13 Anterior Pituitary Hormones

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From: Endocrinology: Basic and Clinical Principles, Second Edition (S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ

13 Anterior Pituitary Hormones

Ilan Shimon, MD and Shlomo Melmed, MD

C^ONTENTS INTRODUCTION

PROOPIOMELANOCORTIN

GROWTH HORMONE

PROLACTIN

THYROID-STIMULATING HORMONE

PSH^AND LH

2.2. POMC Gene

The human POMC gene (Fig. 1) is an 8-kb single- copy gene located on chromosome 2p25. It consists of a 400 to 700-bp promoter, three exons, and two introns.

The majority of the 266-amino-acid POMC precursor protein is encoded by exon 3, which contains all the known peptide products of the gene, including the 39-amino-acid adrenocorticotropin (ACTH), α-mel- anocyte-stimulating hormone (α-MSH),β-MSH,β-lipo- tropin (β-LPH), β-endorphin, and corticotropin-like intermediate lobe peptide (CLIP). Human POMC is digested at either Lys-Arg or Arg-Arg residues by two endopeptidases: prohormone convertase 1 (PC1), abundant in anterior pituitary corticotropes, and PC2, expressed in the brain as well as in pancreatic islet cells, but absent from the anterior pituitary. This tissue-specific enzyme distribution correlates well with the different enzymatic cleavage of the prohormone to its products. Thus, in the pituitary, corticotrope POMC is cleaved into ACTH, β-LPH, and other peptides including N-terminal glycopeptide and joining peptide, and in the brain, ACTH is further cleaved to α-MSH and CLIP (Fig. 1). Exon 1 is not translated and there is only 45% nucleotide sequence homology in exon 1 and the promoter region of the human, bovine, and other mammals. By contrast, exons 2 and 3 bear 80–95% homology among humans, other mammals, and mice. The

1. INTRODUCTION

The human anterior pituitary gland contains at least five distinct hormone-producing cell populations, expressing six different hormones (Table 1): proopiomelanocortin (POMC), growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH).

2. PROOPIOMELANOCORTIN 2.1. Embryology and Cytogenesis

of Corticotropes

The corticotrope is the first cell type to develop in the human fetal pituitary, as early as 6 wk of gestation, and 2 wk later adrenocorticotropic hormone (ACTH) is detectable by radioimmunoassay (RIA) of both the fetal pituitary and fetal blood. The corticotropes constitute between 15 and 20% of the adenohypophyseal cell population, are initially identified by their basophilic staining, and express strong granular cytoplasmic immunopositivity for ACTH and for other fragments of the POMC molecule. By electron micros- copy (EM) the cells are oval with spherical eccentric nuclei, and a large membrane-bound lysosomal structure, the “enigmatic body.”

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Table 1 Expression of Human Anterior Pituitary Hormone Gene and Protein Action POMCGHPRLTSHFSHLH CellCorticotropeSomatotropeLactotropeThyrotropeGonadotropeGonadotrope Fetal appearance6 wk8 wk12 wk12 wk12 wk12 wk Chromosomal gene locus2p17q6α: 6q; β: 1pβ: 11pβ: 19q ProteinPolypeptidePolypeptidePolypeptideGlycoproteinGlycoproteinGlycoprotein α,β-subunits α,β-subunits α,β-subunits Protein length (kDa)ACTH: 4.52223283428.5 Amino acid no.266 (ACTH-39)191199211210204 Normal rangeACTH, 4–22 pmol/L<0.5μg/LbM < 15; F < 20 μg/L0.1–5 mU/LM: 5–20 IU/L;M: 5–20 IU/L; F: (basal) 5–20 IU/LF: (basal) 5–20 IU/L Secretion Regulators StimulatorsCRH, AVPGHRH, ghrelinEstrogen, TRHTRHGnRH, estrogenGnRH, estrogen InhibitorsGlucocorticoidsSomatostatin, IGF-1Dopamine, T3T3, T4, dopamine,Estrogen, inhibinEstrogen, inhibin somatostatin, glucocorticoids ReceptorGSTD aSingle transmembraneSingle transmembraneGSTDaGSTDaGSTDa LocationAdrenalLiver. other tissuesBreast, other tissuesThyroidOvaryTestis Tropic effectsSteroid productionIGF-1 production,Milk productionT3, T4 synthesisTestosterone synthesis,Testosterone synthesis, growth inductionand secretion follicle growth follicle growth aGSTD = Gs protein coupled with seven transmembrane domains. bIntegrated over 24 h.

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main regulators of POMC transcription are corticotropin-releasing hormone (CRH) and glucocorticoids, which exert opposite effects on POMC transcription rate. CRH increases POMC mRNA and protein content via cyclic adenosine monophosphate (cAMP), and the glucocorticoid-negative feedback effect is probably mediated through binding of the glucocorticoid recep- tor complex to cis-acting POMC promoter sequences.

POMC transcription is also stimulated by β-adrenergic catecholamines and insulin-induced hypoglycemia, but suppressed by arginine vasopressin (AVP).

2.3. Regulation of ACTH Secretion

The endogenous circadian rhythm of ACTH pulsatile secretion leads to a parallel diurnal pattern of gluco-

corticoid release. Both ACTH and cortisol are high in the early morning and decline throughout the day, main- taining lower levels during the night. This rhythmicity of ACTH pulse amplitude may be controlled by the concomitant diurnal variation of CRH secretion. CRH and AVP are the main secretagogues of ACTH, and glucocorticoids inhibit its secretion (Fig. 2). CRH, by binding to CRH receptors on the corticotrope, stimulates ACTH synthesis as well as release. In addition, physical stress, exercise, acute illness and hypoglycemia increase ACTH levels. Inflammatory cytokines—

tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, and leukemia inhibitory factor (LIF)—stimulate pituitary corticotropin secretion, and oxytocin, opiates, and somatostatin inhibit ACTH release.

Fig. 1. Schematic structure of POMC gene, mRNA, and protein products. (A) The gene (top) contains the promoter, three exons (thick bars; translated regions are stippled), separated by two introns (diagonal lines). The mRNA transcript (middle) consists of the three exons and the polyadenylation site. The POMC precursor protein (bottom) consists of the signal peptide, N-terminal glycopeptide, joining peptide, ACTH, and β-LPH. (B) Tissue-specific enzymatic cleavage of POMC to its products. POMC is cleaved into N- terminal glycopeptide and joining peptide in the pituitary coticotropes and the brain. In brain, ACTH is further cleaved to α-MSH and CLIP, and β-LPH is digested into γ-LPH and β-endorphin. KR = dibasic amino acids at proteolytic cleavage sites. (From Holm and Majzoub [adrenocorticotropin] in Melmed, 1995.)

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2.4. ACTH Receptor Gene

ACTH receptors have been detected in human adrenal glands, in adrenal tumors, on human mononuclear leukocytes, and on rat lymphocytes. The ACTH receptor gene encodes a 297-amino-acid protein that belongs to the G_sprotein–coupled superfamily of receptors containing seven transmembrane domains. The effect of ACTH is mediated by adenylate cyclase activation, cAMP production, and PKA induction in the adrenal.

2.5. ACTH Action

ACTH stimulates steroidogenesis in the adrenocorti- cal cells. Lipoprotein uptake from the plasma is enhanced, and steroid hormone enzyme gene transcription is increased. The prolonged effects of ACTH may promote growth of adrenal size and act with β-LPH on melanocytes to increase skin pigmentation.

2.6. ACTH Hypersecretion

2.6.1. PITUITARY ADENOMA

ACTH-producing tumors are usually monoclonal, well-differentiated microadenomas. About 10–15% of all pituitary adenomas are clinically active ACTH-pro-

ducing tumors, and 5% are silent corticotrope adenomas.

In addition to POMC glycoprotein, secretory granules in tumor cells stain for ACTH, β-endorphin, β-LPH, and N-terminal peptide. Some corticotrope adenomas contain altered forms of gastrin, cholecystokinin, and also vasoactive intestinal peptide (VIP), neurophysin, α-subunit, and chromogranin A. Cell cultures of corticotrope adenomas secrete ACTH in response to CRH and AVP, and ACTH is suppressible by glucocorticoids, but to a lesser extent than with normal corticotropes.

2.6.2. ECTOPIC ACTH SECRETION

Small-cell carcinomas of the lung, and bronchial and thymic carcinoids, can produce ACTH, leading to Cushing syndrome. These tumors express other neuro- endocrine markers including chromogranins, synapto- physin, and neurotensin. POMC mRNA from nonpituitary tumors may be longer than normal or pituitary tumor POMC mRNA. In addition, CLIP and β-MSH are detected in ectopic tumors, indicating an alternative POMC processing. POMC mRNA and ACTH are not suppressed in small-cell lung cell lines by glucocorticoids.

Fig. 2. Regulation of POMC/ACTH expression in pituitary corticotropes. CRH binds to its G_sprotein–coupled seven-transmembrane domain receptor and activates adenylyl cyclase and cAMP generation to promote POMC through protein kinase A (PKA) activation.

Vasopressin binds to the V_1breceptor in the anterior pituitary and via PKC and Ca⁺²mobilization potentiates the CRH-stimulated increase in cAMP. Inflammatory cytokines, including TNF-α, IL-1, IL-6, and LIF stimulate POMC expression and ACTH secretion.

Cortisol binds to its nuclear receptor and suppresses POMC transcription. CREB = CRE binding protein; MAP kinase = mitogen- activated protein kinase; PI₃kinase = phosphatidylinositol 3-kinase; ATP = adenosine triphosphate; SSTR = somatostatin receptor.

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2.7. ACTH Hyposecretion

BT:Pituitary failure due to irradiation, hypophysec- tomy, large macroadenomas, pituitary apoplexy, trauma, postpartum necrosis, hypophysitis, glucocorticoid withdrawal, or CRH deficiency results in ACTH hyposecretion and hypocortisolism. This is usually a late manifestation of pituitary failure and indicates severly compromised pituitary function.

2.8. ACTH Receptor Defects

Familial glucocorticoid deficiency is a rare autoso- mal recessive disorder of adrenal unresponsiveness to ACTH, characterized by glucocorticoid deficiency in the presence of elevated circulating ACTH and normal mineralocorticoid production. Affected children usually have hypoglycemic episodes, hyperpigmentation, failure to thrive, and chronic asthenia. They have no cortisol or aldosterone responses to exogenous ACTH.

Recently, homozygous and compound heterozygous point mutations have been reported in the ACTH receptor gene.

2.9. Clinical Testing

The low-dose (1 mg) overnight dexamethasone suppression test and the measurement of 24-h urinary-free cortisol are the standard screening tests for Cushing syndrome. A 48-h low-dose dexamethasone suppression test (2 mg/d) is usually performed to diagnose Cushing syndrome, and a high-dose (8 mg/d) suppression test will differentiate a pituitary from a nonpituitary tumor source of ACTH. Once the diagnosis of Cushing syndrome is made, measuring ACTH concentration in plasma is important for etiologic evaluation. The normal levels of ACTH are 4–22 pmol/L. In Cushing disease, ACTH is moderately increased, to 10–50 pmol/L, and ectopic ACTH syndrome usually results in highly elevated levels. Patients with adrenal adenoma have low or undetectable ACTH. The CRH stimulation test may serve to differentiate patients with Cushing disease who have exaggerated ACTH and cortisol response to CRH from patients with ectopic ACTH-producing tumors that, in general, do not respond furthur to CRH. To screen the functional adrenal reserve for cortisol production, the rapid cortrosyn (ACTH 1-24, 1–250 μg) stimulation test is used. Patients with ACTH hyposecretion have a blunted cortisol response to administration of cortrosyn.

2.10. Clinical Syndromes

2.10.1. C^USHING S^YNDROME

In 1932, Harvey Cushing described a syndrome resulting from long-term exposure to glucocorticoids. Most patients (70%) have pituitary corticotrope adenomas

(Cushing disease). Other etiologies include ectopic ACTH (12%); cortisol-producing adrenal adenoma, carcinoma, and hyperplasia (18%); and the rare ectopic CRH syndrome. Prolonged administration of glucocorticoids produces a similar syndrome. Patients have a typical habitus including “moon facies,” “buffalo hump,” truncal obesity, and cutaneous striae, as well as muscle weakness, osteoporosis, impaired glucose tol- erance, hirsutism, acne, hypertension, depression, and ovarian dysfunction. Usually the clinical presentation is insidious, but the ectopic syndrome associated with small-cell lung carcinoma may be acute, with rapid onset of hypertension, edema, hypokalemia, glucose intolerance, and hyperpigmentation. When a probable diagnosis of Cushing disease is made, the most direct way to demonstrate pituitary ACTH hypersecretion is by catheterization of the inferior petrosal venous sinuses, which drain the pituitary. ACTH measurements in petrosal and peripheral venous plasma before and after CRH stimulation can document a central- to-peripheral gradient of ACTH in blood draining an adenoma. High-resolution magnetic resonance imag- ing of the sella turcica, enhanced by gadolinium, is useful in determining the location of corticotrope adenomas with a sensitivity of 2 mm. The treatment of choice is transsphenoidal adenomectomy, and the cure rate is 70–80%.

2.10.2 HYPOCORTISOLISM

Hypocortisolism can be either primary (Addison disease), secondary to pituitary ACTH deficiency, or ter- tiary resulting from CRH deficiency. Primary adrenal insufficiency can result from autoimmune adrenocorti- cal destruction, acquired immunodeficiency syndrome, tuberculosis, bilateral hemorrhage, and metastatic disease. Clinically, patients present with fatigue, weakness, nausea and vomiting, weight loss, hypotension, hypoglycemia, and hyperpigmentation (only in primary hypocortisolism). Treatment of patients with Addison disease includes glucocorticoids and mineralocorti- coids, whereas patients with secondary adrenal insufficiency do not require mineralocorticoid replacement.

GROWTH HORMONE 3.1. Embryology and Cytogenesis

of Somatotropes

Somatotropes that contain GH immunoreactivity are identified at 8 wk of gestation, and circulating GH is measurable in fetal serum at the end of the first trimes- ter. Somatotropes comprise 40–50% of pituitary cells and are located in the lateral wings of the gland. These acidophilic cells reveal intense cytoplasmic immunopositivity for GH.

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3.2. GH Gene

The human GH genomic locus contains a cluster of five highly conserved genes and spans approx 66 kb on the long arm of chromosome 17 (q22-24). All these genes have the same basic structure consisting of five exons separated by four introns. The hGH gene codes for a 22-kDa protein (Fig. 3) containing 191 amino acids and is exclusively expressed in somatotropes, whereas the others are expressed in placental tissue.

The GH promoter, 300 bp of 5'-flanking DNA, con- tains cis-elements that mediate both pituitary- and hormone-specific signaling. Pit-1, a 33-kDa tissue- specific transcription factor, binds to specific sites on the promoter. This factor is expressed in lactotropes, somatotropes, and thyrotropes and is critical for GH, PRL, and TSH-β gene transcription. GH-releasing hormone (GHRH) stimulates GH transcription, and insulin-like growth factor-1 (IGF-1) inhibits GH mRNA expression (Fig. 4).

3.3. GH Secretion

GH is secreted as a 22-kDa single-chain polypeptide hormone, or a less abundant 20-kDa monomer, formed by alternative mRNA splicing. The secretion is pulsatile, with low or undetectable basal levels between

Fig. 4. Regulation of GH expression in the somatotrope. GHRH and somatostain bind to specific G protein–coupled seven-transmem- brane domain receptors (GHRH receptor; SSTRs 2 and 5, respectively). GHRH activates adenylyl cyclase to generate cAMP, whereas somatostatin receptor binding suppresses cAMP production. GHRH stimulation activates PKA and CREB phosphorylation. The transcription factors c-fos and Pit-1 then induce GH transcription. Somatostatin, coupled to the G_iprotein, suppresses GH secretion.

Ghrelin binds to the GH secretagogue receptor, induces adenylyl cyclase to generate cAMP, and activates phospholipase C (PLC) signaling, leading to protein kinase C induction and Ca⁺²release. IGF-1, through binding to its receptor, exerts negative feedback on GH expression via the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways.

Fig. 3. Primary structure of human GH. GH is a 191-amino-acid single-chain 22-kDa polypeptide with two intramolecular disulfide bonds. (From Fryklund et al., 1986.)

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peaks. In children, maximum GH secretory peaks are detected within 1 h of the onset of deep sleep. Soma- tostatin (SRIF) and GHRH interact to generate pulsatile GH release, and SRIF appears to be the primary regulator of GH pulses in response to physiologic stimuli.

GHRH stimulates GH synthesis and secretion, whereas SRIF, as well as IGF-1, inhibits GH secretion (Fig. 4).

Ghrelin, a recently discovered orexigenic factor, is a potent GH secretagogue produced by the neuroendo- crine cells of the stomach. Ghrelin stimulates GH secretion through binding to its specific receptor, the GH secretagogue receptor, in the hypothalamus and pituitary. Thyrotropin-releasing hormone (TRH) does not stimulate GH secretion in normal subjects but induces GH release in patients with acromegaly.

3.4. GH Receptor and Binding Proteins

In addition to the liver, which contains the highest concentration of GH receptors, many other tissues express these receptors. The human GH receptor is a 620- amino-acid protein (130 kDa) with an extracellular hormone-binding domain of 246 amino acids, a single transmembrane region, and a cytoplasmic domain of 350 residues. The human GH receptor gene has been assigned to chromosome 5p13. The GH-binding proteins, soluble short forms (60 kDa) of the hepatic GH receptor and identical to the extracellular domain, bind half of circulating GH. They prolong GH plasma half- life by decreasing the GH metabolic clearance rate and also inhibit GH binding to surface receptors by ligand competition.

3.5. GH Action

GH acts both directly, via its own receptor, and indirectly, via IGF-1, on peripheral target tissues. Longitu- dinal bone growth–promoting actions on the chon- drocytes in the epiphyseal growth plate are probably stimulated indirectly by GH, through local as well as hepatic-derived circulating IGF-1. GH itself has chronic antiinsulin effects that may result in glucose intolerance. When administered to GH-deficient adults, the hormone increases muscle volume and lean body mass, significantly decreases body fat, as well as improves physical performance and psychologic well-being in these patients.

3.6. Clinical Testing

GH immunoradiometric assays (IRMA), employing a double monoclonal antibody sandwich system, are now widely used because of their sensitivity and accu- racy, compared with RIAs. Random GH measurements are not helpful in the diagnosis of GH hypersecretory or deficiency states because of the pulsatile nature of pitu-

itary GH secretion, and integrated measurements over time are required. Serum IGF-1 levels are invariably high in acromegaly and correlate better with the clinical manifestations of hypersomatotrophism than single GH measurements. Therefore, high IGF-1 levels are highly specific for diagnosing acromegaly. IGF-1 is less helpful in the evaluation of GH deficiency (GHD), because its levels are low in infancy and the normal range over- laps with values measured in GH-deficient children.

3.6.1. PROVOCATIVE TESTS

Provocative tests are dynamic tests that assess GH reserve in the evaluation of GHD, by pharmacologic stimulation of the somatotropes. The diagnosis of GHD in children is determined by an inadequate GH response to at least two separate provocative tests.

3.6.2. G^ROWTH HÔRMONE–RÊLEASING HÔRMONE The GHRH test (1 μg/kg, intravenously) may help in the diagnosis of GH insufficiency, when GH does not increase within 60 min subsequent to injection. If the somatotropes are first primed with intermittent GHRH pulses, the acute GHRH test may sometimes distinguish between hypothalamic and pituitary GH deficiency.

3.6.3. INSULIN-INDUCED HYPOGLYCEMIA

Insulin-induced hypoglycemia is the most reliable provocative stimulus for the diagnosis of GHD. Cloni- dine, arginine, L-dopa, and propranolol are other stimu- lants used in the evaluation of GH reserve. The diagnosis of GHD in adults is currently being reevaluated because the sensitive new IRMAs indicate “normal” integrated GH levels of <0.5 μg/L.

3.6.4. S^UPPRESSION T^ESTS

In patients with acromegaly, the elevated GH levels fail to suppress (<1 μg/L) after an oral glucose load.

3.7. GH Hypersecretion: Acromegaly

More than 95% of patients with acromegaly harbor a pituitary adenoma; two-thirds have pure GH-cell tumors; and the others have plurihormonal tumors, usually expressing PRL in addition to GH. These patients have elevated GH and IGF-1 levels and normal GHRH concentrations. Ectopic acromegaly may be central owing to excess GHRH production by functional hypothalamic tumors, or by peripheral rare extrapituitary GH-secreting tumors (pancreas) and the more common tumors secreting GHRH (carcinoid, pancreas, small-cell lung cancer). Patients with ectopic acromegaly disclose normal (central) or elevated (peripheral) GHRH levels. The clinical manifestations of acromegaly include generalized visceromegaly with enlargement of the tongue, bones, salivary glands, thyroid,

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heart, and soft organs; characteristic facial features of wide spacing of the teeth, large fleshy nose and frontal bossing; and skeletal overgrowth leading to mandibu- lar overgrowth with prognathism, and increased hand, foot, and hat size. Patients present with voice deepen- ing, headaches, arthropathy and carpal tunnel syndrome, muscle weakness and fatigue, oily skin and hyperhydrosis, hypertension and left ventricular hypertrophy, sleep apnea, menstrual abnormalities, depression, and glucose intolerance. Patients with acromegaly have a significant increase in overall mortality owing to cardiovascular disorders, malig- nancy, and respiratory disease. Selective transsphenoidal resection is the indicated treatment for GH- secreting pituitary adenoma. Octreotide and lanreotide (cyclic somatostatin analogs) significantly attenuates GH and IGF-1 levels in most patients, and chronic administration is accompanied by marked clinical improvement. Pegvisomant (GH antagonist) was shown recently to normalize IGF-1 levels in most patients with acromegaly, when administered daily.

However, this drug further increases GH (albeit bio- logically inactivated) in treated patients, and the long- term effects on adenoma size are still unknown.

3.8. GH Hyposecretion

GHD in children may be isolated or combined with deficiencies of other pituitary hormones. Its incidence approaches 1:5000 to 1:10,000, and only between 25 and 30% of affected children exhibit identifiable under- lying disorders. Several types of hereditary GHD with different modes of inheritance have been described.

Molecular defects include GH gene deletion or lack of synthesis or secretion of GHRH, and excess somatostatin secretion has also been postulated. A group of children with growth failure may secrete an immunore- active but a bioinactive GH molecule. Point mutations or major deletion of the Pit-1 gene results in strains of dwarf mice that fail to develop pituitary somatotrope, lactotrope, and thyrotrope cells. This may be a potential mechanism for human GHD. Children with GHD are short and fail to grow at a normal rate, and this is usually noted by 12–18 mo of age. Patients tend to be over- weight for their height but are normally proportioned.

These children have low stimulated GH levels and low IGF-binding protein-3 (IGFBP-3). IGF-1 levels are normally very low before 3 yr of age and do not correlate with stimulated GH levels. GH replacement with recombinant human GH (rhGH) should be started as early as possible, because total height gain is inversely proportional to pretreatment chronologic and bone age.

Adult GHD may be isolated or owing to panhypopitu- itarism from several causes. These include pituitary

apoplexy, large pituitary tumors, surgical trauma, hemo- chromatosis, hypophysitis, and other sellar lesions. GH- deficient adults have altered body composition with increased fat and decreased muscle volume and strength, lower psychosocial achievement, and altered glucose and lipid metabolism. These patients have low stimulated GH, low or normal IGF-1 and low IGFBP-3. The clinical effects of rhGH treatment in adults include changes in body composition and lipid profile and improvement in quality of life.

3.9. GH Receptor Defects (Laron Dwarfism)

GHD may be owing to failure of the liver and peripheral tissues to generate IGF-1 in response to GH. The genetic defect appears to be in the GH receptor itself, but the clinical characteristics of Laron dwarfism are identical to those in GH-deficient children. Basal and stimulated GH levels are high, but IGF-1 values are low and do not respond to GH therapy. Successful response of Laron dwarfs to recombinant IGF-1 therapy has been reported.

4. PROLACTIN

4.1. Embryology and Cytogenesis of Lactotropes

Lactotropes are the last cells to differentiate in the human fetal pituitary. PRL is found only at 12 wk of gestation and until 24 wk is localized in mammosoma- totropes. These cells produce both GH and PRL and appear to be the source of differentiated lactotropes, which are found only after that time. The lactotropes are acidophilic cells, contain PRL-immunostained secretory granules, and constitute 15% of adenohypophysial cells. However, in multiparous women they represent up to a third of the cells, and during pregnancy and lactation they may constitute 70% of the pituitary cells.

4.2. Prolactin Gene

The human PRL gene is approx 10 kb long, consists of five exons separated by four large introns, and encodes the 199-amino-acid PRL peptide (23 kDa) (Fig. 5). The gene, located on chromosome 6, has two regions respon- sible for lactotrope-specific transcription activation—

a proximal promoter (–422 to +33) and a distal enhancer element (–1831 to –1530 bp), both contain- ing specific binding sites for Pit-1. PRL is homologous to GH and placental lactogen, and they are thought to have arisen from a common original ancestral gene.

Sequence homology among human PRL, bovine, and other mammals is in the range of 70–80%. Dopamine, the major PRL inhibitory factor, acts through the D2 dopamine receptor (D2R), to decrease intracellular cAMP, PRL gene transcription, synthesis, and release,

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mediated by the phosphoinositide/calcium pathway (Fig. 6). VIP induces PRL synthesis and secretion, whereas glucocorticoids and thyroid hormones exert an inhibitory effect on PRL transcription and secre-

tion. Estrogens, by binding of the estrogen receptor to the PRL enhancer element, and TRH through the phosphoinositide-PKC pathway, induce PRL transcription and secretion (Fig. 6).

Fig. 5. Schematic structure of PRL gene, mRNA, and protein. The PRL gene (top) consists of the promoter, and five exons (1–5), separated by four introns (A–D). The PRL protein is a 199-amino-acid 23-kDa polypeptide with three intraprotein disulfide bonds.

(From Molitch [Prolactin] in Melmed, 1995.)

Fig. 6. Regulation of PRL expression in the lactotrope. Dopamine is the predominant physiologic inhibitor of PRL. Dopamine binds to the D2R, coupled to G_iprotein, and suppresses cAMP, PKA, and intracellular calcium, thus inhibiting Pit-1 and PRL expression.

Estrogens (stimulation) and glucocorticoids and thyroid hormones (suppression) mediate their effects on PRL via binding of their nuclear receptors to response elements on the PRL promoter. TRH binds a G protein–coupled seven-transmembrane domain receptor, activates the G_sstimulatory protein, increases cAMP, and induces the phosphoinositide-PKC pathway that also increases intracellular calcium concentrations. This may result in increased transcriptional activity of Pit-1. IP₃= inositol triphosphate; DAG = diacylgylcerol.

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4.3. PRL Secretion

PRL is under tonic inhibitory hypothalamic control and is secreted in pulses with an increase in amplitude during sleep. Basal levels increase throughout the course of pregnancy, up to 10-fold by term. In the postpartum period, basal PRL levels remain elevated in lactating women, and suckling triggers a rapid release of PRL.

TRH is a pharmacologic stimulator of PRL release, and dopamine is the predominant physiologic inhibitor factor (Fig. 6).

4.4. PRL Receptor

The human PRL receptor is a 598-amino-acid protein encoded by a gene on chromosome 5 (p13-p14). The receptor contains a long extracellular region, a single transmembrane region, and a short cytoplasmic domain.

There is a high sequence homology among the human, rat, and rabbit PRL receptors, and between the human PRL and GH receptors, which are colocalized to the same area on chromosome 5. PRL receptors are widely distributed, and their hormonal regulation is tissue specific. In the mammary gland, high progesterone levels during pregnancy limit PRL receptor numbers, and early in lactation the numbers increase markedly. Testoster- one increases and estrogens decrease PRL receptor levels in the prostate. In most organs studied, PRL is able to up- and downregulate the level of its own receptor.

4.5. PRL Action

PRL contributes to breast development during pregnancy, with estrogen, progesterone, and placental lactogen. After delivery PRL stimulates milk production.

Hyperprolactinemia suppresses gonadotropin-releasing hormone (GnRH) pulses at the hypothalamus, pulsatile secretion of pituitary gonadotropins, and ovarian release of progesterone and estrogen. In men testosterone levels are low. PRL may induce mild glucose intolerance and has a role as an immune modulator.

4.6. Immunoradiometric Assay

Most assays for human PRL are based on the double- antibody method. Normal concentrations are slightly higher (<20 μg/L) in women than in men (<15 μg/L).

4.7. Hyperprolactinemia

The differential diagnosis of hyperprolactinemia includes PRL-secreting pituitary adenomas, pituitary stalk compression blocking dopamine access (owing to large nonfunctioning adenomas), acromegaly, chronic breast stimulation, pregnancy, hypothyroidism, chronic renal failure, hypothalamic disorders, and medications (estrogens, phenothiazines, methyldopa, metoclopra- mide, and verapamil). PRL serum levels >200 μg/L are

usually associated with prolactinoma. Hyperprolac- tinemia presents as amenorrhea and galactorrhea in women, and impotence and infertility in men.

4.8. PRL-Secreting Pituitary Adenomas

Prolactinomas are the most common hormone-secreting pituitary adenomas. Many tumors secrete both GH and PRL. These monoclonal tumors are classified as microprolactinomas (<10 mm, 90% occurring in women), or macroprolactinomas (>10 mm, 60% occurring in men).

In macroadenomas, the clinical presentation of hyperprolactinemia may be associated with mass effect signs of headaches and visual field disturbances. Most patients (70%) are successfully treated with dopamine agonists (bromocriptine and cabergoline). Transsphe- noidal surgery is reserved for drug-resistant tumors.

4.9. Immune System Interaction.

Lymphocytes express PRL receptors. Hypoprolac- tinemic states are associated with impaired lymphocyte proliferation, decreased macrophage activation, and other manifestations of immunosuppression, which can be restored with PRL treatment in rats.

5. THYROID-STIMULATING HORMONE 5.1. Embryology and Cytogenesis

of Thyrotropes

Differentiated thyrotropes are found in the fetal pituitary at 12 wk of gestation, when TSH β-subunits are immunolocalized and also found in the circulation.

However, TSH levels remain low until wk 18, when the fetal levels increase significantly. Thyrotropes comprise only 5% of the anterior pituitary cell population.

5.2. TSH Gene

Human TSH is a 211-amino-acid glycoprotein with a molecular mass of 28 kDa, which is structurally related to LH, FSH, and human chorionic gonadotropin (hCG).

They are composed of a heterodimer of two noncovalently linked subunits, αand β. The α-subunits of all four glycoproteins are identical and are encoded by a 13.5-kb gene (Fig. 7), located on chromosome 6q21- 23, containing four exons and three introns. The α-subunit is expressed in thyrotropes, gonadotropes, and placental cells, but cell-specific expression is depen- dent on different regions of the promoter. The β-subunits of the glycoproteins define tissue specificity despite a 75% similarity in their primary structure and cysteine residues. The TSH β-subunit gene is 4.9 kb in size, is located on chromosome 1p22, and consists of three exons and two introns. The pituitary transcription factor Pit-1 binds to the β-subunit promoter but is not required for α-subunit gene expression. Both α- and β-

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subunit transcription are induced by TRH and inhibited by triiodothyronine (T₃) and dopamine (Fig. 8).

5.3. TSH Secretion

TSH pulsatile secretion occurs every 2–3 h, and is superimposed upon basal hormone release from the pituitary. TSH has a circadian pattern of secretion, with nocturnal levels measured up to twice daytime levels.

TSH secretion is enhanced by TRH, while T₃, thyroxine (T₄), dopamine, SRIF and glucocorticoids suppress TSH secretion (Figure 8).

5.4. TSH Receptor

The TSH receptor is located on the plasma membrane of thyroid follicular cells. It consists of a polypeptide chain of 764 amino acids containing a 398-amino-acid extracellular domain, seven transmembrane segments, and a short intracellular domain of 82 amino acids.

Receptor-ligand binding activates Gs protein and adeny-

late cyclase cascade. The TSH receptor gene is located on chromosome 14 (q31) and consists of 10 exons. The extracellular domain and parts of the transmembrane domain contribute to TSH binding, and binding specifity is conferred by the TSH β-subunit, although LH and hCG can activate the human TSH receptor to a certain degree. Germ-line mutations in the transmembrane domain of the TSH receptor gene, resulting in consti- tutive cAMP activation, were reported in congenital hyperthyroidism, and somatic mutations in this domain were also found in patients with hyperfunctioning thyroid adenomas. Moreover, resistance to thyrotropin, caused by mutations in the extracellular domain of the TSH receptor gene, has been described.

5.5. TSH Action

TSH induces morphologic changes of the follicular cells; causes thyroid gland enlargement owing to hyperplasia and hypertrophy; and stimulates iodide uptake Fig. 7. Schematic structures of human α, hLH-β, hCG-β, and hFSH-β genes: dark areas = untranslated regions; stippled areas = signal peptides; unshaded areas = mature proteins; hatched area = 5' untranslated sequence; solid triangles = introns; pair of arrows in top diagram = cAMP regulatory element; arrow in bottom diagram = polyadenylation site used by some FSH-β transcripts. (From Gharib et al., 1990.)

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and organification, thyroglobulin gene transcription, and thyroid hormone secretion.

5.6. Clinical Testing

RIA for TSH, the “first-generation assay,” uses labeled antigen and is useful in distinguishing elevated TSH levels in primary hypothyroidism from normal euthyroid values but is unable to differentiate euthyroid and hyperthyroid subjects. The new sensitive immuno- metric assays employ labeled antibody and a “capture antibody” in sandwich formation and clearly discrimi- nate euthyroid from hyperthyroid patients. TRH (200–

500 μg, intravenously or intramuscularly) stimulates TSH release in euthyroid subjects. Hyperthyroid patients have no response, and primary hypothyroid patients demonstrate augmented TSH response to TRH stimulation. However, elevated basal TSH levels in TSH-secreting pituitary tumors fail to respond to TRH, and patients with hypothyroidism secondary to pituitary or hypothalamic disease have attenuated TSH response.

5.7. TSH Hypersecretion

Most cases of elevated serum TSH levels are a result of primary thyroid failure. Thyroid hormone resistance is a rare syndrome that includes clinical euthyroidism, elevated levels of thyroid hormones, and inappropriately normal to slightly increased TSH levels. TSH-producing pituitary adenomas comprise <1% of all pituitary tumors and secrete the TSH α- and β-subunits chracteristic of the normal thyrotropes. However, the α- subunit is synthesized in excess of the β-subunit, a useful characteristic in the diagnosis of these tumors. TSH secretion by these tumors fails to respond to TRH stimulation or to normal thyroid hormone–negative feedback.

However, somatostatin suppresses TSH release from the tumors. Most patients present with hyperthyroidism and diffuse goiter. TSH is elevated or inappropriately normal in the presence of elevated thyroid hormones.

Cosecretion of other pituitary hormones—GH, PRL, and FSH—is common, resulting in acromegaly, and Fig. 8. Regulation of TSH expression in the thyrotrope. Both α- and β-subunit transcription and TSH secretion are induced by TRH and inhibited by thyroid hormones and dopamine. TRH, via binding to a G_sprotein–coupled seven-transmembrane domain receptor, activates PLC, which hydrolyzes phosphoinositol 4,5-bisphosphate to DAG and IP₃, activating PKC, and stimulating α-subunit and β-TSH transcription. Increased cAMP also enhances α- and β-subunit transcription. Dopamine and somatostatin decrease TSH by receptors coupling to the G_iprotein, reducing adenylyl cyclase activity. Dopamine inhibits TSH transcription, production, and secretion, whereas somatostatin suppresses secretion. Both T₄and T₃are potent feedback inhibitors of TSH. The intracellular monodeiodination pituitary of T₄to T₃contributes to T₃content in the thyrotrope, where T₃binds to specific nuclear receptors, inhibiting transcription of TSH subunit genes. Glucocorticoids and cytokines (TNF, IL-6) also decrease TSH secretion.

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amenorrhea, galactorrhea, and impotence. Because these tumors are usually large macroadenomas, local intracranial mass effects are common. Transsphenoidal pituitary surgery is the preferred initial approach, but most patients are not cured and require adjuvant medi- cal or radiation therapy. The use of somatostatin analogs, octreotide and lanreotide, is a successful treatment option for these tumors. Thyroid ablation or surgery is not recommended because of the potential risk of pituitary tumor expansion, owing to release of the thyrotrope cells from negative feedback inhibition.

6. FSH AND LH

6.1. Embryology and Cytogenesis of Gonadotropes

Gonadotropes are found in the fetal pituitary at 12 wk of gestation, when β-subunits are immunolocalized and first detected in blood. Gonadotropes represent up to 10% of the pituitary cell population. These basophilic cells reveal cytoplasmic positivity for FSH and LH, usually both in the same cell. However, some cells contain only one of the two hormones.

6.2. FSH and LH Genes

The gonadotropins, FSH and LH, are members of the glycoprotein hormone family and share many struc- tural similarities with TSH and hCG. The α-subunits of all four members of this hormone family are identical, and the β-subunits share considerable amino acid homology with one another, indicating evolution from a common precursor. The FSH- and LH-β-subunits are both expressed in the gonadotropes and possess three exons and two introns (Fig. 7). FSH-β-subunit, located on the short arm of human chromosome 11p13, is highly conserved among different species. LH β-gene, approx 1.5 kb in length, is one of the hCG β-like gene cluster, arranged on human chromosome 19q, and encodes a 121-amino-acid mature protein. Human FSH is a 34- kDa protein with 210 amino acids, and LH is a 28.5- kDa protein consisting of 204 amino acid residues.

GnRH pulses increase transcription rates of all three gonadotropin subunits: α, LH-β,and FSH-β. Testoster- one increases FSH-β mRNA levels in pituitary cell cultures but has no effect on LH-β mRNA. Estrogen negatively regulates transcription of all three subunits.

However, estrogen exerts a positive feedback effect at the pituitary level under several physiologic conditions and increases LH-β mRNA.

6.3. Regulation of Secretion

GnRH is the major regulator of gonadotropin secretion from pituitary cells. The frequency and amplitude of GnRH pulses are critical for stimulating LH and FSH

output. Estrogens can exert both stimulatory and inhibitory effects on gonadotropin secretion, depending on the dose, duration, and other physiologic factors. Test- osterone inhibits in vivo serum FSH levels, but its direct effects on FSH release at the pituitary level are stimulatory. Another regulator of FSH secretion is inhibin, a gonadal peptide produced by Sertoli cells that inhibits FSH release and, under some conditions, may also regulate LH output.

6.4. Gonadotropin Receptor

FSH, LH, and TSH receptors have similar structure and belong to the subfamily of G_sprotein–coupled receptors having seven hydrophobic transmembrane segments.

The specific, high-affinity interaction between hormone and receptor is owing to the large extracellular N-terminal domain of the receptor (LH: 340 amino acids). The LH receptor is a single 75-kDa polypeptide of 700 amino acids. FSH receptor is also a single polypeptide, consisting of four subunits of similar mass (60 kDa).

6.5. Gonadotropin Action

In the male, LH binds to receptors on the Leydig cells and stimulates testosterone synthesis. High intrates- ticular testosterone levels are important for spermatoge- nesis. FSH is probably essential for the maturation process of the spermatids. In the female, LH and FSH are major regulators of ovarian steroid production. FSH plays a critical role in follicle growth and cytodifferen- tiation of granulosa cells.

6.6. Clinical Testing

RIAs of FSH and LH suffer from limited sensitivity and specificity owing to crossreactivity of free α-subunits and other pituitary glycoprotein hormones. The two site-directed IRMA and nonisotopic asays are more sensitive measurements with no α-crossreactivity and are extremely useful in studying physiologic events characterized by low gonadotropin levels. The GnRH (25–100 μg, intravenously) stimulation test has limited usefulness in the diagnosis of hypothalamic- pituitary disorders. Patients with primary testicular failure exhibit an exaggerated increase in serum FSH and LH response within 30 min after injection. How- ever, patients with hypothalamic and pituitary disorders cannot be distinguished. Repetitive administration of GnRH pulses may normalize gonadotropin responses in patients with hypothalamic disease, indicating pituitary integrity.

6.7. Gonadotrope-Cell Tumors

Gonadotrope adenomas are the most common pituitary adenomas. In the past, they were called “nonse- creting adenomas,” because the gonadotropins and their

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subunits produced by them are either not released or inefficiently secreted, and they usually do not produce a distinct clinical syndrome. These tumors produce supranormal (up to 10 times normal) serum levels of α- and FSH-β-subunits, but rarely of LH-β. Usually, the subunits are not secreted in the same proportions. Some gonadotrope adenomas produce α-subunits but not intact FSH or LH. Administration of TRH to patients with gonadotrope adenomas usually results in secretion of gonadotropins or their subunits, compared with no stimulation in healthy subjects. Most of the “nonsecret- ing adenomas” immunostain for intact FSH and LH, or α-, FSH-β-, and LH-β-subunits. Mass effects, including optic chiasm pressure and other neurologic symptoms, may be the first symptoms of large gonadotrope tumors.

Excessive secretion of FSH or LH may actually downregulate the axis.

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