Endocrinology Quiz 1 1-4

3a. Hypothalamus-Pituitary Axis

1. Describe the structure of the hypothalamus and pituitary and their vascularization. 2. Explain the functional relationship between the hypothalamus and the posterior pituitary. 3. List the major stimuli for the release of anti-diuretic hormone and oxytocin. 4. Explain the functional relationship between the hypothalamus and the anterior pituitary. 5. List the target tissues and major actions of the anterior pituitary hormones. 6. Explain what controls prolactin secretion. 7. List the hypothalamus releasing factors and their targets. 8. Explain the concept of negative- and positive-feedback controls. 9. Describe the major classes of endocrine disorders at the level of the hypothalamus, anterior pituitary and target tissues.

Pineal Gland - Melatonin

The pineal gland is a midline structure located on the posterior aspect of the third ventricle. Developmentally it arises from the roof of the diencephalon. It accumulates calcium deposits with age which are physiologically insignificant. Calcifications are seen in histological sections as corpora arenacea or brain sand. In sufficiently high-resolution imaging modalities, such as x-rays and MRI, the calcifications are used by radiologists as a stereotaxic mark, roughly at the center of the brain. The pineal gland produces the hormone melatonin which has the primary function of regulating circadian rhythms. Melatonin production is typically low during the day and high at nighttime, and is adjusted through exposure to light. Numerous physiological functions are entrained to circadian rhythmicity, including the secretion of endocrine hormones prolactin, growth hormone, insulin, cortisol and TSH. In animals, melatonin more obviously controls important seasonally varying features of physiology, including fat storage, body temperature, general activity and reproductive behavior. Such of these seasonal variations that may persist in humans are often obscured by our relatively modern tendency to live in artificial light. The connection of the pineal to light-sensing photoreceptors is indirect. The pineal arises from the roof of the diencephalon (epithalamus) in a region evolutionarily linked to the formation of pituitary and optic stalks. However, in humans the physiological connection with the eye is made through the suprachiasmatic nucleus of the hypothalamus, which displays oscillatory activity with high discharge rates during daylight. In humans, such day-night neuronal oscillations will "free-run" in the absence of photic stimulation. The hormone melatonin is released directly into blood and cerebrospinal fluid. Ltryptophan is the important precursor used by pinealocytes to produce melatonin. Melatonin is highly fat-soluble and thus able to move easily through cell membranes. It is broken down by the liver. Receptors for melatonin exist in numerous organs throughout the body, and in the suprachiasmatic nucleus. In animals, the pars tuberalis is a major site of melatonin receptors, where they are thought to regulate seasonal behaviors, but the function of this area in humans is unclear. Two receptor subtypes are known. Functionally, the most obvious role of melatonin is to induce sleep. The plasma level of melatonin required to trigger sleep is fairly constant, though diurnal changes in receptor sensitivity do occur. As a pharmaceutical, melatonin can be used to ameliorate blindness-induced insomnia, jet-lag, and similar sleep disorders, but monitoring to ensure a correct physiological dosage is important to achieving a desirable effect. Historically, associations between pineal-destroying tumors and precocious puberty have been described. Also described are cases of delayed puberty together with high melatonin levels. Such cases suggest a possible role for melatonin in Endo/Repro 4 - 5 [ determining the onset of puberty, although much further study of this function of melatonin in humans is needed.

2) Enzyme linked receptors with single transmembrane domains (e) The MAP kinase pathways (for information, not for memorization) The MAP (mitogen activated protein) kinase pathways refer to cascades of protein kinase reactions that have central roles in signaling cell proliferation in response to growth factor stimulation of eukaryotic cells. Let's consider a well- characterized pathway that uses a MAP kinase, called ERK (extracellular signal- regulated kinase) and can be activated following signaling by insulin as well as other factors. This cascade employs guanine nucleotide binding proteins (Ras proteins) that are anchored to the cytoplasmic side of the cell membrane. Like G-proteins, Ras is predominately bound to GDP under resting conditions. Exchange of GDP with GTP is mediated by guanine nucleotide exchange factors that promote dissociation of GDP from Ras, much like ligand-bound serpentine receptors increase displacement of GDP from the α-subunit of Gproteins. Ras-GTP activates Raf, a serine/threonine kinase that in turn phosphorylates MEK; MEK then phosphorylates and activates ERK that further activates downstream substrates in both the cytoplasm and the nucleusto mediate the cell response (Fig. 4). This cascade is initiated by recruitment to the cell membrane of an adapter protein called Grb2 that binds either directly to an activated tyrosine kinase receptor or to a phosphorylated substrate of the receptor. Grb2 serves as a docking site for the guanine nucleotide exchange factor SOS that is required for Ras activation (Fig.4) From growth factor binding to the final response, each step in the pathway not only provides amplification of the signal but also serves as a potential site for regulation.

(a) Tyrosine Kinase receptors G-proteins do not mediate all biological responses initiated by agonist bound surface membrane receptors. Many receptors (eg. those for insulin, epidermal growth factor [EGF] and platelet derived growth factor [PDGF]) have intrinsic tyrosine kinase activity in their cytoplasmic domains. Signal transduction requires dimerization of the agonist-receptor complexes and phosphorylation of tyrosine residues of the receptor itself. This autophosphorylation increases both the receptor's kinase activity and its affinity for an array of substrates that associate with the receptor's cytoplasmic domain and are subsequently phosphorylated. These phosphorylated substrates participate in the biological response induced by the agonist. (b) Serine/threonine kinases This class of receptors resembles tyrosine kinase receptors in that after ligand binding, they dimerize and phosphorylate cytoplasmic substrates called "Smads". After they are phophorylated, Smads move to the nucleus and participate with other gene regulatory proteins in influencing transcription of target genes. Mullerian inhibitory substance and inhibin, hormones that you will learn about later in this course, interact with receptors that are members of this family. (c) Guanylate cyclase receptors The receptor for atrial natriuretic peptide (ANP) has been shown to have guanylate cyclase activity in its cytoplasmic domain. Binding of ANP to its receptor increases cytoplasmic levels of cGMP that mediate actions of the hormone. Some of these actions result from cGMP activation of protein kinase G. (d) Cytokine receptor family Receptors for growth hormone and prolactin belong to a large superfamily of receptors that include erythropoeitin and other hematopoietic growth factors. This family of receptors lacks intrinsic enzymatic activity. However, they associate with soluble cytoplasmic tyrosine kinases (JAKs) after they bind ligand and dimerize. When JAKs interact with the cytoplasmic domains of the dimerized receptor, they become activated and phosphorylate factors called STATs (signal transducers and activators of transcription) that move to the nucleus and regulate transcription of specific genes.

Qs

1. A number of diseases result from an insufficiency or lack of enzymes or other proteins involved in steroid hormone synthesis. Many of these diseases can be treated by giving the patient the end product (steroid hormone) of the disrupted pathway(s). Which steroid hormone(s) would you give a patient who lacked: A. 17 alpha hydroxylase B. 11 beta hydroxylase C. Aromatase D. Aldosterone synthase E. DHEA F. StAR 2. 21-alpha-hydroxylase (CYP21) deficiency is the most common form of congenital adrenal hyperplasia (CAH), and results in masculinization of external genitalia in females and early virilization in males. Taking the biosynthetic pathways into consideration, what is the most likely explanation for this? 3. Cushing syndrome is caused by sustained and pronounced hypersecretion of the glucocorticoid cortisol. It can occur through a number of mechanisms including excess production of the adrenocorticotropic hormone (ACTH - produced in the pituitary) and ACTHproducing tumors. Surgery is one way to treat these two disease mechanisms, but in some cases that is not an option. If you were to design a drug to help such patients what would you do?

3b. Growth Hormone and Endocrinology of Growth

1. Describe the effects of growth hormone and its relationship to insulin-like growth factors. 2. Explain the control of growth hormone secretion. 3. Describe the effects of other hormones on growth. 4. Describe factors that determine the target height

4. Anatomy and Embryology of Endocrine Organs

1. Describe the structure and origin of major endocrine cells and glands. 2. Compare the pineal gland with other endocrine systems. 3. Describe the embryology of the pituitary, adrenal gland and pancreas. 4. Describe the relevance of fat depots to endocrine signaling and immune function.

qs

1. What is the difference between long-loop and short-loop negative feedback? 2. What is the functional difference between the anterior and the posterior pituitary? 3. What is unique about prolactin secretion?

Figure 3. Steroid hormone synthesis begins in the inner mitochondrial membrane Figure 4. Overview of Steroid Hormone Biosynthesis Pathways (dashed arrows indicate alternate pathways of synthesis). 17--hydroxylase (CYP17) an important enzyme with two activities 17--hydroxylase (CYP17) - this enzyme has two activities: 1. Hydroxylation activity: It catalyzes the hydroxylation of carbon 17 (C17) of progesterone or pregnenolone. 2. C17-C20 lyase activity: It can catalyze the cleavage of the 2-carbon side chain of progesterone or pregnenolone at C17. These two activities allow the biosynthesis of steroid hormones to occur via two separate pathways; one that allows 17 hydroxylated steroids (precursors of cortisol; C21) to retain their side chains and one where the side chain is cleaved (precursors of sex hormones; C19).

5. 11-deoxycortisol is then transported back into the inner mitochondrial membrane, where it is hydroxylated by 11--hydroxylase to form cortisol. 6. Cortisol then exits the cell.

Modulation of Cellular Responses Hormone dependent responses are affected both by availability of free hormone and by the sensitivity of cells to a given level of hormone.

A) Factors influencing hormone availability. Secretion rates, uptake and degradation rates influence circulating free hormone levels, as well as the concentrations of plasma carrier proteins that transport steroids and thyroid hormones. Since free hormone, the biologically active form is in equilibrium with carrier-bound hormone, increases or decreases in carrier protein concentrations can result in reciprocal changes of free hormone levels. In the rat, α-fetoprotein, a plasma protein that binds testosterone and estradiol with high affinity, gradually declines from very high values in the newborn to very low values just before puberty. In this manner, increasing quantities of free sex hormone are available as the animal develops. Negative feedback loops play a central role in regulating rates of hormone secretion. A hormone-stimulated tissue may produce a product that turns off further hormone secretion. For example, thyroid hormone production stimulated by pituitary TSH (thyroid stimulating hormone) inhibits further TSH release. Alternatively, a hormone-activated tissue may correct a physiological disturbance, thereby removing the original stimulus for hormone secretion. Insulin release in response to elevated blood glucose levels, for example, stimulates glucose uptake by liver and muscle, thus returning blood glucose to normal values. B) Factors influencing cell sensitivity 1) Negative cooperativity In some systems (e.g. insulin binding to lymphocytes) the binding of a hormone molecule to a receptor influences the affinity of neighboring binding sites. When increasing receptor occupancy decreases the affinity of remaining receptors for hormone, the process is called negative cooperativity. This mechanism may modulate hormone action by providing high cell sensitivity (receptor affinity) at low hormone concentrations and low sensitivity at high hormone concentrations. 2) Down regulation Exposure of responsive cells to high concentrations of hormone has in some systems been demonstrated to decrease the number of surface membrane receptors. Loss of binding sites has been called down regulation. This phenomenon may protect the cell from intense stimulation by chronically elevated hormone levels. For example, decreased sensitivity of responsive cells to insulin has been demonstrated in clinical obesities characterized by high circulating insulin levels. 3) Other Decreases in target cell sensitivity have been observed to result from other factors as well: e.g. uncoupling of receptor from G-protein or decreases in the activity of one or more enzymes several steps removed from the hormone-receptor interaction. Endo/Repro 1 - 11 Uncoupling of receptor from G-protein has been observed in the β-adrenergic system in which phosphorylation of the cytoplasmic domains of the receptor reduces its affinity for G-protein. Phosphorylation is mediated by β-adrenergic receptor kinase, or β-ARK, which associates with free βγ at the membrane and is positioned to phosphorylate the receptor. A cytoplasmic component, β-arrestin, then binds to the phosphorylated receptor and blocks its interaction with G-protein. The higher the hormone concentration, the greater the concentration of βγ in the membrane, and the greater is the potential for desensitization.

Mechanism of Action of Hormones

A) Receptors for peptides, glycoproteins and amino acid derivatives. Peptide hormones and amines are too polar to passively diffuse through lipoprotein membranes and too large to pass through membrane pores. They initiate their response at the outer surface of target cells by binding to glycoprotein receptors anchored within the plasma membrane. Membrane receptors for these water soluble hormones can be generally assigned to one of two groups: a) those predicted to have 7 membrane-spanning domains (serpentine receptors) and b) those with a single membrane-spanning domain.

Intro

A. Overview There are five classes of steroid hormones. Two are corticosteroids: Glucocorticoids and mineralocorticoids, which are produced primarily in the adrenal cortex. Three are sex steroid hormones: Testosterone, which is produced primarily in the testes, and estrogens and progesterone, which are produced primarily in the ovaries. However, the sex hormones are also produced in lesser amounts in the adipose tissue and adrenal glands in males and females, and by the placenta during pregnancy. Steroid hormones differ from peptide hormones in that they are not encoded by genes. All steroid hormones are made from cholesterol (Figure 1). They are lipophilic and thus can pass through membranes, leaving the cell shortly after synthesis and traveling through the blood stream bound to carrier proteins to target tissues.

B. Biosynthesis of Steroid Hormones

All steroid hormones are made from cholesterol derived from two sources: Receptor mediated uptake of Low Density Lipoprotein (LDL) by the LDL-receptor, which is converted to esterified cholesterol and then free cholesterol. Cholesterol can also be synthesized in a multistep process from acetyl CoA. A key rate-limiting enzyme in this process is HMG-CoA reductase (3-hydroxy-3- methyl-glutaryl-CoA reductase), which is inhibited by cholesterol in a negative feedback mechanism. The first step in the synthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by the enzyme 20α,22 desmolase (Figure 2). This enzyme is also known as CYP11A (new name) and P450SCC (old name). The new name for this enzyme (as well as the other enzymes involved in steroid hormone biosynthesis) is based on the nomenclature for the genes that code for enzymes in the cytochrome P450 family (CYP) that are involved in the synthesis and metabolism of a wide range of molecules.

An example of a Receptor Defect: Androgen insensitivity syndrome

Androgen insensitivity syndrome is a dramatic example of the impact of a receptor defect. In this disorder,r an individual fails to synthesize a functional androgen receptor. A consequence is that during development, the testis of an XY individual produces testosterone, but those target tissues that depend on this hormone for differentiation, i.e., the vas deferens, seminal vesicles, seminiferous tubules, fail to develop. The testosterone metabolite, dihydrotestosterone, required for differentiation of the prostate and formation of male external genitalia, is also ineffective because it uses the same receptor. The result is the birth of a baby that looks like and is raised as a female. Endo/Repro 1 - 12 At puberty. the pituitary gland begins to increase the production of luteinizing hormone (LH) that stimulates the testis to produce more testosterone. The normal regulatory feedback loop whereby elevated levels of testosterone inhibit further secretion of LH, however, is inoperative in the absence of a functional testosterone receptor. Testosterone continues to rise to levels that far exceed those of normal males at puberty, but no male secondary sex characteristics develop. Paradoxically, the individual develops the phenotype of a mature female, because of the conversion of a small percentage of testosterone to estrogen by aromatase, an enzyme found in peripheral tissues. The receptor disorder is usually detected at puberty when menstruation fails to occur.

Endocrine disorders

As you may have guessed by the 3-hormones sequence, the three sites of production (hypothalamus, pituitary and third endocrine or target gland) and the requirement for receptors on the target tissue, the potential for dysfunction can be multiple. There are three major categories of endocrine disorders: hyposecretion, hypersecretion and hyporesponsiveness of the target cells. 1. Hyposecretion: may be primary or secondary. Primary: The endocrine target gland secretes too little hormone, because it is not functioning normally. For example, decreased cortisol secretion by adrenal gland because of the genetic absence of a steroid-forming enzyme, or decreased secretion of thyroid hormones (T.H.) by thyroid gland for lack of iodine. Secondary: The endocrine target gland may be fine but its tropic hormone, produced by the pituitary, is too low. For example hyposecretion of T.H. due to low TSH secretion by pituitary 2. Hypersecretion: may also be primary or secondary. Primary: Dysfunctional gland secreting too much hormone. The most common cause is tumor of the target gland. For example hypersecretion of T.H. due to tumor of the thyroid gland. Secondary: Excessive stimulation by tropic hormone. For example hypersecretion of ACTH due to a tumor of the corticotropes (Cushing's disease). 3. Hyporesponsiveness: the target cells do not respond to the third hormone. Three major causes: a. Lack or deficiency of receptors for the hormone. For example in type II diabetes, insulin is produced but few insulin receptors exist. (No insulin is produced in type I diabetes). b. Post-receptor defect in target cells. Normal receptor but activated receptor unable to cause, for example, formation of a 2nd messenger (e.g. cAMP) or open a membrane channel. Lack of metabolic activation of a hormone. For example, normal secretion of testosterone (T) and normal receptors for T and dihydrotestosterone (DHT, metabolite of T active in target cells), but lack of enzyme (5α-reductase) that converts T to DHT. This results in a lack of virilization of the urogenital sinus and the external genitalia during embryogenesis (congenital 5α-reductase deficiency). In hyporesponsiveness the plasma hormone concentration is normal or elevated, but the response to administered hormone is low. Hypophysectomy in experimental animals is not deadly, but widespread changes are predictable in terms of known hormonal function of the gland, especially lack of secretion of tropic hormones. Most of the target gland atrophy: thyroid, adrenal, gonads; sexual cycles stop. The most dramatic effect is that growth is inhibited

Structure and Location of Endocrine Organs

By definition, an endocrine cell is one that secretes its product into a connective tissue layer for the purpose of signaling to a distant site. Closely related are the concepts of paracrine secretion, in which a cell affects its close neighbors, and autocrine secretion, where the secretion affects the original cell itself. (As an example, a neurotransmitter that binds to a receptor on the cell that released it may cause feedback inhibition of more neurotransmitter release by that cell.) Transport of an endocrine product may rely simply on diffusion, but may also (especially in the case of steroid hormones) rely on carrier proteins to bind, concentrate and transport them. The target of an endocrine hormone is a specific receptor that may be located on a cell membrane, or intracellularly in the target cell. All endocrine cells have an epithelial origin. In adult epithelia such as skin, GI system and lung, endocrine cells remain a scattered, minority cell type within the epithelium. Many endocrine organs, such as the anterior pituitary, parathyroid, endocrine pancreas and others consist of bundles of cells that delaminate from their epithelium of origin. These cells effectively lose their apical surface, but stick together in gland-like structures absent of any lumen. There are at least two examples of organs in which the endocrine cells form a new epithelioid structure, i.e. cells with a functional apical/basal polarity of their surface. In the thyroid, cells delaminate from an embryonic epithelium, but an apical cell surface is retained in cells which then organize to form closed spherical structures known as thyroid follicles. The follicular lumen, from which there is no exit except back through the surrounding endocrine cells, is used to store an intermediary compound that binds the trace element iodine. Doing so enables the organism to survive through relatively long periods of dietary iodine deficiency. In the ovary, the follicular / granulosa cells that surround a developing oocyte also retain an apical/basal orientation. The basalsurface, asin all endocrine glands, separates the endocrine cells from the surrounding tissue by elaborating a basement membrane, which in the ovarian follicle effectively contributes to the physical and biochemical isolation of the oocyte.

Embryology of Adrenal Gland

Each adrenal gland consists of a cortex derived from mesenchyme and a medulla into which neuronal crest cells migrate. The cortex is formed from coelomic mesoderm adjacent to the dorsal mesentery beginning at about week 4. This region of proliferating tissue begins as small buds of mesenchyme that separate from the epithelium. Migrating neural crest cells invade the developing buds and venous sinusoids form internally. The neural crest cells form the chromaffin cells in the interior of the gland. Capillaries that penetrate the cortex in a radial manner join the internal sinusoids. The early mesenchyme forms a fetal cortex that includes the zona glomerulosa and a fetal cortex that functions during fetal life. The zona glomerulosa and an underdeveloped zone fasciculata are present at birth but the zona reticularis proliferates after birth and is present by year 3. The final phase of adrenal development usually precedes the onset of puberty by a couple of years. Neural crest cells that form the medulla are similar in morphology, location and secretions to ganglia from the sympathetic chain, leading to the hypothesis that the adrenal medulla originates in evolution from a "modified sympathetic Endo/Repro 4 - 7 [ ganglion." In fact, endo-adenogenic tissue persists along the paravertebral and para-aortic axes through fetal life, and is postulated to contribute to vascular tone in the fetus before the adrenal medulla is fully developed. These collections of endocrine tissue are known as the Organs of Zuckerkandel, and are important to remember as a potential source of neuroblastomas and paragangliomas. These rare but deadly diseases often masquerade as pheochromocytoma (adrenal medullary tumor) and will elude the unprepared physician, as surgical excision of the these minor organs is required to maximize patient survival

The brain and endocrine axes: Hypothalamus, Pineal, Pituitary

Endocrine axis describes the traditional system by which the brain exerts endocrine control on a given body system. A canonical axis starts in the hypothalamus with a secretion of a releasing hormone. These hormones cause the release of a pituitary adenohypophyseal hormone which then targets a peripheral endocrine gland producing a third hormone controlling a body function such as metabolism or reproductive cycle. Hormone levels in the blood typically serve as negative feedback for pituitary and/or hypothalamus. Blood-brain barrier: Throughout the brain, the blood brain barrier is formed by tight junctions among vascular endothelial cells, the endothelial basal lamina, and astrocytic end feet forming a cellular barrier that separates the connective tissue space surrounding the vasculature from the extracellular space surrounding neurons and glia. In addition, capillaries throughout the brain are continuous, requiring most substances to rely on regulated caveolar transport for passage. The circumventricular structures, especially choroid plexus and pineal gland, the posterior pituitary and median eminence, are exceptions to the general structure of the blood-brain barrier. Capillaries in these regions are fenestrated, and blood [ Endo/Repro 4 - 4 flow is typically higher in these regions than in the rest of the brain. The choroid plexus takes advantage of the efflux of plasma from such vessels to provide the volume of CSF. Also in these regions, a specialized cell, the tanycyte, works to separate plasma and CSF compartments. In the case of the pituitary, blood flow is largely in the outward direction - that is, once encountering a region of fenestrated capillaries, blood does not flow back into central brain regions. This vascular arrangement serves to concentrate hypothalamic releasing hormones in the adenohypophysis, above their levels found in circulating blood.

Mode of Action of GH: Somatomedins

GH exerts its effects indirectly through mediation by chemical messengers whose synthesis and release are induced by GH: the polypeptides IGF I and -II (insulin like growth factor I and II), or somatomedin C. Of the two, IGF-I appears to be the most important mediator of the actions of GH. IGF-II may be involved during fetal development, although insulin also plays an important role (see further). GH acts on liver and other tissues to secrete IGF I into the blood and locally. In general plasma concentrations of IGF-I reflect the availability of GH and/or the rate of growth. Children whose growth is more rapid than average have higher than average concentrations of IGF-I, whereas children at the lower extreme of normal growth have lower values. Children or adults who are resistant to GH because of a receptor defect have low plasma concentrations of IGF-I despite high concentrations of GH. Growth of these children is restored to nearly normal rates following daily administration of IGF-I. Therefore in patients with a GH receptor defect treatment with IGF-I reproduces most of the effects of GH on body size and composition. The IGF receptor has tyrosine kinase activity, similar to the insulin receptor. Overwhelming evidence indicates that IGF-I stimulates cell division in cartilage and many other tissues and accounts for much and perhaps all of the growth-promoting actions of GH. The original somatomedin hypothesis postulated an important role for plasma IGF-I (secreted principally by the liver) in promoting growth. Recent evidence, however, suggests that IGF-I acts locally in an autocrine or paracrine manner to stimulate cell division and bone growth and that IGF-I in the circulation plays only a minor role, if any, in stimulating growth. The current Endo/Repro 3b - 3 view is that GH acts directly on both the liver and its peripheral target tissues to promote IGF-I production. The liver is the main source of IGF-I in blood, and its function is to regulate GH secretion by negative feedback. Both IGF-I and II are present in blood at relatively high concentrations throughout life and circulate in blood tightly bound to IGF binding proteins. Consequently, the IGFs do not readily escape from the vascular compartment and have half lives in blood of about 15 hours.

Control of GH Secretion

GH is secreted in episodic bursts, with the largest secretion associated with the early hours of sleep. Whereas males secrete most of their GH during sleep (particularly during a period called deep slow wave sleep), females secrete more GH during the day (Fig. 1). Little information or diagnostic insight can, therefore, be obtained from a single random measurement of the GH concentration in blood. A more meaningful evaluation is to obtain a 24-hour integrated concentration of GH in blood. In addition to spontaneous pulses, secretory episodes are induced by stress, exercise, fasting, low plasma glucose, and certain amino acids like arginine and leucine. These signals can be used as provocative tests for judging competence of the GH secretory apparatus. GH secretion, though most active during the adolescent growth spurt, persists throughout life. Changes in GH secretion with age primarily reflect changes in the magnitude of secretory pulses. As quality of sleep deteriorates in the elderly, the amount of GH decreases, and this may be related to loss of lean body mass in later life. This effect may be pronounced in men, since they secrete most of their GH in the early hours of sleep. Effect of GHRH and somatostatin: GH secretion is stimulated by GHRH and inhibited by somatostatin (S-S). GHRH provides the primary drive for GH synthesis; in its absence secretion of GH ceases. Somatostatin reduces or blocks secretion of GH in response to GHRH but has little influence on promoting GH synthesis in Endo/Repro 3b - 4 the absence of GHRH. GH secretion is controlled by classical long- and short-loop negative-feedback mechanisms (Fig. 2). During sleep for example it is unclear whether GHRH secretion increases or S-S decreases. Pulsatility appears to be the result of intermittent secretion of both GHRH and S-S. Plasma IGF-I can also inhibit GH secretion by the pituitary by inhibiting the stimulatory effect of GHRH. Receptors for GHRH, S-S and IGF-I are present on the surface of somatotropes and control the level of cAMP which mediates GH synthesis and secretion.

Genetic Factors and Target Height

Genetic factors influence final height. Correlation is found between Mid-Parental Height (MPH) and the child's height. There is a heritable pattern to birth length, postnatal increase in length, and the intrinsic rate of change in growth. These effects are sex-specific. Fig. 5 shows determination of target height in a case of familial short stature. An example is given of a 10-year-old boy who is 49 inches tall (124 cm, all measurements are actually done in cm), his mother is 61 inches tall and the father is 63 inches tall. The Mid-Parental Height is calculated by adding the father's height to Endo/Repro 3b - 7 the mother's height and the sum is divided by two; 2.5 inches are added to convert to the equivalent percentile on a boy's chart (2.5 inches would be subtracted for a girl). The mother and father height are plotted at the far right of the chart where adult heights are displayed; the result is the Target Height or MPH (~65 inches in this case). The limits of 2 SD above and below the target height are displayed by plotting 2 SD (approximately 4 inches above and below the target height). This process is equivalent to moving the 50th percentile for the U.S. population to a conceptual 50th percentile for the family under consideration. It is evident that the height of the child, while below the third percentile, is within the bounds of the percentiles described by 2 SD from the Target Height and the child appears to fit within the genetic pattern of the family. The growth velocity and the degree of skeletal maturation are some of the factors necessary to evaluate this analysis in more detail. Another trait that can be inherited (or can also be idiopathic) is of constitutional delay, in which the growth curves can deviate from the normal pattern shown above. This is the most common cause of short stature and pubertal delay. Such a child may follow or fall below the average growth curve in earlier years, but instead of following the curve at lower level, as in familial short stature, with constitutional delay the growth crosses and falls below the normal curves, especially during the period of normal pubertal growth. This is not a pathological condition, Endo/Repro 3b - 8 but a variant of normal growth. The child will normally undergo a late puberty with a growth spurt and catch up with his cohort. The most common problem for such children is usually psychological as they do not follow the growth of their peers, and if this is a sufficient problem it may warrant treatment.

The Concept of Feedback Control

Given that hypothalamic releasing factors control anterior pituitary function, what controls secretion of the releasing factors? There are two types of control on hypothalamic neurons, neural and hormonal. Neural control includes all kinds of input from other areas of brain (e.g. effect of stress). In the case of hormonal secretion control, the end-product, in this case the secreted hormone, exerts a negative-feedback upon the hypothalamic-pituitary system to reduce its own secretion (Fig. 4). Some feedback loops are positive, e.g. increased secretion of LH by estrogen. In Fig. 4, the situation in which hormone-3 exerts influence upon the pituitary and/or hypothalamus is referred to as long-loop negative-feedback. In this case hormone-3 can directly act on pituitary to make it less responsive to hormone-1 (releasing factor) and also on the hypothalamus to reduce the frequency of action Endo/Repro 3a - 8 potentials in neurons secreting hormone-1. Fig. 5 shows an example of negativefeedback control for the CRH-ACTH-Cortisol sequence. In cases where no hormone-3 is produced, hormone-2 will exert control over its own release through a short-loop feedback (Fig. 4). For example, prolactin and growth hormone do not influence an endocrine gland (but see further for GH), they therefore exert a feedback control directly over the hypothalamus.

The Hypothalamo-Hypophysis Axis and Control of Hormone Release The pituitary and the endocrine glands are not autonomous; the pituitary is under influence of the CNS. For example, stress and certain emotions are known to influence secretion of hormones (e.g. ACTH; psychological amenorrhea). This influence of the CNS on the pituitary is through humoral factors released by the hypothalamus. Recall that hypothalamic neurons terminate in median eminence and neural stalk and that the anterior pituitary receives its blood supply through the portal vein. The hypothalamus influences the pituitary through blood-borne factors called hypothalamic releasing hormones (or factors). Each releasing hormone is secreted by a particular group of hypothalamic neurons and influences the release of one or more anterior pituitary hormones (Fig. 3). Some releasing hormones are stimulatory while others are inhibitory. For those affected by more than one releasing hormone (e.g. GH, prolactin) the pituitary response depends upon the relative amount of opposing hormones

Gonadotropin Releasing Hormone (GnRH or LHRH) is a decapeptide. It is concentrated in the medial basal hypothalamic nuclei. It stimulates pituitary synthesis and secretion of LH and FSH. Response to GnRH is modulated by the sex steroids. High, steady levels (as opposed to cyclical levels) of GnRH block gonadal steroidogenesis (possible contraceptive?). GnRH binds to specific pituitary cell plasma membrane receptors and activates phospholipase C (PLC). Growth Hormone Releasing Hormone (GHRH) is a relatively large polypeptide (40-44 AA) which stimulates GH synthesis and release from pituitary directly. It was initially isolated from a human pancreatic tumor. Somatostatin (S-S, GIH) is a tetradecapeptide which inhibits release of GH from normal pituitary and from GH-secreting pituitary tumors. It is also released by D cells of islets of Langerhans. Thyrotropin Releasing Hormone (TRH) is a tripeptide. It stimulates pituitary synthesis and secretion of thyroid stimulating hormone (TSH) and prolactin. TRH-induced TSH response decreases with age in men but not in women. TRH binds to specific plasma membrane receptors on the pituitary thyrotropes and activates phospholipase C. TRH is in greatest concentration in the hypothalamus. Endo/Repro 3a - 7 Prolactin Inhibiting Factor (PIF) is most probably the neurotransmitter dopamine (DA). It tonically inhibits the secretion of prolactin. Prolactin is unique among the anterior pituitary hormones in that its secretion is increased rather than decreased when the vascular connection between the pituitary gland and the hypothalamus is interrupted. Corticotropin Releasing Hormone (CRH) is a large polypeptide (41 AA) which stimulates pituitary ACTH synthesis and release. There may be more than one hypothalamic factor that stimulates ACTH release. Vasopressin may stimulate ACTH release directly or indirectly via CRH.

Intracellular receptors Steroid hormones (and thyroid hormone, vitamin D and vitamin A derivatives) act by regulating gene expression in responsive cells. Since these ligands are lipid soluble, they presumably can passively diffuse across cell membranes. They bind to receptors that are located in the cytoplasm or the nucleus of a target cell and form hormone/receptor complexes that bind to DNA. Hormone binding to steroid receptors first causes the displacement of associated proteins, such as heat shock proteins (eg. HSP90) that prevent the receptors from interacting with DNA, when ligand is absent. Newly formed hormone/receptor complexes then dimerize and, in the case of cytoplasmic receptor/hormone pairs, translocate to the nucleus. There they interact with DNA at sites called hormone response elements (HREs).

HREs have palindromic sequences (inverted repeats), ie. 5'-3' sequence identities in complementary strands of DNA, or they have direct repeats. The dimerization of the hormone-receptor complexes apparently favors the interaction of each of the receptor subunits with one of the two identical half-sites in the HRE. Binding of the receptor-hormone complex to HREs may initiate or suppress transcription of nearby genes under HRE control. In contrast to steroid hormone receptors, those that bind thyroid hormone, vitamin D or vitamin A derivatives associate with hormone response elements even in the absence of hormone. These receptors are not associated with HSP90, but with nuclear proteins that modulate transcription of downstream genes. Upon hormone binding conformational changes in the receptor cause these nuclear proteins to dissociate and to be replaced with others that promote activation or suppression of transcription of target genes. Intracellular receptors share regions of strong sequence homology. The receptors have three structural domains: a C-terminal hormone binding region, a central, highly conserved DNA binding domain, and a variable N-terminal domain that participates in recruitment of transcription factors. The DNA binding domains, which are rich in cysteine residues and recognize specific HREs, are proposed to be unmasked upon interaction of the hormone-binding region with its appropriate ligand. (See Fig.5)

Single epithelial cells You have seen previously how single endocrine cells can control the function of an organ system and provide feedback to the brain.

In the lung, the neuroendocrine cell (DNES cell, or small granule cell) is the first cell type to differentiate during development. They are recognizable in the pseudoglandular period and are thought to play an important role in controlling cell division and differentiation. Their function in the adult lung is less clear; evidence suggeststhey serve as protectors of the stem cell niche, and are important in the regulation of airway inflammation. Their most obvious clinical relevance is as the cell of origin in small cell lung cancer, which represents 15-20% of all primary lung cancers. Another 15-20% of (non-small cell) lung cancers display "neuroendocrine differentiation", meaning they acquire morphologies reminiscent of endocrine cells. Endo/Repro 4 - 3 [ In the GI system, the enteroendocrine cell (EEC) represents a diverse population of cells arranged in an organ-specific manner that control elements of digestion, motility and satiety. Examples are: gastrin (G cells, pyloric stomach) regulation of gastric acid secretion and epithelial proliferation CCK (intestinal epithelium, neurons) control of digestion in duodenum, targeting pancreas and gall bladder somatostatin (D cells, pylorus, duodenum, pancreatic islets, neurons) inhibition of insulin, glucagon & growth-hormone ghrelin (gastric body) regulation of appetite

Hormone Classification Hormones are chemical substances synthesized and secreted by one group of cells to influence one or more other groups of cells. They include peptides, glycoproteins, steroids and amino acid derivatives (Table l).

Properties of Hormone-Receptor Interactions To initiate a response, hormones must bind to receptor molecules found on or in target cells. This interaction has several characteristics: 1) It is highly specific. The physiological concentrations of circulating hormones range from 10-7 to l0- 12M. A receptor must therefore recognize one chemical messenger out of a vast excess of other molecular species. 2) In many cases, it is a simple, bimolecular, reversible reaction. H + R ↔ HR, where H=free hormone, R=free receptor, and HR=hormone receptor complex or "occupied" receptor. 3) It is saturable. Since there are finite numbers of receptors in cells or on their surfaces, there is a maximum hormone binding capacity. If we assume that a biological response is related to the number of hormone-receptor complexes formed (i.e. to receptor occupancy), then there must also be a maximum biological response. In some responsive cells, the biological response, expressed as a percentage of the maximal response, is equal to the percentage of receptors occupied with hormone. Endo/Repro 1 - 3 Examples exist, however, in which the biological response may be maximal when only a fraction of the receptors are occupied. Those extra or spare receptors increase the sensitivity of a cell to a given level of hormone. 4) It is of high affinity. In order for hormone-receptor complexes to form in the presence of very low circulating hormone levels, the equilibrium association constant (KA) for the reaction H + R ↔ HR must be very high. KA = [HR]/[H][R] where [HR] represents the concentration of hormone receptor complex (ie. occupied receptor) and [H] and [R] are the concentrations of free hormone and free (unoccupied) receptor, respectively. KA's typically are about 1010M-1, but may be as low as 107M-1 or as high as 1012 M-1. Frequently, the hormone/receptor interaction is defined by its KD, the equilibrium dissociation constant. KD = 1/KA = [H][R]/[HR]. The latter expression is easily used to calculate the receptor occupancy for any given free hormone concentration [H]. If, for example, the KD is 10-10M and the free hormone concentration [H] is also 10-10M, [R]/[HR] = 1, and [R] and [HR] are equal. Thus, when [H] = KD, half of the receptors will be bound to hormone and half will be free. A ten-fold increase in the free hormone concentration above the KD results in receptor occupancy of roughly 90%. A free hormone concentration that is 1/10th the KD would result in close to 10% occupancy. Nearly the full range of receptor occupancy, therefore, can be obtained by varying the free hormone concentration by one order of magnitude above or below the KD. 5) It occurs only in responsive tissue. In general specific binding of a hormone is found only in tissues known to be sensitive to that hormone.

Hormone Inactivation Internalization of hormone-receptor complexes

Some ligands (eg. insulin, EGF, nerve growth factor), together with their receptors, have been shown to be internalized by a mechanism called receptor mediated endocytosis. Hormone-receptor complexes are found to cluster in regions of the membrane called coated pits that invaginate and pinch off from the membrane to form coated vesicles. The membrane coat is a network that is formed largely by clathrin, a protein that lies against the inner surface of the membrane. Protein complexes, adaptins, are also found in the coat and are thought to recognize cytoplasmic domains of the receptors and to trap them within the coated pit. Although this process generally targets the hormones (and in some cases, the receptors) to the lysosome, where they are degraded, it has been suggested that internalized hormones, receptors or their degradation products may, in some cases, act within the cell to further mediate a biological response. Receptors that are not degraded can be recycled to the cell surface. Peptide hormones are thought to be inactivated by proteases on the cell surfaces of target tissues. Some hormones are internalized, as described above, and transported to lysosomes where they are degraded. Steroid hormones are inactivated in the liver, where enzymes in the smooth endoplasmic reticulum convert them to polar derivatives that are filtered but not reabsorbed by the kidney.

Fat depots Adipocytes are an important source of endocrine hormones that influence energy metabolism. These include leptin and adiponectin. Adipose tissue is stored in specific locations throughout the body. It has recently been appreciated that adipocytes do not represent a homogenous group of cells. Adipocytes show regional variation in their properties, such as gene expression hormone production and hormonal responsivity. Insulin sensitivity of adipocytes, for example, is known to vary greatly with their anatomical location. Sex steroids regulate the insulin sensitivity of adipocytes, leading to sex differences in, for example, rates of type II diabetes. Replication potential also varies greatly by location, being greater in subcutaneous than in visceral regions. Recently, it has been recognized that adipose tissue inflammation correlates with obesity, and plays a role in comorbidities. Obesity causes apoptosis in adipocytes, leading to chronic production of inflammatory cytokines and a generalized inflammatory state. Obesity is also known to reduce the levels of adiponectin. Adiponectin reducesinsulin resistance, is anti-inflammatory, and has been associated with lower risk of atherosclerosis and diabetes.

Some major sites of adipocyte accumulation are recognized: Subcutaneous depot - fat of the hypodermal layer. Subcutaneous fat is the major component of the dimples and folds that are colloquially referred to as cellulite. This depot is relatively increased in women, with aging, with inactivity, and with pregnancy, and also has a genetic factor. Visceral depots: omental depot - Omental fat is a subset of visceral fat. The omentum is the tissue connecting the stomach to the peritoneal wall, through which runs the vasculature, lymphatics and nervous supply to and from the gut. The omental fat depot typically hangs down anteriorly and produces much of the girth increase of obesity. mesenteric depot - Mesenteric fat is the other major subset of visceral fat. The mesentery is the tissue connecting the intestines to the peritoneal wall. perirenal depot - immediately surrounding the kidney capsule, renal pelvis and adrenal gland, often infiltrating the renal sinus, out to the level of Gerota's fascia. epicardial depot - primarily in the subepicardial layer of the heart, and surrounding the major coronary vasculature. Important in coronary disease. retroperitoneal depot - in the plane between the parietal peritoneum and adjacent structures, separated from perirenal depot by Gerota's fascia. gonadal depot - surrounding the perimetrium in females, or epididymis in males. It's relative importance in humans is not well studied, but this depot in lab animals, particularly mice, is better studied and known to have unique properties. Some organs such as bone marrow and thymus involute, meaning their stromal tissue is nearly completely replaced by adipose tissue. And some tissues show a gradual adipocyte infiltration with aging or inactivity, for example with the accumulation of intermuscular fat. [ Endo/Repro 4 - 6 Liposuction, the surgical removal of adipose tissue, has both cosmetic and medical uses. Cosmetically, it is best used for "body sculpting" in which areas of subcutaneous fat are targeted. Liposuction for weight loss is best used as a "last resort" choice in the morbidly obese when needed to acutely improve their cardiovascular physiology. Weight loss due to liposuction is usually temporary, so successful treatment of obesity relies on treating the underlying condition; diet and exercise if the cause is overeating, or endocrine therapy if the cause is imbalance of metabolic hormone

The first step in the synthesis of all steroid hormones is the conversion of cholesterol to prenenolone

Synthesis begins in the mitochondria of steroidogenic tissues and occurs in the inner mitochondrial membrane (see step 1 in Figure 3) Cholesterol is transported into the mitochondria and moved from the outer mitochondrial membrane to the inner mitochondrial membrane via Steroidogenic Acute Regulatory Protein (StAR). How StAR facilitates this transport is unclear although it appears to act on the outside of the mitochondria and its entry into the mitochondria ends its function. The cholesterol transport into the mitochondria by StAR is a rate-limiting step in steroid hormone biosynthesis, and defects in this process result in a number of conditions such as lipoid congenital adrenal hyperplasia (CAH) and familial glucocorticoid deficiency type 3 (also known as nonclassic lipoid CAH). A series of enzymatic steps in the mitochondria and endoplasmic reticulum then convert pregnenolone into all of the other steroid hormones and their intermediates (Figure 4).

Synthesis of Aldosterone (Figure 6)

Synthesis of Aldosterone (Figure 6) Synthesis of aldosterone occurs in zona glomerulosa of adrenal cortex. 1. As described for cortisol biosynthesis, cholesterol is converted in 2 steps to progesterone. 2. Progesterone is then converted to aldosterone through hydroxylation and oxidation steps catalyzed by 21--hydroxylase and the CYP11B system i.e. progesterone is hydroxylated to 11-deoxycorticosterone (DOC) by 21--hydroxylase (CYP21) which is then hydroxylated by 11--hydroxylase (CYP11B1) before being oxidized by aldosterone synthase (CYP11B2) to form aldosterone, which then exits the cell. Figure 6. Synthesis of Aldosterone. Modified from: Chapter 41. The Diversity of the Endocrine System, Harper's Illustrated Biochemistry, 29e, 2012

Testosterone Metabolism (Figure 9) Steroid Hormone Receptors as Drug Targets Androgen Receptor Antagonists e.g. Flutamide for treatment of prostate cancer Progesterone Receptor Antagonists e.g. RU486 for contraception Aldosterone Receptor Antagonists e.g. Spironolactone for fluid retention (edema) in patients with congestive heart failure, liver cirrhosis, or nephrotic syndrome. Also used to treat high blood pressure, heart failure, and low blood potassium (hypokalemia).

Testosterone can be converted in its target tissues to the active hormone DHT (dihydrotestosterone) by 5-reductase 5-reductase inhibitors are used in the treatment of enlarged prostate gland (benign prostatic hyperplasia) and male pattern hair loss. Estrogens are made from androgens and involve the conversion of androstenedione or testosterone by aromatase (CYP19)(Figure 10). They are produced in the ovary, as well as the skeletal muscle and adipose tissue. Estradiol is the most potent ovarian estrogen whereas estrone is mostly produced in extra ovarian tissues. Steroid Hormone Signaling - The Estrogen Receptor (Figure 11) The estrogen receptor is located in the cytoplasm, masked by the heat shock protein, HSP90. When estradiol (E2) enters the cell it displaces HSP90 and binds to the estrogen receptor. This hormone-receptor complex translocates into the nucleus, dimerizes and binds to the estrogen response element (ERE) as a homodimer. There it enhances gene transcription by recruitment of coactivator proteins. In the absence of ligand, the estrogen receptor cannot bind to the DNA.

Sex Hormone Biosynthesis - Two examples

Testosterone: Biosynthesis of an Androgen Testosterone is primarily produced in the testes, but its synthesis also occurs in the zona reticularis of adrenal cortex. Following the conversion of cholesterol to pregnenolone, the rate limiting step, there are several routes to testosterone (see Figure 7). The major pathways are the: 1. 5 pathway (the main pathway in the testes): Pregnenolone is hydroxylated by 17-- hydroxylase (CYP17) to form 17--hydroxy pregnenolone, which in turn is also acted on by 17--hydroxylase (CYP17) to form dehydroepiandrosterone (DHEA). DHEA is then converted to androstenedione by 3--hydroxy steroid dehydrogenase. 17--hydroxy steroid dehydrogenase then converts androstenedione to testosterone. 2. 4 pathway: Pregnenolone is converted to progesterone by 3--hydroxy steroid dehydrogenase. Progesterone is then converted to 17--hydroxy progesterone by 17--hydroxylase (CYP17), which is hydroxylated by 17--hydroxylase (CYP17) to form androstenedione. 17--hydroxy steroid dehydrogenase then converts androstenedione to testosterone.

B. ANTERIOR PITUITARY Prolactin: major hormone responsible for lactogenesis. Single chain polypeptide of l98 AA. Secretion is under tonic inhibitory control by dopamine. Stress, exercise, estrogen, suckling, and pregnancy are associated with increasing circulating levels. Levels increase with sleep but there is no circadian rhythm. Induces synthesis of casein and lactalbumin in mammary glands. Prolactin stimulates breast development (in a supportive role with estrogen). Prolactin does not regulate a secondary endocrine gland, but receptors for prolactin exist in the adrenal cortex. Excess prolactin secretion may lead to galactorrhea (milk discharge from the nipple) and inhibition of GnRH secretion and ovulation. Prolactin deficiency results in failure to lactate. Pro-opiomelanocortin: ACTH (39 AA) and ß-lipotropin (91 AA) are part of a 3lK MW pro-hormone, pro-opiomelanocortin (POMC). The 3lK precursor undergoes proteolytic cleavage into those hormones. The carboxyl terminal is ß-lipotropin which contains γ-lipotropin, ß-MSH, ß-endorphin, γ-endorphin, α-endorphin, and enkephalin. The 3l K pro-hormone is a glycoprotein. The amino terminal fragment has no known function but does contain γ-MSH, which may modulate adrenal cortical synthesis of mineralocorticoids. ACTH has a circadian rhythm. Corticotropin releasing hormone (CRH) stimulates ACTH synthesis and secretion. Hypoglycemia, stress, pyrogen and low glucocorticoid levels increase ACTH release. ß-lipotropin induces pigmentation and may affect adrenal steroid secretion. However, ACTH is the most important human pigmentary hormone. FSH, LH, and TSH are pituitary glycoprotein hormones. Human chorionic gonadotropin (hCG, MW 46,000) is a placental glycoprotein hormone with biologic activity indistinguishable from that of LH. The glycoprotein hormones share a common quaternary structure of two dissimilar subunits, designated α and ß. The α subunits have essentially the same amino acid sequences, but differ in their degrees of glycosylation. The ß subunits confer biologic and immunologic specificities of the hormones. The subunits alone are devoid of significant intrinsic biologic activity; their half-lives are of the order of l0-30 minutes. FSH, LH, and TSH have plasma half-lives of 30 min - 2 hrs. Human CG has a plasma half-life of 24-30 hours. All these anterior pituitary hormones help maintain the size and blood flow of target glands; hypophysectomy normally causes atrophy of these organs.

The anterior pituitary secretes several tropic (or trophic) hormones (Table I). Fig. 3 shows the target tissues stimulated by the anterior pituitary hormones. Growth Hormone: The most abundant of the pituitary hormones. Somatotropes account for about 40-50% of the total number of pituitary cells. GH is a singlechain polypeptide that is homologous with prolactin and human placental lactogen. Endo/Repro 3a - 5 Normal plasma basal levels are 5 ng/ml. The growth promoting actions of GH on muscle and the skeleton are insulin-like, but GH's long term effects on carbohydrate metabolism and lipolytic effects are opposite to those of insulin. GH induces liver and other tissues to secrete Insulin-like Growth Factor I (IGF-I) or somatomedins (MW 8000) which circulate bound to a protein complex (MW 120,000). Prolactin and insulin may stimulate somatomedin release from liver as well. Growth hormone does not regulate a secondary endocrine organ, but GH receptors exist in the adrenal cortex. GH is discussed in detail the Endocrinology of Growth lecture.

Extremes of GH Secretion

The effects of GH hypersecretion (caused by a pituitary tumor), depend on the age. Before puberty, it causes gigantism, since GH promotes bone lengthening. After puberty it causes acromegaly, a syndrome characterized by disfiguring bone thickening (since bone can no longer grow longitudinally, it thickens), enlargement of hands and feet (acral parts), protrusion of lower jaw, increased body hair and glucose intolerance. GH excess can be treated with somatostatin analogs (e.g. octreotide), which inhibit GH secretion. A deficiency in GH in children results in failure to grow, short stature, mild obesity, and delayed puberty. GH deficiency can be caused by lack of somatotropes, lack of GHRH, failure to generate IGF in liver, or GH receptor deficiency. However regardless of the level of GH in plasma, optimal height will be attained only if children are properly nourished. 1. What is the difference between constitutional delay and familial short stature? 2. What does it mean to fit within the genetic pattern of the family? 3. What is the role of estrogen on growth and cessation of growth in males and females?

Functional Relationship to Hypothalamus

The hypothalamus (and hence the brain) influences the pituitary secretion of hormones through a complex arrangement of neural and blood supply (Fig. 2). For the posterior pituitary, axons having their cell bodies in the hypothalamus terminate in a capillary plexus supplied by the inferior hypophyseal artery. Peptide hormones synthesized in these cell bodies, travel as neurosecretory granules that are stored in nerve terminals lying in the posterior pituitary. These granules, released by nerve impulses transmitted to the posterior pituitary by the hypothalamus, enter the peripheral circulation via the capillary plexus. Therefore, a single cell performs hormone synthesis, storage and release. Fig. 2. Anatomical and functional relationships between hypothalamus, the pituitary gland, and its blood supply. Note that the adenohypophysis has no direct arterial supply but receives blood from veins that first drains neural tissue in the median eminence. OCT, oxytocin; see Table 1 for other hormones. Adapted from Berne and Levy (1996). In contrast, the anterior pituitary is a collection of endocrine cells regulated by blood borne stimuli originating in neural tissue of hypothalamus. Cell bodies of particular hypothalamic neurons synthesize releasing or inhibiting hormones that travel to and are stored in the median eminence near the capillary plexus of the superior hypophyseal artery (Fig. 2). Upon stimulation, releasing or inhibiting hormones enter the capillary plexus, travel down the portal veins and exit from a secondary capillary plexus to reach their specific endocrine target cells in the adenohypophysis. These cells respond by increasing or decreasing their output of tropic hormones, which in turn enter the same capillary plexus to reach the peripheral circulation.

Embryology of Pancreas

The pancreas normally has two lobes which originate as separate dorsal and ventral buds of the GI tract in the embryo. The ventral bud also originates the common bile duct, gall bladder and liver. Pancreatic tissue in the dorsal and ventral buds normally comes together to form one organ, but two outflow tracts are typically retained. The duct of Santorini is the original outflow of the dorsal bud into the duodenum, while the duct of Wirsung, drains together with the bile through the ampulla of Vater and sphincter of Oddi. Typically these two ducts are anastomosed within the pancreas, but there may be important individual variations in how secretions ultimately reach the duodenum. The endocrine tissue of the pancreas, the Islets of Langerhans, or pancreatic islets, arise initially from individual cells in the pancreatic (future exocrine) epithelium. These cells delaminate and coalesce during development following regions of lower oxygen tension in the organ. There, they differentiate into the major types of islet cells, producing glucagon, insulin, somatostatin, etc. Of relevance, an individual pancreatic islet cell cycles through each hormone in the sequence during its development. For example, a delta-cell destined to produce somatostatin will, in the fetus, transiently produce first gastrin, then glucagon, insulin, and finally somatostatin. Note that gastrin, which is produced largely by stomach parietal cells in the adult, is also the first hormone produced during development by pancreatic islet cells. Adult gastrin-producing tumors, termed gastrinomas, may arise in stomach or duodenum, but they are most dangerous when they have their origin in pancreatic islet cells. Presumably these tumors represent a de-differentiation of these cells back to a precursor state. The disease caused by excessive gastrin secretion is known as Zollinger-Ellison syndrome (ZES).

Embryology of Pituitary Gland

The pituitary consists of two embryologically and morphologically distinct glands, the neurohypophysis and the adenohypophysis. The gland remains connected to the hypothalamus by the pituitary or hypophyseal stalk. After neurulation the adenohypophysis forms from ectoderm in the roof of the stomodeum. The invagination of this pharyngeal epithelium forms the pouch of Rathke. The pouch grows toward the developing neural tube and becomes constricted by continued proliferation of mesenchyme to form a closed vesicle that remains for a time connected with the ectoderm by a solid cord of cells. The anterior aspect of Rathke's pouch enlarges and becomes the anterior pituitary, or pars distalis, while the posterior aspect of Rathke's pouch remains thin and becomes the pars intermedia that fuses with the adjoining part of the neurohypophysis. The original lumen of Rathke's pouch remains first as a cleft but eventually becomes a very narrow space between the anterior and posterior lobes. Two small diverticula of Rathke's pouch grow along the infundibulum and fuse to surround it; this constitutes the pars tuberalis of the adult pituitary. The neurohypophysis forms from neuroectoderm of the floor of the developing forebrain. It begins as a hollow diverticulum elongating toward the stomodeum from the floor of the neural plate. This forms an infundibular sac and its walls continue to increase in thickness until the cavity is obliterated except at its upper end where it persists as the infundibular recess of the brain's third ventricle. The distal end enlarges to form the pars nervosa. At seven years of age the pituitary gland is about half the weight of the adult gland; it attains its adult weight at puberty. The pituitary gland is typically larger in both weight and size in females than in males.

The Pituitary Gland The nervous system and the endocrine system are the two major controlling systems in the body. The nervous system is a rapidly responding system that regulates the activities of muscle and secretory cells by means of nerve impulses and neurotransmitters. The endocrine system is a slower responding system that influences virtually all cells by means of hormones. These two systems closely interact by several means, one of which uses the hypothalamus as a relay to regulate the activity of the pituitary (or hypophysis).

The pituitary gland lies in a pocket of bone (the sella turcica) at the base of the brain, just below the hypothalamus, to which it is connected by a stalk containing nerve fibers and small blood vessels. Fig. 1. The pituitary gland or hypophysis. Adapted from Berne and Levy (1996). The pituitary is composed of two adjacent lobes (Fig. 1): the posterior lobe or neurohypophysis and the anterior lobe or adenohypophysis. The posterior lobe secretes two hormones, actually made by the hypothalamus but stored in the posterior pituitary: oxytocin and ADH (antidiuretic hormone). The anterior lobe secretes at least six hormones. All of this is accomplished by a mass of only 500 mg of pituitary tissue in association with 10 g of hypothalamus. To understand how each part of the pituitary secretes which hormone, it is important to know how the pituitary develops. The pituitary develops embryonically from two different ectodermal regions, the floor of the brain and the roof of the mouth. The floor of the brain in the region of the hypothalamus develops into the neurohypophysis, which consists of a structure called pars nervosa connected to the brain by a stalk like process, the infundibulum. A small elevation, the median eminence, connects the infundibulum to the brain (Fig. 1). The roof of the mouth loses its connection with the mouth and becomes the adenohypophysis.

A. POSTERIOR PITUITARY

The posterior pituitary secretes two small peptides (9 AA each), closely related: ADH (also called arginine vasopressin, AVP) and oxytocin (OCT). As neural hormones, they originate from and are secreted by different nerve cells. Like other peptide hormones, they are synthesized as part of larger precursor molecules, prepro-hormones, which include a characteristic neurophysin (whose biological function is unknown), associated with them. ADH: Originates primarily in the supraoptic nuclei. Primary role is to conserve body water and regulate the tonicity of body fluids. The target cells reached by ADH are renal cells (primarily collecting ducts) responsible for reabsorbing free water. Water deprivation stimulates ADH secretion resulting in decreased water clearance and enhanced conservation of water. A water load produces the opposite effect, decreased ADH secretion, and increased water clearance. Lack of ADH secretion leads to diabetes insipidus. The change in osmolality is sensed by specialized osmoreceptor neurons with connections to ADH nerve cells. ADH (also known as Arginine Vasopressin, AVP) constricts vascular smooth muscle (via a V1 receptor and an IP3/Ca2+ mechanism). Oxytocin: Originates primarily in the paraventricular nuclei. Primary role is to eject milk from lactating mammary glands in the breast. Suckling by infant, the major stimulus for oxytocin release, stimulates sensory receptors in the nipple; the resulting nerve impulses are transmitted to hypothalamus and cause oxytocin release from neurosecretory vesicles in posterior pituitary. Oxytocin thus released in blood causes contraction of myoepithelial cells of mammary gland and ejection of milk. Oxytocin also has a powerful action on smooth muscle of the uterus during parturition (labor). Dilation of the cervix during labor is the primary stimulus for oxytocin release, which then enhances contraction of uterine muscle (positive-feedback). Oxytocin is also used clinically to induce labor and control postpartum hemorrhage. [Note: in humans, oxytocin levels are low during the initial labor but increase as labor progresses. So oxytocin itself may not be responsible for initiating labor.]

Other Important Endocrine Organ Embryologies

The thyroid gland originates from the endoderm in the region of the first pharyngeal arch, the "primitive pharynx", in a region which also contains the median tongue bud. These cells delaminate, but initially retain a lumen called the thyroglossal duct. From there, it descends to a more caudal level, meeting parathyroid tissue that arisesin the third and fourth pharyngeal pouches. If thyroidectomy is being considered, one should consider that thyroid tissue may aberrantly be retained in the adult in locations along the track of the thyroglossal duct, i.e. rostrally from the thyroid up towards the pharynx and tongue.

Fig. 5. (a) Schematic model of the interaction between steroid hormone receptors and DNA. Receptors bind to DNA as dimers. (DBD) DNA binding domain; (S) Steroid hormone binding domain. (b) The amino acid sequence of the estrogen receptor DBDis shown in the 'zinc-finger' representation. Asterisks indicate residues that interact with DNA base pairs, filled boxes, those making phosphate contacts, and filled circles, those involved in dimer formation. (modified from Schwabe et al, Structure 1:187[1993])

There is a growing body of evidence that suggests that steroid hormones may have non-genomic actions within the cell. In some cases a hormone will elicit responses too rapid to be explained by nuclear events. Indeed, aldosterone has been shown to have effects on red blood cells that lack a nucleus and on fibroblasts in knockout mice that lack the aldosterone receptor! The non-genomic responses of aldosterone and other steroid hormones are speculated to be mediated Endo/Repro 1 - 10 by receptors that are associated with the membrane; research is currently aimed at identifying these non-traditional receptors.

Introduc

This lecture provides an overview of the physiology of growth. Growth Hormone is a pituitary product that has no single easily identifiable target endocrine gland; its function is considered more fully in this lecture and in the associated discussion. Some pathological conditions are considered in the lecture and in the subsequent discussion in the context of developing a better understanding of the regulatory processes at work. Growth is a complex phenomenon that is affected by growth hormone, but also by thyroid hormones, androgens, estrogens, glucocorticoids, insulin and other factors such as parental height, nutrition and psychological stress. [In this section, growth is meant to indicate linear growth and not developmental, neurological, etc. growth]. GH is the single most important hormone required for postnatal growth. Attainment of adult size is absolutely dependent on GH. GH acts by stimulating mitosis of many target tissues. GH: promotes bone lengthening by stimulating maturation and mitosis of chondrocytes (cells that lay down new cartilage). promotes protein synthesis in tissue, especially muscle, and organs (increases amino acid uptake). This facilitates tissue enlargement. stimulates gluconeogenesis by increasing hepatic glucose output and inhibiting glucose uptake (anti-insulin effect). Thus GH is diabetogenic. stimulates lipolysis (increases fat metabolism).

Effect of other Hormones on Growth:

Thyroid hormones: Linear growth is stunted in children suffering from unremediated deficiency of thyroid hormones. Although treatment of hypothyroid children with thyroid hormones results in rapid growth and maturation of bone, thyroid hormones have little, if any, linear growth-promoting effect in the absence of GH. Failure to grow in thyroid-deficient individuals is largely due to a decrease in GH synthesis and number of GHRH receptors on somatotropes. Insulin: During fetal growth, it is insulin, not GH or thyroid hormones, that serves as growth-promoting hormone. Pancreatic cell mass is determined by locally produced IGF-II, which is the predominant IGF that is expressed in the tissues of embryos and fetuses. Insulin is closely related to IGF-I and -II and can activate IGFI receptors (Table I). Optimal concentrations of insulin are required to maintain normal growth during postnatal life. But insulin cannot sustain a normal rate of growth in the absence of GH. Gonadal hormones and pubertal growth spurt: Onset of sexual maturation is accompanied by dramatic acceleration of growth. The adolescent growth spurt is attributable to sex steroids from the gonads and, perhaps, the adrenals. While gonadal steroids promote linear growth, they also accelerate closure of the epiphyses and, therefore, limit the final height that can be obtained. Hypogonadal individuals tend to be tall with long arms and legs. Recent observations have shown that it is estrogens rather than androgens that are responsible for both acceleration of growth at puberty and maturation of the epiphyseal plates. Androgens are precursors of estrogens and are converted to estrogens by an aromatase in gonads and peripheral tissues. Children of either sex lacking the aromatase do not experience a pubertal growth despite higher than normal levels of androgens. The well-established growth-promoting effect of androgens Endo/Repro 3b - 6 administered to children whose epiphyses are not yet fused is likely attributable to androgen conversion to estrogen. Estrogen increases in the plasma of both girls and boys early in puberty. Nevertheless the maximal rate of growth achieved in adolescence is greater in males than in females, and a supportive growth-promoting role for androgens is not ruled out. Most of the increase in height stimulated by estrogen or androgens at puberty is due to a resulting increased secretion of GH and a consequent increase in IGF-I (Fig. 3). The short stature of Pygmies seems to be caused by a genetic inability to produce IGF-I. [Pygmies actually have normal GH and IGF-I levels before puberty, but there is no IGF-I increase at time of puberty and thus no pubertal growth spurt. The problem seems to stem from GH resistance at puberty.] Glucocorticoids: Glucocorticoids are required for synthesis of GH and normal growth. But excessive glucocorticoids decrease GH secretion, and, given their catabolic effect in muscle, glucocorticoids also antagonize the effect of GH.

1) G-protein linked receptors Receptors with 7 transmembrane domains (serpentine receptors) are coupled to membrane associated guanine nucleotide binding proteins called regulatory or G-proteins. G-proteins are responsible for coupling receptor recognition of many hormones to the modulation of certain "effectors", e.g. adenylate cyclase, phospholipase C, ion channels. As you recall, G-proteins are actually heterotrimers comprised of three subunits, α, β and γ. The α- subunit (Gα) is a GTPase; it cleaves GTP to GDP and Pi. Since the products of hydrolysis dissociate very slowly from the α-subunit of the heterotrimer in unstimulated cells, Gα is found predominately bound to GDP in the absence of hormone. In the presence of hormone, however, the occupied Endo/Repro 1 - 4 receptor interacts with G-protein and facilitates the release of bound GDP. Cytoplasmic GTP then occupies the nucleotide binding site of Gα and causes this subunit to dissociate from both the hormone-receptor complex and βγ. Gα is then positioned to interact with a given effector, turning it on (or off). The influence of Gα-GTP on the effector lasts until GTP is cleaved to its products. Gα-GDP then dissociates from the effector (returning it to its basal level of activity) and reassociates with βγ. The cycle is repeated when another hormone molecule binds to receptor. This scheme is depicted in Fig.1. Although for many years the α component was viewed as the sole G-protein transducing subunit, recent evidence suggests that in some systems the βγ dimer may influence effector function as well.

a. G-protein coupling to adenylase cyclase Many hormone-receptor interactions lead to activation of adenylate cyclase, a membrane protein that catalyzes the formation of cyclic AMP (cAMP, 3'-5' AMP) from ATP at the inner membrane surface. Cyclase activation is mediated by the regulatory protein Gsα, a stimulatory G-protein. The α-subunit of G-protein bound to GTP activates adenylate cyclase; G- protein in complex with GDP does not. The proportion of G-protein in the GTP state, and hence adenylate cyclase in the active form, depends on the rate of exchange of GTP for GDP and the rate of hydrolysis of bound GTP. Cyclic AMP, produced by adenylate cyclase, activates cytoplasmic protein kinase A (PKA). In its inactive form PKA consists of regulatory and catalytic subunits. Binding of cAMP to the regulatory subunits activates the catalytic subunits by causing them to dissociate from the complex (Fig. 2.). The response of a cell to the second messenger cAMP results from the modulation of the activity of various enzymes subsequent to their phosphorylation. The nature of the response depends on the array of substrates available to the kinase. Thus, phosphorylation of lipase in the epinephrine-stimulated adipocyte leads to triglyceride hydrolysis; phosphorylation of glycogen synthetase in the epinephrine-stimulated liver cell inhibits glycogen synthesis. PKA can influence the activity of specific proteins that preexist in the cytoplasm, but it also can affect transcription of target genes. The catalytic subunit can enter the nucleus where it phosphorylates CREB, cyclic AMP response element binding protein. CREB then binds to "response elements", sequences of DNA that influence transcription of nearby genes (see Section B below). Because an enzyme, such as adenylate cyclase, generates many copies of its products, and these products activate downstream enzymes, a signal initiated by a single ligand (binding molecule) can be significantly amplified in signal transduction cascades. Since there is potential for many downstream enzymes to be regulated as well, a large number of cell processes may be influenced following receptor-ligand interaction. Some ligands that use cAMP as a second messenger are listed in Table II. Endo/Repro 1 - 6 b) G-protein coupling to Phospholipase C Turnover of membrane associated inositol phospholipids, particularly phosphatidylinositol bisphosphate (PIP2), appears to be a key event in signal transduction of a number of other hormones and neurotransmitters, e.g. angiotensin II, cholecystokinin, gastrin, and norepinephrine (α-l). Hormone receptor interaction in these systems activates a G-protein, Gqα, which activates phospholipase C rather than adenylate cyclase. Phospholipase C cleaves PIP2 to diacylglycerol and inositol l,4,5-trisphosphate (IP3). Both products are intracellular mediators. IP3 mobilizes Ca+2 from vesicular storage sites; diacylglycerol, which is retained in the membrane, associates with cytoplasmic protein kinase C and in the presence of Ca +2 activates it (Fig. 3). Fig. 3. Inositol phospholipids and signal transduction. Inositol phospholipids are normally very minor components of membranes. (PLC, phopholipase C; PIP2, phosphatidylinositol 4,5- bisphosphate; DAG, 1,2- diacylglycerol; IP3, inositol 1,4,5- trisphosphate;C kinase; protein kinase C; P, phosphoryl group; CAM, calmodulin, and R1 and R2, fatty acyl groups). Modified from Turnover of Inositol Phospholipids and Signal Transduction, PIP2 can participate in another distinct signaling pathway following its phosphorylation by phosphoinositol (PI) 3-kinase to form phosphatidyl inositol 3,4,5-trisphosphate (PIP3). These phospholipids interact with protein kinases, called PDKs, as well as an enzyme called Akt. When these enzymes are recruited to the cell membrane, PDK phosphorylates and activates Akt, and Akt will itself phosphorylate target proteins, such as transcription factors, to bring about physiological responses. Different forms of PI 3-kinase, the enzyme that triggers this cascade, can be activated by G-proteins and by tyrosine kinase receptors (see below). Insulin action, for example, is mediated in part by PI 3-kinase activation. c. G-protein coupling to other effectors Other examples of G-protein family members are those that inhibit adenylate cyclase and activate K+ channels (Giα) or stimulate (in photoreceptors) cGMP phosphodiesterase (transducin or Gtα).

Merkel cells

in the skin are also part of the neuroendocrine system. Merkel cells are mysterious in both function and origin. Since their discovery, they have been postulated to play a role in sensation of light touch. Nerve endings are found in close approximation to their basal surface. The origin of Merkel cells is debated to be either epidermal or from neural crest. Merkel cells are most numerous in palmar hand, feet, plantar toes, lip and masticatory mucosa. Though their endocrine function is unclear, they have the morphological feature of storing and secreting small amines in dense, basal granules. The disease Merkel cell carcinoma, also called endocrine carcinoma of the skin, is assumed to originate in Merkel cell precursors. Though rare, (~200 cases/year in USA) it is rapidly increasing in prevalence, and can be lethal, and therefore should be distinguished from the more common basal cell carcinoma and melanoma. Merkelcell.org is a website devoted to this rare condition. Neuroendocrine cells may exist in other organs such as breast, prostate, bladder, and may very occasionally be a source of primary cancer in these organs. In addition, some cancers have a propensity for differentiation to a neuroendocrine phenotype. Such secondary differentiation is commonly seen in prostate cancer.