E2 alone. KP stimulation therefore offers a possible route to reverse HA. In fact, subcutaneous infusion of KP (0.03, 0.10

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30

Serum LH (iU/L)

1.0 nmol/kg/hr

10 0.3 nmol/kg/hr 0.1 nmol/kg/hr

5 Vehicle

Mean serum LH (iU/L)

15

20 1.00 nmol/kg/hr

10 0.10 nmol/kg/hr 0.03 nmol/kg/hr Vehicle

0

0 60

0

120 180 240 300 360 420 480 Time (mins)

Figure 3.16 Effectsof KP-54 infusions on serum LH in healthy women during the early follicular phase of the menstrual cycle. Data are collated from all participants (n=4) in response to 8-hr infusions of vehicle and KP-54 (subcutaneous; 0.1, 0.3 and 1.0 nmol/kg/h). Mean serum LH results for each time point (from all four participants) during the 8-hr study are presented as a time profile. The two highest doses (0.3 and 1.0 nmol/kg/h) significantly increased LH secretion (p15 mm) were seen on ultrasound. Subcutaneous hCG injection (5000 IU) was given to induce oocyte maturation and 36 hours later 20 oocytes were retrieved without any complication. However, 4 days after the retrieval she developed nausea, vomiting and abdominal tenderness; she had also gained 5 kgs in weight and noticed an increase in her abdominal circumference. An endovaginal

ultrasound was conducted that revealed bilateral ovarian enlargement (right = 11.5 cm and left = 12.2 cm) and free fluid. Based on these findings, the patient was diagnosed with moderate OHSS. She was admitted to hospital for observation and fluid management; she made a full recovery over the next 2 weeks. Efforts have been made to overcome the problems with hCG stimulation by using a GnRH agonist (Thomsen and Humaidan, 2015), but Jayasena et al. (2014a) suggested that KP stimulation might also safely trigger oocyte maturation in patients at high risk for OHSS. In a randomized study of 60 women at high risk of OHSS, KP stimulation was associated with oocyte maturation in 95% of the women and pregnancy rates of 62–85% were achieved with different doses of KP. More importantly, no woman developed OHSS (Abbara et al., 2015; Table 3.3; see also Abbara et al., 2017). In summary, KP safely stimulates final oocyte maturation and high implantation rates in IVF patients at high risk of OHSS. The authors conclude that larger, randomized studies are needed so that KP stimulation can be compared with other triggers (such as hCG) to optimize treatment of patients at high risk of developing OHSS during IVF treatment (Abbara et al., 2018).

3.5 Chapter Summary This chapter provides a basic introduction to the physiology and neuroendocrine control systems of the human menstrual cycle. A major focus is on the ability of sex hormones, such as E2, to exert feedback on the hypothalamic regulation of GnRH neuron secretion. GnRH represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. GnRH neurons are also the locus of a system that generates pulsatile

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secretion of LH, a pattern that is crucially important for sexual maturation and subsequent fertility. That human fertility is dependent on GnRH neurons is emphasized using a case study of Kallmann syndrome, in which GnRH neurons are absent from the hypothalamus. GnRH secretion is regulated through E2 feedback but also by a variety of neurotransmitters and neuropeptides, and their receptors. Detailed supportive neuroanatomical evidence, obtained from human brain tissue, has revealed close apposition of nerve terminals containing these factors with GnRH neuron axons and dendrites. Two well-described neurochemical systems are used as examples to illustrate the extensive clinical importance of drugs that inhibit LH secretion and those that stimulate the reproductive system. Opioids, including endogenous opioids such as β-endorphin, exert powerful inhibitory effects on LH secretion. KP, on the other hand, is the most potent LH secretagogue known. Opioid abuse as a result of addiction to drugs, such as heroin, results in hypogonadism, one form of which is the disruption of menstrual function. Chronic opioid treatment for pain also inhibits the reproductive systems of both men and women. Of equal concern, hypogonadism-induced symptoms include hypocortisolism, GH deficiency, infertility, osteoporosis, mood disorders, fatigue and hyperalgesia. The inhibitory influence of hypothalamic EOPs, in men and women, is revealed through the stimulatory influence of antagonists such as naloxone and naltrexone. This effect has been employed in attempts to reverse some aspects of infertility, such as PCOS and functional HA. The human hypothalamus contains neurons that express the neuropeptide KP, encoded by the KISS1 gene. KISS1 R are also localized to this brain region. KP is co-localized and co-released with two other neuropeptides: DYN, an opioid, and NKB. These cells have been labeled as KNDy neurons and they play a critical role in controlling reproductive function. NKB/DYN act in an autocrine fashion to control KP release. Alternate, reciprocal, signaling by NKB and DYN is a possible locus of pulsatile LH secretion. Injection or infusion of KP potently stimulates the pulsatile secretion of LH and is particularly effective in the early follicular phase of the menstrual cycle. In clinical terms KP has been used in attempts to reverse the effects of functional amenorrhea by increasing pulsatile release of LH. KP also shows promise as a trigger for oocyte maturation in IVF, particularly in women who are at high risk for OHSS.

3.6 Review Questions 1. Which of the following statements regarding opioids are correct? a. G protein-coupled opioid receptors are expressed in the hypothalamus. b. Opioid antagonist therapy has been shown to improve ovulation in women with polycystic ovary syndrome (PCOS). c. Chronic opioid therapy is associated with pituitary dysfunction. d. Hypothalamic opioid receptors have affinity for endogenous opioids only. e. Opioids lower serum luteinizing hormone (LH) in premenopausal as well as postmenopausal women. 2. Kallmann’s syndrome is associated with which of the following abnormalities? a. A mutation in the PROKR2 gene b. Absence of a sense of smell c. High serum LH, follicle stimulating hormone (FSH) and low testosterone levels d. An increased arm span-to-trunk ratio e. Small testes 3. A 19-year-old female presented with amenorrhea for 6 months. She was known to have anorexia nervosa since she was 12 and had previously responded to counseling. However, after the separation of her parents, she had a relapse of symptoms. Her body mass index, which was previously 18.9 kg/m2, had dropped to 15.6 kg/m2 and she stopped menstruating. Which of the following abnormalities would you expect on her blood test? a. High serum LH and FSH and low serum estradiol (E2) b. High serum LH but low FSH c. Low serum LH and FSH and low E2 d. High serum LH and FSH and high E2 e. Normal LH, FSH and E2 levels 4. Regarding opioid therapy for non-cancer pain, which of the following statements are correct? a. Both acute and chronic opioid therapy can cause hypogonadism. b. Chronic opioid therapy can lead to testicular atrophy. c. Opioids primarily act on ovaries to reduce E2 production.

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Chapter 3: Neuroendocrinology of Female Reproduction

d. Hypogonadism is generally seen with oral opioids whereas injections are safe. e. Naloxone can reverse some of the hypogonadal effects of morphine. 5. Regarding infertility management, which of the following statements are correct? a. A midcycle human chorionic gonadotropin (hCG) injection as part of an in vitro fertilization protocol can induce oocyte maturation by mimicking the LH surge in women. b. hCG injection in men can induce testicular growth. c. Testosterone treatment alone in men can potentially lower LH and FSH and thus cause low sperm count. d. The prolonged LH-like effect of hCG can lead to ovarian hyperstimulation syndrome. e. Weight reduction can improve fertility in obese women suffering from PCOS. 6. A 32-year-old female underwent pituitary tumor surgery for a non-functioning adenoma. After surgery, she became amenorrheic and her blood work showed undetectable LH and FSH as well as low E2 levels. Which of the following is an appropriate treatment choice for her if she is seeking fertility? a. E2 alone; given so it peaks twice each month, near the middle of the cycle. b. Progesterone alone; given so it peaks once each month at midcycle. c. E2, and progesterone, taken on a regular monthly schedule (two peaks of estrogen, one of progesterone). d. Injections of equal amounts of both FSH and LH every day each month. e. Pulses of gonadotropin releasing hormone (GnRH) administered every 1–2 hr by a portable, programmable infusion pump. 7. Kisspeptin (KP) is a brain neuropeptide implicated in the regulation of human fertility. Which of the following statements are correct? a. KP induces LH secretion by directly stimulating the anterior pituitary gland. b. KP is a powerful stimulant for LH secretion. c. KP binds to GnRH receptors. d. KP is co-localized in neurons with neurokinin B.

e. Unlike GnRH, KP infusion can stimulate LH release without tachyphylaxis. 8. GnRH is the final common pathway that enables the brain to induce puberty and regulate the menstrual cycle. Which of the following statements are correct? a. In patients with delayed puberty, sexual maturation can be induced with continuous GnRH agonist treatment. b. Precocious puberty may be treated with a GnRH long-acting agonist. c. KP injections induce pulsatile LH secretion in women with hypogonadotropic hypogonadism. d. GnRH agonists are used in the treatment of some breast tumors. e. Kisspeptin acts by increasing the secretion of GnRH.

Further Reading Besser G M & Thorner M O. (2002). Comprehensive Clinical Endocrinology, 3rd Edition (St. Louis, MO: Mosby). Boehm U, Bouloux P-M, Dattani M T et al. (2015). European Consensus Statement on congenital hypogonadotropic hypogonadism – pathogenesis, diagnosis and treatment. Nat Revs Endocr 11, 547–564. Caronia L M, Martin C, Welt C K et al. (2011). A genetic basis for functional hypothalamic amenorrhea. N Engl J Med 364, 215–25. Christian C A & Moenter S M. (2010). The neurobiology of preovulatory and estradiol-induced gonadotropin-releasing hormone surges. Endocr Revs 31, 544–577. Gordon C M, Ackerman K E, Berga S L et al. (2017). Functional hypothalamic amenorrhea: An endocrine society clinical practice guideline. J Clin Endocr Metab 102, 1413–1439. Grumbach M M. (2002). The neuroendocrinology of human puberty revisited. Horm Res 57 (Suppl 2), 2–14. Hall J E. (2014). Neuroendocrine control of the menstrual cycle. Yen and Jaffe’s Reproductive Endocrinology, 7th Edition; Strauss J F & Barbieri R L, Eds. (Philadelphia, PA: Elsevier), 141–156 Herbison A E. (2015). Physiology of the adult gonadotropin-releasing hormone neuronal network. Knobil and Neill’s Physiology of Reproduction, 4th Edition; Plant T M & Zeleznik, A J, Eds. (New York: Elsevier), 399–467. Pinilla L, Aguilar E, Dieguez C, Millar R P & Tena-Sempere M. (2012). Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiol Revs 92, 1235–1316.

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Stamou M I, Cox K H & Crowley W F Jr. (2015). Discovering genes essential to the hypothalamic regulation of human reproduction using a human disease model: Adjusting to life in the “-omics” era. Endocr Revs 36, 603–621. Vuong C, Van Uum S H M, O’Dell L E, Lutfy K & Friedman T C. (2010). The effects of opioids and opioid analogs on animal and human endocrine systems. Endocr Revs 31, 98–132. Wierman M E, Kiseljak-Vassiliades K & Tobet S. (2011). Gonadotropin-releasing hormone (GnRH) neuron migration: initiation, maintenance and cessation as critical steps to ensure normal reproductive function. Fronts Neuroendocr 32, 43–52. Wilkinson M & Brown R E. (2015). An Introduction to Neuroendocrinology, 2nd Edition (Cambridge: Cambridge University Press).

References Abbara A, Jayasena C N, Christopoulos G et al. (2015). Efficacy of kisspeptin-54 to trigger oocyte maturation in women at high risk of ovarian hyperstimulation syndrome (OHSS) during in vitro fertilization (IVF) therapy. J Clin Endocr Metab 100, 3322–3331. Abbara A, Clarke S, Islam R et al. (2017). A second dose of kisspeptin-54 improves oocyte maturation in women at high risk of ovarian hyperstimulation syndrome: a phase 2 randomized controlled trial. Hum Reprod 32, 1915–1924 Abbara A, Islam R, Clarke S A et al. (2018). Clinical parameters of ovarian hyperstimulation syndrome (OHSS) following different hormonal triggers of oocyte maturation in IVF treatment. Clin Endocr 88, 920–927. Abs R, Verhelst J, Maeyaert J et al. (2000). Endocrine consequences of long-term intrathecal administration of opioids. J Clin Endocr Metab 85, 2215–2222. Ahmed M I, Duleba A J, El Shahat O, Ibrahim M E & Salem A. (2008). Naltrexone treatment in clomiphene-resistant women with polycystic ovary syndrome. Human Reprod 23, 2564–2569. Antunes J L, Carmel P W, Housepian E M & Ferin M. (1978). Luteinizing hormone-releasing hormone in human pituitary blood. J Neurosurg 49, 382–386. Azziz R, Marin C, Hoq L, Badamgarav E & Song P. (2005). Health care-related economic burden of the polycystic ovary syndrome during the reproductive life span. J Clin Endocr Metab 90, 4650–4658.

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Balasubramanian R, Dwyer A, Seminara S B, Pitteloud N, Kaiser U B & Crowley W F Jr. (2010). Human GnRH deficiency: a unique disease model to unravel the ontogeny of GnRH neurons. Neuroendocr 92, 81–99.

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4

Neuroendocrine Regulation of Appetite and Body Weight

Anorexia nervosa and obesity constitute two extremes of dysfunctional body weight control. Anorexia nervosa is characterized by low body weight and global endocrine abnormalities, especially of the hypothalamic–pituitary axis, as well as altered adipokine function and appetite-regulating hormone levels (Schorr and Miller, 2017). These same systems are implicated in the pathology of obesity. How the brain regulates appetite and body weight has become a vital public health issue, often described as a global obesity epidemic (Schwartz et al., 2017). Obesity is strongly associated with a decrease in the quality of life and an escalating incidence of cardiovascular disease, type 2 diabetes, osteoarthritis, certain cancers, hypertension, stroke and Alzheimer’s disease (World Obesity Federation, 2017; Goodarzi, 2018). A comprehensive and detailed study, assembling data from 195 countries, examined the prevalence of overweight and obesity among children and youth (30) in 2015. High BMI was responsible for 4.0 million deaths globally. The prevalence of obesity has doubled since 1980 in more than 70 countries, and increased in many others. In Canada, for example, recent statistics indicate that 6.9 million people (>18 years of age) are obese and 9.4 million are overweight (BMI=25–29) (Stats Canada, 2017). In the United Kingdom, in 2015, 58% of women and 68% of men were overweight or obese (GOV.UK, 2017). The United States has the highest incidence of childhood obesity (12.7%). The World Obesity Federation estimates that the global cost of treating obesity-related illnesses will approach US$1.2 trillion per year by 2025. Notwithstanding these alarming figures, it is worth noting that the use of BMI as a risk indicator of health may be questionable; that is,

up to 50% of adults deemed overweight, and 29% of obese adults – as determined by BMI – were cardiometabolically healthy. For example, heavily muscled individuals with increased body weight will have BMI values that may be interpreted as overweight or obese. Conversely, 30% of individuals classified as healthy by BMI scores were in fact metabolically unhealthy (Dhurandhar, 2016; Tomiyama et al., 2016). This misclassification prompted the suggestion that excess body fat – largely deposited in the abdominal region – may be a superior indicator of metabolic ill health (Maffetone et al., 2017). A prevailing perspective is that obesity is caused by individual greed and self-indulgence in the face of an abundance of hyperpalatable, energy-dense and low-cost food, as well as a sedentary lifestyle. Fundamentally, the pathogenesis of obesity appears to be straightforward: calories are consumed in amounts that exceed ongoing energy expenditure. Nonetheless, not all people faced with such abundance eat too much or become obese and there are now efforts to regard obesity as a disease (Editorial, 2017; World Obesity Federation, 2017). Considerable progress has been made in our understanding of the neuroendocrine pathways that regulate appetite and the control of body weight, and it is also clear that pronounced genetic components may sometimes underlie a susceptibility to becoming overweight and obese (Yeo, 2017). For example, studies in twin pairs and in adopted children revealed that a large percentage of the risk for obesity is heritable (Stunkard et al., 1990; Schwartz et al., 2017). The search for specific genes through genome-wide analysis revealed more than 100 sites (loci) associated with BMI, as well as strong support for hypothalamic genes that underlie regulation of body mass and the susceptibility to obesity (Locke et al., 2015; Pigeyre et al., 2016). More than 30 obesity syndromes are known, and gene mutations for leptin, leptin receptor, melanocortin-4 receptor (MC4 R) and pro-opiomelanocortin (POMC) induce

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Figure 4.1 Obesity and the incidence of some common chronic diseases. Adapted and redrawn from the original (World Obesity Federation, 2017).

hyperphagia and obesity (Heymsfield and Wadden, 2017). Although rare, these examples will be used in the following sections to provide insights into human appetite control (see Section 4.3).

4.1 The Neural Control of Appetite

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The major neural centers for the regulation of appetite are located in the hypothalamus and brain stem. Although our present understanding of this system is inevitably based on animal experiments, these control pathways appear to largely conform to the available knowledge from human data. Figure 4.2 outlines in general terms the homeostatic regulation of food intake. The brain, and especially the hypothalamus, integrates signals (e.g., leptin) from adipose tissue (long-term energy storage) and short-term meal-related signals (nutrients, and gut-derived hormones such as ghrelin) to regulate food intake. In normal-weight

humans, weight gain as a result of forced overeating inhibits the rewarding aspects of food while enhancing satiety, resulting in reduced food intake, increased energy expenditure and a return to normal body weight (Schwartz et al., 2017). In contrast, food deprivation induces central adaptive responses that amplify the rewarding properties of food and desensitizes the response to satiety signals. The net result is an increase in food consumption (Morton et al., 2014). Figure 4.3 illustrates in more detail some of the hypothalamic neurochemical pathways known to regulate food intake (Schwartz and Morton, 2002). The clinical relevance of these systems will be described in the following sections, and a case will be described illustrating hypothalamic lesion-induced hyperphagia (see Uher and Treasure, 2005). An informative online poster (Dietrich and Horvath, 2010) illustrates much of the information to follow: http://www.nature.com/nrn/posters/feeding.

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Leptin Deficiency Reward

Food intake

Satiety

Short-term satiety signals

Nutrient related

Ghrelin Adiposity-related (long term)

Blood glucose

GLP-1 PYY 3–36 CCK

Liver Stomach

Insulin Leptin Adipose tissue

CCK GI tract GLP-1 PYY3–36

Figure 4.2 Hormonal and nutrient regulation of food intake. The brain, and especially the hypothalamus, integrates signals from long-term energy stores (e.g., leptin from adipose tissue) and shortterm meal-related signals (nutrients, and gut-derived satiety signals such as ghrelin) to regulate food intake. Overfeeding inhibits the rewarding properties of food while enhancing meal-induced satiety, thereby reducing food intake. In response to energy deprivation, central adaptive responses are engaged that increase the rewarding properties of food and reduce the response to satiety signals, collectively resulting in increased food consumption until deficient fat stores are replenished. Reproduced with permission (Morton et al., 2014). Abbreviations: CCK, cholecystokinin; GI, gastrointestinal; GLP-1, glucagon-like peptide-1; PYY3-36, peptide YY.

4.2 Leptin: A Fat-Derived Peptide Hormone Adipose tissue is a large endocrine organ that secretes a new family of hormones called adipokines (Fasshauer and Blüher, 2015). This section will focus on leptin as the prototypical adipokine, although many more adipokines have now been identified (Beall et al., 2017). Since its discovery in 1994, leptin has generated thousands of publications and is implicated in many neural, reproductive, metabolic, immune, endocrine and neuroendocrine systems (Farooqi and O’Rahilly, 2014; Friedman, 2016). The generally accepted view of leptin’s mode of action is its ability to reduce food intake and to maintain body fat levels. It does this by instructing the hypothalamus, which contains leptin receptors, to reduce appetite and increase energy expenditure (Figure 4.3).

The crucial importance of leptin in the control of body weight is revealed in humans carrying mutations that effectively produce leptin deficiency; that is, either mutations in the leptin gene – that reduce biologically active leptin – or in the leptin receptor gene that abolish the effects of leptin. Mutations in the leptin receptor signaling pathway are also known, and these will be described in Section 4.3. Mutations in the leptin gene are rare, but affected children are severely obese and exhibit intense hyperphagia and aggressive behavior when food is denied. The earliest patients discovered with leptin gene mutations had no circulating leptin and they responded to leptin treatment with a remarkable reduction in fat levels and body weight (in children: Farooqi et al., 2002; Farooqi and O’Rahilly, 2014; in adults: Licinio et al., 2004). Rather than mutations in the leptin gene, a complete deletion of the gene has also been reported (Ozsu et al., 2017). This child was grossly obese by the age of 6 months and exhibited intense hyperphagia. The effect of leptin deficiency in children is revealed in Figure 4.4. Other clinical features include high rates of childhood infection, hyperinsulinemia, type 2 diabetes as adults, hypothyroidism (low thyroxine [T4], high thyroid stimulating hormone [TSH]), absence of puberty and hypogonadotropic hypogonadism. The assay of serum leptin levels would seem to be an effective test in patients with severe early-onset obesity; an undetectable serum leptin level being suggestive of congenital leptin deficiency. However, another form of leptin gene mutation leads to high levels of immunoreactive serum leptin that has no biological activity; that is, it does not bind to leptin receptors (Wabitsch et al., 2015). Treatment of these patients with recombinant human leptin normalized weight loss and eating behavior. A similar clinical picture is seen in patients with a mutation in the leptin receptor; that is, signaling at the mutant receptor is impaired, so that these patients are effectively leptin free even though leptin levels are high (Farooqi et al., 2007). They are also characterized by severe obesity, hyperphagia, abnormal immune function and delayed puberty due to hypogonadotropic hypogonadism. The elevated serum leptin levels are typical of the obese state and reflect the increase in fat mass.

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Chapter 4: Neuroendocrine Regulation of Appetite and Body Weight

Neuron NPY

α–MSH

Food intake

Energy expenditure

AgRP

INCREASE APPETITE

NPY

DECREASE APPETITE Arcuate nucleus

NPY/ AgRP Third ventricle

POMC –

+

Melanocortin (MC4R) receptor for MSH – Ghrelin

+ GHRELIN PYY3–36 GLP-1

PYY/GLP-1/CCK

STOMACH INSULIN AMYLIN

CCK

NPY

LEPTIN Insulin or leptin or amylin GI TRACT

Duodenum PANCREAS ADIPOSE TISSUE

Figure 4.3 Neuroendocrine control of food intake. The figure illustrates the crucial role of the hypothalamic arcuate nucleus in regulating food intake. Leptin and insulin (co-released with amylin) circulate in the blood at concentrations proportional to body fat mass. They decrease appetite by inhibiting neurons that produce the molecules NPY and AgRP, at the same time stimulating α-MSH (POMC) neurons in the arcuate nucleus. NPY and AgRP stimulate eating, and α-MSH inhibits eating, via other neurons (top). Activation of NPY/AgRP-expressing neurons inhibits POMC neurons, either directly through NPY receptors or by AgRP blocking the effects of α-MSH. The hormone ghrelin stimulates appetite by activating the NPY/AgRP-expressing neurons. PYY3-36 and GLP-1, released from the small intestine, inhibit these neurons and thereby decrease appetite. Reproduced with permission (Schwartz and Morton, 2002). Abbreviations: AgRP, agouti-related peptide; CCK, cholecystokinin; GI, gastrointestinal; GLP-1, glucagon-like peptide-1; MSH, melanocyte-stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; PYY, peptide YY.

Leptin and Amenorrhea

56

Leptin deficiency – in terms of low serum levels – is also encountered in certain conditions where the leptin gene is functional and leptin receptors are normal. For example, anorexia nervosa patients have abnormally low serum leptin levels, as do some patients with hypothalamic amenorrhea. The critical importance of leptin for normal reproductive function was clearly demonstrated in those leptin-deficient children described earlier (Figure 4.4). Leptin treatment resolved the problems of hyperphagia and obesity but, significantly, allowed these children to enter puberty and to exhibit pulsatile gonadotropin secretion (Chou and Mantzoros, 2014; Farooqi and O’Rahilly, 2014). The assumption here is that leptin had a positive effect on the hypothalamic regulation of pituitary

gonadotropin secretion. This may also be true in leptin-treated adult leptin-deficient males, where testosterone and free testosterone reached normal adult values along with the appearance of secondary sexual characteristics (Paz-Filho et al., 2015). In women with an intact leptin gene, and in the absence of organic disease or ovarian failure, hypothalamic amenorrhea is most commonly associated with stress, weight loss or intense athletic exercise (see Section 3.3). Such conditions disrupt normal neuroendocrine processes, induce premature osteoporosis, decrease fat mass and reduce serum leptin levels, resulting in anovulation (Chou and Mantzoros, 2014; Paz-Filho et al., 2015). In two clinical trials, leptin replacement in amenorrheic women reduced body weight and fat mass and significantly elevated luteinizing hormone

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Chapter 4: Neuroendocrine Regulation of Appetite and Body Weight

human leptin increases nocturnal LH secretion in patients with lipodystrophy, suggesting that leptin has a direct effect on the hypothalamic–pituitary system (Abel et al., 2016).

Obesity and Leptin Excess

Figure 4.4 Leptin treatment of an obese child. Left panel, a 3-yearold boy with congenital leptin deficiency (body weight: 42 kg). On the right, the same boy, after four years of daily subcutaneous injections of recombinant human leptin. Leptin induced a striking decrease in body fat (body weight: 32 kg). Reproduced with permission (Farooqi and O’Rahilly, 2014).

(LH), LH pulse frequency and estradiol concentrations. The effect of leptin on LH secretion in two amenorrheic patients is illustrated in Figure 4.5 (Welt et al., 2004). Evidence of ovulation and increased bone health in some patients confirm the involvement of the hypothalamus (Chou and Mantzoros, 2014; Kyriakidis et al., 2016).

Lipodystrophy and Leptin Deficiency Lipodystrophy – the complete or partial absence of fat tissue – is a rare disorder associated with severe insulin resistance, diabetes and hyperphagia (Fiorenza et al., 2011). Most patients with generalized lipodystrophy are leptin-deficient and physiological replacement doses of leptin induce improvements in insulin sensitivity, glucose tolerance and levels of fasting glucose (Paz-Filho et al., 2015). Lipodystrophic patients also exhibit reproductive dysfunction such as amenorrhea, reduced fertility, polycystic ovarian syndrome, hyperandrogenism (in adults) and central hypogonadism in children (Brown et al., 2016). Recombinant human leptin treatment of adult women with this syndrome decreased free testosterone levels, improved insulin sensitivity and induced normal menstruation (Musso et al., 2005). It is possible that leptin replacement acts on the neuroendocrine hypothalamus; that is, recombinant

In contrast to those obese patients with deficient leptin – that is, because of leptin gene mutations (see earlier in the chapter) – obesity in the general population is characterized by increased serum leptin levels due to the enlarged adipose tissue mass. Figure 4.6A illustrates that serum leptin concentrations are positively correlated with percent of body fat and are significantly higher in obese compared with normal-weight individuals (31.3+/-24.1 vs. 7.5+/-9.3 ng/mL; p