Growth Hormone–Releasing Hormone (GHRH)

PreproGHRH is a polypeptide chain of 108 amino acids (12.4 kDa). Removal of the 20 amino acid signal (leader) sequence yields the 88 amino acid proGHRH. Cleavage of the 11 amino acid N-terminal pro-sequence and the 31 amino acid C-terminal pro-sequence, with release of 2 free amino acids (positions 76-77 with reference to preproGHRH), produces the 44 amino acid mature GHRH. The terminal leucine of GHRH is amidated. Alternative splicing of the mRNA (isoform 2) produces a preproGHRH protein of 107 amino acids, which is missing amino acid 103.

Somatotropin Release–Inhibiting Hormone

Somatotropin release–inhibiting hormone (SRIH) also is known as somatostatin.¹⁹⁵ It is widely distributed throughout the body (CNS, gut, and delta cells of the islets of Langerhan) and produces multiple physiologic effects. Somatostatin receptors are widely distributed (see later). Somatostatin that functions as SRIH is produced by the paraventricular nucleus of the hypothalamus. In the islets, somatostatin suppresses glucagon and insulin secretion, whereas somatostatin release is stimulated by both of these hormones. In this way, delta cell somatostatin modulates islet function by smoothing out extremes in the secretion of glucagon and insulin to maintain a stable blood glucose concentration. Somatostatin in the gut is found in highest concentration in the duodenum and jejunum.

Expression of the somatostatin gene produces the 116 amino acid polypeptide preprosomatostatin. Cleavage of the signal sequence (24 amino acids) produces prosomatostatin (92 amino acids), and subsequent cleavage of the N-terminal pro-sequence (64 amino acids) yields a 28 amino acid form of somatostatin (SST-28). SST-28 has an intrachain disulfide bond between amino acids 17 and 28. In many tissues, SST-28 undergoes cleavage to a 14 amino acid form (SST-14) through removal of the N-terminal 14 amino acid sequence by the enzymes prohormone convertase 1/prohormone convertase 2 (PC1/PC2) and carboxypeptidase E (CPE). SST-14 is the major form of somtatostatin in the CNS and delta cells, whereas SST-28 is the major form in the gastrointestinal tract. SST-28 is also the major circulating form of somatostatin. Therefore, somatostatin measurements in peripheral blood do not reflect SRIH secretion. Somatostatin is highly conserved in nature: all vertebrates have the identical sequence for SST-14.¹⁹⁵

In addition to GH suppression, somatostatin also suppresses TRH, TSH, CRH, and ACTH. However, the effect of somatostatin on regulation of the adrenal cortical and thyroid axes ordinarily is minor. In the gastrointestinal tract, somatostatin reduces the secretion of multiple hormones including (1) gastrin, (2) secretin, (3) cholecystokinin, (4) vasoactive intestinal polypeptide, (5) motilin, (6) neurotensin, and (7) pepsin and reduces gastric pH, intestinal motility, ion and nutrient absorption, and proliferation of the mucosa (see Chapter 51). Calcitonin, catecholamines, renin, and pancreatic exocrine function are suppressed by somatostatin. Somatostatin analogs (e.g., octreotide) are used pharmacologically to suppress a variety of hormone overproduction conditions.²⁸

Growth Hormone–Releasing Hormone Receptor (GHRHR)

The anterior pituitary somatotroph GHRH receptor (GHRHR) is a member of family B-III of the G-coupled receptor superfamily (the “secretin” family).⁷ Receptors for (1) secretin, (2) vasoactive intestinal polypeptide (VIP), (3) parathyroid hormone (PTH), and (4) calcitonin share partial sequence identity with GHRHR.

PreGHRHR is a 423 amino acid polypeptide that is converted to the mature 401 amino acid form of GHRHR by removal of the 22 amino acid signal peptide. The N-terminal extracellular domain is 110 amino acids. GHRHR has seven transmembrane domains and a 42 amino acid cytoplasmic domain. Amino acid 50 may be glycosylated.

Somatostatin (SRIH) Receptor

Throughout the body, there are five receptors for somatostatin (SSTR1 through SSTR5; Table 53-3). Each receptor is encoded by a gene located on a separate chromosome. SSTR2 has two alternatively spliced isoforms. All of the SSTR receptors have seven transmembrane domains, and are coupled with a pertussis toxin–sensitive G protein. SSTR3, SSTR4, and SSTR5 are expressed in the pituitary.

Growth Hormone (GH)

GH has two disulfide bridges (amino acids 54 and 165, and amino acids 182 and 189).⁵⁹ Structurally, GH has four main alpha-helices, and within the connecting loops three mini-helices. Two circulating forms of GH are present: a 22 kDa form that is a 191 amino acid chain (full-length GH) representing 85 to 90% of circulating GH, and a 20 kDa GH that lacks amino acids 32 through 46.¹⁸⁴ The 20 kDa form of GH results from alternative splicing of the GH mRNA transcript. In addition to the 22 kDa and 20 kDa forms, circulating GH exists as aggregates and oligomers. “Big GH” is a dimer of GH monomers, and “big-big GH” is GH associated with its binding protein (GHBP). GHBP is the external domain of the GH receptor (GHR), which binds GH with high affinity and is produced by cleavage of the GHR. Approximately 55% of all circulating GH forms are monomeric; big GH and big-big GH represent ≈27% and ≈18% of circulating GH, respectively. Approximately 50% of GH is not bound to GHBP, ≈45% is bound to GHBP, and the remaining 5% of GH is bound to low-affinity binding proteins. Considering the multiple forms of GH, it is not surprising that significant analytical biases are observed between different immunoassays for GH.

The gene for GH (chromosome 17q24.2) is a member of the GH subfamily that includes (−5′ to 3′ direction) (1) GH (the GH1 gene), (2) a chorionic somatomammotropin (human placental lactogen) pseudogene designated CSHP, (3) a chorionic somatomammotropin-A designated CSH1, (4) the placentally produced 22 kDa GH variant (GH-V; gene designation GH2), and (5) chorionic somatomammotropin-B (gene designation CSH2).⁴⁰ Somatomammotropin (hPL) is a placental hormone with growth-promoting properties. Prolactin (199 amino acids) shares a homologous amino acid sequence with GH, but prolactin, encoded on chromosome 6p22, is not part of the GH complex on 17q24.2.

GH has both direct and indirect activity. Its direct actions will be described in the following sections.¹¹⁸ The indirect activity of GH is mediated by IGF-I. To initiate its direct and indirect activity, GH binds to tissue receptors (GHR) that appear to be expressed by all tissues.

Growth Hormone Receptor (GHR)

The GHR is a member of the class 1 hematopoietic cytokine family.¹⁴² Other members of this family include receptors for erythropoietin, granulocyte-macrophage colony-stimulating factor, and various interferons. Structurally, the GHR is a single-chain, 620 amino acid protein (130 kDa). PreGHR includes an 18 amino acid leader sequence. The GHR structure includes an extracellular domain (246 amino acids), a transmembrane domain (24 amino acids), and a cytoplasmic domain (350 amino acids).³¹ When the extracellular portion of the GHR is shed, the 55 kDa GHBP moiety is released into the circulation.

Four isoforms of the GHR are expressed by alternative splicing of the nascent mRNA. Isoform 1 is the full-length receptor. Isoform 2 differs in the sequence of amino acids 292-297 (with reference to preGHR) and lacks amino acids 298-638. Isoform 3 differs in the sequence of amino acids 292-294 and lacks amino acids 295-638. An alanine at position four is replaced by aspartic acid, and amino acids 25 to 46 are missing in isoform 4.

The GHR exists as a cell surface dimer in its inactive state (Figure 53-3). When GH binds to the GHR, the receptor recruits or activates 120 kDa Janus-associated kinase enzymes (JAK2, a type of adapter tyrosine kinase). JAK2 then exerts tyrosine kinase activity by phosphorylating itself and the GHR.¹³² Phosphorylation activates JAK2, which triggers several intracellular pathways involving (1) STATs (signal transducers and activators of transcription), (2) the insulin receptor substrate (IRS), (3) phosphatidylinositol 3′-kinase (PI3K), and (4) a mitogen-activated protein kinase (MAPK). STAT5b is involved, but it is unclear whether STAT5a is also involved. In its role as a transcription factor, phosphorylated, dimerized STAT5b enters the nucleus to promote gene transcription. Independent of JAK2, GHR signaling can be effected via Src, which is a tyrosine kinase. (Note: Src is the Rous sarcoma virus proto-oncogene.)

Insulin-like Growth Factors

IGF-I is a member of the insulin-related peptide family whose other members include IGF-II, insulin, and relaxin. Stimulated by hCG during pregnancy, relaxin is produced by the corpus luteum verum (the corpus luteum of pregnancy), the decidua (the uterine lining during pregnancy), and the placenta. Relaxin increases collagenase activity to soften and lengthen the cervix and pubic symphysis and facilitate parturition. Relaxin also reduces uterine contractility by inhibiting myosin kinase activity. In humans, there are three nonallelic relaxin genes: RLN1, RLN2, and RLN3.

Proinsulin, IGF-I, IGF-II, and relaxin are all composed of two domains (A and B) joined by a connecting domain. The connecting domains vary in sequence and length much more than the A and B domains. The connecting domain of proinsulin is cleaved to release C-peptide (connecting peptide), producing insulin A and B chains. Insulin, IGF-I, IGF-II, and relaxin have two disulfide bridges. IGF-I and IGF-II share 62% homology, and they each share 50% homology with insulin.

The 110 amino acid sequence of preproinsulin includes a signal peptide (amino acids 1-24), the insulin B chain (amino acids 25-54), C-peptide (amino acids 57-87), and the insulin A chain (amino acids 90-110). The two interchain disulfide bonds are between amino acids 31 and 96, and 43 and 109. The single intrachain disulfide bond is between amino acids 95 and 100.

Two forms of prepro-IGF-I are expressed as a consequence of alternative mRNA splicing: IGF-IA and IGF-IB. The IGF-IA preprohormone is 153 amino acids, including a signal peptide (amino acids 1-21), an N-terminal propeptide (amino acids 22-48), IGF-I (70 amino acids), and a C-terminal propeptide (the E domain, amino acids 119-153). The IGF-I polypeptide includes the B domain (amino acids 49-77), the C (connecting) domain (amino acids 78-89), the A domain (amino acids 90-110), and the D domain (amino acids 111-118). Three disulfide bonds are present between amino acids 54 and 96, 66 and 109, and 95 and 100. IGF-I is not glycosylated.

The 195 amino acid IGF-IB preprohormone is composed of a signal peptide (amino acids 1-21), a propeptide (amino acids 22-48), IGF-I (70 amino acids), and another propeptide region (the E domain, amino acids 119-195). Differences in the D versus E domain of the prepro-IGF-I distinguish IGF-IA from IGF-IB; the tertiary structure of the IGF-I proteins and the placement of disulfide bonds are identical between IGF-IA and IGF-IB.

The IGF-II preprohormone comprises 180 amino acids. The first 24 residues constitute the signal (or leader) peptide. After their removal, IGF-II is derived from pro-IGF-II after cleavage of the C-terminal E peptide (amino acids 92-180). Therefore, amino acids 25 to 91 include the B region (amino acids 25-52), the C region (amino acids 53-64), the A region (amino acids 65-85), and the D region (amino acids 86-91) of IGF-II. Isoform I is the full-length prepro-IGF-II (180 amino acids). Formed by alternative splicing, isoform II lacks amino acid 25 (alanine) and is therefore 179 amino acids in length. IGF-II is glycosylated at amino acid 99 and has three intrachain disulfide bonds between amino acids 33 and 71, 45 and 84, and 70 and 75. A potential glycosylation site is amino acid 163.

Prorelaxin is a 185 amino acid protein that contains a signal sequence (amino acids 1-22), a B chain (amino acids 23-53), a connecting propeptide (amino acids 56-158), and an A chain (amino acids 163-185). A-B interchain disulfide bonds can occur between amino acids 35 and 172, and 47 and 185. An additional disulfide bridge may occur between amino acids 171 and 176.

IGF-I and IGF-II circulate together with an IGF binding protein, IGFBP (specifically IGFBP-3), and the acid-labile subunit (ALS) to form a 150 kDa trimeric protein complex. About 75 to 80% of IGF-I/IGFBP-3 complexes are trimeric; the remaining IGF-I/IGFBP-3 complexes are dimeric and may include other IGFBPs. Less than 1% of the total IGF-I is free (the biologically active form). The binding affinity of IGF-I for the insulin receptor is low (≈7% of insulin affinity), but circulating IGF-I concentrations exceed insulin by three orders of magnitude. Without binding proteins, therefore, IGF-I could cause potentially devastating hypoglycemia.

The trimeric IGF-I-IGFBP-3-ALS complexes do not normally cross capillary membranes because of their size. However, the 50 kDa binary complex and free IGF-I are able to enter the interstitium, where binding to type I IGF receptors can occur.

Most of the circulating IGF-I is produced by hepatocytes.¹³⁹ However, IGF-I is also produced locally throughout the body and thus acts as a paracrine and an autocrine hormone. The possible endocrine (systemic) influence of IGF-I on growth is discussed later.

Similar to IGF-I, IGF-II does not normally produce hypoglycemia. However, reports have described tumors that secrete a larger than normal form of IGF-II. This big IGF-II did not bind normally to IGFBP-3, and therefore the free IGF-II concentration was greatly elevated. As a result of IGF-II binding to the insulin receptor, the clinical syndrome was similar to hypoglycemia caused by hyperinsulinism (i.e., free fatty acids were not elevated, ketones were absent, and lactate and alanine were not elevated), yet insulin itself was suppressed because the beta cells were normal. Removal of the tumor resolved the patient’s hypoglycemia.⁴⁴

Insulin-like Growth Factor Binding Proteins

Because of their very high affinity (K_d 10⁻¹⁰ to 10⁻¹¹ M) for IGFs, IGFBPs are regarded as inhibitors of IGF action.¹³⁶ IGFBPs have higher affinity for IGFs than the IGF receptors. Currently under study is the possibility that IGFBPs may directly influence growth. Receptors for IGFBPs have been described. Proteolysis of IGFBPs releases IGF-I; therefore IGFBP proteases can influence free IGF-I concentrations. Table 53-4 summarizes the features of the seven known IGFBPs.

Receptors for Insulin-like Growth Factors

Two types of receptors for IGFs have been identified: the type I IGF receptor and the type II IGF receptor (Figure 53-4).¹³⁴ Structurally, the type I IGF receptor is similar to the insulin receptor. Because this receptor does not exclusively bind IGF-I, the terminology “IGF-I receptor” is not recommended. The type I receptor is derived from a single precursor protein of 1367 amino acids that includes the 30 amino acid signal peptide. The 706 amino acid (130 kDa) alpha chain is extracellular and is bound by a disulfide bond to the transmembrane 90 kDa (627 amino acids) beta chain. Cleavage of the alpha and beta chains releases a tetrapeptide (707 to 710: arginine-lysine-arginine-arginine). Beta chain amino acids 906 to 929 form a transmembrane domain. The receptor exists as a homodimer (beta-alpha-alpha-beta) with the two alpha chains bound to each other by two disulfide bonds. Similar to the type I IGF receptor, the insulin receptor is a homodimer of two 135 kDa alpha chains and two 95 kDa beta chains.

Tyrosine kinase activity in the cytoplasmic portion of the type I IGF receptor beta chain results from binding of an IGF molecule to the cysteine-rich portion of the alpha chains (amino acids 148-302), which causes conformational changes in both alpha and beta chains. Intracellular signaling involves autophosphorylation and phosphorylation of the 185 kDa insulin receptor substrate 1 (IRS1), which is the predominant target of the active type I IGF receptor. The type I IGF receptor binds IGF-I with higher affinity than IGF-II, and affinity for insulin is lower than the affinity of the receptor for IGF-I or IGF-II. The affinities of the insulin receptor are the opposite: insulin >> IGF-II > IGF-I.

The type II IGF receptor is structurally dissimilar from the type I IGF receptor and the insulin receptor. The 270 kDa, 2451 amino acid, type II IGF receptor is a monomeric protein that is similar to the epidermal growth factor (EGF) receptor. (Note: The leader sequence is 40 amino acids.) The EGF receptor itself is also known as ErbB1 or HER1. The external portion of the receptor is 2264 amino acids, the transmembrane domain is 23 amino acids, and the cytoplasmic domain is 164 amino acids. Beginning at the N-terminus, thirteen ≈150 amino acid repeats and a 47 amino acid fibronectin type II domain are followed by two more repeats. Evidence indicates at least five glycosylation sites: amino acids 112 (with preference for the pre-type II IGF receptor), 581, 626, 747, and 1246. Two disulfide bonds are found between amino acids 1903 and 1927, and between amino acids 1917 and 1942.

Ligand binding to EGF receptors results in dimerization and tyrosine kinase activation. The external ligand-binding domain of EGFs is composed of numerous short amino acid repeats. The type II IGF receptor removes IGF-II from the circulation, and its engagement leads to activation of transforming growth factor (TGF)-beta. The type II IGF receptor binds mannose-6-phosphate, in addition to IGFs, permitting the uptake and intracellular movement of mannose-6-phosphate–containing lysosomal enzymes. The binding sites for IGFs and mannose-6-phosphate are found on different parts of the receptor. The affinities of the type II IGF receptor are as follows: IGF-II >> IGF-I > insulin. Because this receptor does not exclusively bind IGF-II, the terminology “IGF-II receptor” is not recommended.

A hybrid receptor consisting of the alpha-beta chain of the insulin receptor and the alpha-beta chain of the type I IGF receptor has been described. It has been suggested that these hybrid receptors may allow cancers to respond to insulin.²³ Increasing evidence suggests that IGF-I may enhance the growth of many tumors. Thus, the casual use of IGF-I injections in children to enhance growth in short but otherwise normal children is problematic and potentially carcinogenic.

Physiologic Actions

GH effects can be classified as indirect or direct.¹⁷³ GH directly raises blood glucose by stimulating gluconeogenesis and reducing insulin sensitivity. Also, it causes adipose tissue lipolysis and the resulting GH-induced elevations in free fatty acids provide an alternative energy source that serves to spare glucose for CNS utilization. Therefore when glucose and free fatty acid concentrations are raised at times of stress, in partnership with epinephrine, glucagon, and cortisol, fuels for the fight-or-flight response are provided. GH has other effects on intermediary metabolism: GH stimulates the uptake of nonesterified fatty acids by muscle and accelerates the mobilization and metabolism of fat from adipose tissue to the liver.

At the epiphysis of growing bone, GH promotes epiphyseal prechondrocyte differentiation. GH directly stimulates the production of IGF-I, IGFBP-3, and the acid labile subunit (ALS). In turn, after entering the interstitium, unbound IGF-I binds to type I IGF receptors.

Indirect effects of GH are mediated through IGF-I production, which (together with GH) is necessary for linear growth in childhood. IGF-I is mitogenic and antiapoptotic. Epiphyseal prechondrocyte differentiation stimulated by GH, along with the local effects of IGF-I (also under the control of GH), stimulates the clonal expansion of differentiating chondrocytes.

Thus, the overall effect of GH is to promote growth in soft tissue, cartilage, and bone. This action results from stimulation of protein synthesis that is induced in part by an increase in amino acid transport through cell membranes. The effects of GH on bone and muscle are exerted both directly and through the effects of IGF-I under the influence of GH. Increased growth of soft tissue and the skeleton is accompanied by changes in electrolyte metabolism, including positive nitrogen and phosphorous balance, a rise in plasma phosphorous concentration, and a fall in blood urea and amino acid concentrations. Additional responses to GH include increased intestinal absorption of calcium and decreased urinary excretion of sodium and potassium. The metabolic changes most likely are caused by increased uptake of these ions by growing tissue.

IGF-I increases glucose oxidation in adipose tissue and stimulates glucose and amino acid transport into diaphragmatic muscle and heart muscle. Synthesis of collagen and proteoglycans is enhanced by IGF-I, which also has positive effects on calcium, magnesium, and potassium homeostasis. The insulin-like effects of this growth factor have been ascribed in part to its structural similarity to insulin.

Evidence indicates that the local effects of IGF-I (autocrine or paracrine) are predominant in stimulating growth, when compared with the systemic effects of IGF-I produced by the liver. When the hepatic IGF-I gene was knocked out in mice (although nonhepatic tissues expressed IGF-I), growth was normal, and IGFBP-3 and ALS concentrations were low. IGF-I may function as a growth factor in utero; however, its secretion in utero is not under the control of GH.

In the absence of GH, IGF-I is not as effective a growth stimulant as it is when both are present. IGF-I treatment alone is not recommended as therapy for GH deficiency; IGF-I therapy is reserved for cases of GH resistance due to GHR deficiency.⁹⁵

GH (through IGF-I) and insulin induce growth in a similar manner because both have protein anabolic effects and stimulate the transport of amino acids into peripheral cells. Their respective effects on glucose homeostasis, however, oppose each other. Most growth-promoting GH effects are delayed rather than immediate and are exerted primarily through IGF-I.

IGF-I concentrations vary widely with age and gender.⁸³ IGF-I rises during childhood, and during puberty, IGF-I concentrations can be two to three times the adult concentration. Following adolescence, IGF-I concentrations show a gradual decline, reaching a steady state in the third decade of life.

GH is not the only determinant of IGF-I concentration in the circulation. Transformation of the GH stimulus to IGF-I production and secretion is modulated by (1) nutrition, (2) the presence or absence of chronic inflammation, (3) thyroid function, (4) glucocorticoids, and (5) sex steroids. IGF-I secretion is reduced by (1) malnutrition, (2) malabsorption (e.g., inflammatory bowel disease), (3) celiac disease, (4) cystic fibrosis, (5) chronic disease, (6) hypothyroidism (e.g., Hashimoto thyroiditis), and (7) sex hormone deficiency during adolescence. Therefore a decreased IGF-I concentration is not necessarily synonymous with GH deficiency.

In cases of acquired GH resistance and genetic GH resistance (GHR mutation or signaling disorder), GH concentrations will rise, and high concentrations of GH produce hyperlipidemia and hyperglycemia.⁷⁵ Cases of acquired GH resistance are not treated with GH or IGF-I, but instead are treated through resolution of the underlying disorder.

The actions and regulation of IGF-II have been debated.¹⁶⁰ IGF-II is believed to be important for intrauterine growth. Mice display intrauterine growth retardation when the IGF-II gene is knocked out. Although IGF-II–producing tumors are very rare, such tumors can produce hypoglycemia. The tumors produce an aberrant form of IGF- II (“big” IGF-II) that does not associate normally with IGFBPs. The production of big IGF-II, in combination with a reduction in an IGFBP concentration, results in higher concentrations of free IGF-II, which causes hypoglycemia by associating with the insulin receptor. Both IGF-I and IGF-II are of great interest to cancer researchers, but a detailed discussion of this topic is beyond the scope of this chapter.

Clinical Significance

Clinically important states of GH excess or deficiency are uncommon and are often difficult to diagnose.⁹⁰ GH concentrations vary widely under normal circumstances; therefore random measurements of GH, in general, are not diagnostically useful. A single GH measurement cannot distinguish between normal fluctuations and the low or high concentrations that are typical of various diseases. GH measurements are best determined as part of dynamic testing: physiologic or pharmacologic provocative stimuli are used to help diagnose GH deficiency, whereas GH suppression (or lack thereof) following glucose administration is useful in evaluating GH excess.⁷¹ IGF-I concentrations often correlate better with the clinical severity of acromegaly than with glucose-suppressed or basal GH concentrations.⁴⁹

In contrast to GH, a single measurement of IGF-I is considered to be an accurate reflection of GH-IGF-I production, irrespective of the time of the day or meals. IGF-I has a much longer half-life than GH, so its concentration is more stable. The half-life of GH is slightly longer than 15 minutes, whereas the half-life of the trimeric IGF-I-IGFBP-3-ALS complex is 17 to 22 hours. The half-life of unbound IGF-I is only 10 to 20 minutes, but the unbound form of the hormone accounts for less than 1% of the total concentration. Serum concentrations of IGF-I are influenced by (1) age, (2) gender, (3) degree of sexual maturity, (4) thyroid status, and (5) nutritional status. As mentioned previously, IGF-I concentrations are low in GH deficiency and in patients with acute or chronic protein or caloric deprivation. In pediatric endocrinology, measurements of IGFBP-3 have been used in addition to IGF-I measurements to assess GH; however, the value of this approach has not been established. The diagnostic use of GH to stimulate IGF-I production is controversial and is not currently included in standard medical practice.¹⁶¹

Growth Hormone Excess

Acromegaly is the rare clinical syndrome in adults that results from GH excess.57,214 Even less common is gigantism, which results from GH excess in childhood. The clinical features of acromegaly involve overgrowth of the skeleton and soft tissue, producing (1) acral enlargement (enlargement of the extremities), (2) organomegaly (enlarged heart and/or liver), (3) facial coarsening, (4) intestinal polyposis, (5) premature cardiovascular disease, (6) hyperhidrosis (increased sweating), (7) skin tags, (8) joint disease, (9) myopathy with weakness, (10) insulin resistance, and often (11) diabetes mellitus. Premature cardiovascular disease is the most common cause of death in individuals with acromegaly. Gigantism is characterized by extreme tall stature, in addition to the clinical features of acromegaly, as pathologic GH excess occurs before epiphyseal fusion is complete.

Most cases of acromegaly (≈95%) result from anterior pituitary GH-secreting tumors (somatotropinomas).⁵⁸ Somatotropinomas are usually large macroadenomas (>10 mm in diameter); approximately 75% of these tumors are visualized by computed tomography (CT) or magnetic resonance imaging (MRI) (the preferred method for pituitary imaging is MRI). Some anterior pituitary tumors secrete both GH and prolactin (somatomammotropinomas). Approximately 5% of acromegaly cases result from GHRH-secreting hypothalamic tumors, and less than 1% of cases result from extrapituitary somatotropinomas, GHRH-secreting islet cells, or lung or breast tumors.

In severe or advanced cases of GH excess, the diagnosis may be nearly certain on the basis of physical appearance alone. However, in less severe or early cases, the physical changes may be subtle and gradual, so a high degree of clinical suspicion is needed to make an early diagnosis. The reversibility of tissue changes depends largely on the duration of the disease. In addition to soft tissue changes, acromegaly may cause severe disability or death from cardiac, pulmonary, and/or neurologic sequelae. The most important requirement for the diagnosis of acromegaly is the demonstration of inappropriate and excessive GH secretion.¹⁵⁰

As many as 10% of patients with active acromegaly have random serum GH concentrations that fall within the normal reference interval. Essentially all patients with acromegaly have an abnormal GH response to oral glucose (53-1). Patients with acromegaly typically show no change in their basal concentration of GH or demonstrate a paradoxical increase in GH⁷⁰; normal individuals, on the other hand, show suppression of GH concentrations to less than 1.0 µg/L after the oral glucose load. Serum IGF-I concentrations are also elevated in active acromegaly, and often correlate better with the clinical severity of acromegaly than do glucose-suppressed or basal GH concentrations.

Growth Hormone Deficiency and Growth Retardation

In children, short stature with a normal growth velocity (≥4 to 5 cm/y) results from (1) familial short stature, (2) primordial growth failure (prenatal-onset growth failure), or (3) constitutional delay in growth and adolescence (delayed maturation). Short stature with a low growth velocity (<4 to 5 cm/y) results from genetic short stature, chronic illness, malnutrition, deprivation (nutritional or psychologic), Turner syndrome in girls, or endocrine disorders (e.g., hypothyroidism, disorders of the GHRH-GH-IGF-I axis, poorly controlled diabetes, rickets, pseudohypoparathyroidism, pseudopseudohypoparathyroidism, and Cushing syndrome).¹⁶⁷

GH deficiency is not a common cause of growth retardation. About one half of children evaluated for growth retardation have no specific organic cause; about 15% have an endocrine disorder, of which approximately half (about 8% of all children with short stature) have GH deficiency. However, short children with low growth velocities with no clear explanation should be screened for GH deficiency once hypothyroidism has been excluded.

GH deficiency in children is characterized by (1) short stature, (2) low growth (3) velocity, (4) immature facial appearance, (5) retarded bone age on radiologic examination, and (6) increased adiposity. In cases of congenital GH deficiency, size at birth is usually normal because in utero, IGF-I does not appear to be under GH control. Micropenis (an abnormally short penis) is evident in some boys with congenital GH deficiency and will resolve with GH replacement in childhood. Adults with GH deficiency experience (1) reduced muscle mass, (2) increased adiposity, (3) osteoporosis with decreased bone density, (4) an increase in fracture risk, (5) decreased quality of life, (6) dyslipidemia, and (7) an increased risk for cardiovascular disease.¹¹³ GH deficiency is probably the most common identifiable abnormality in adults with large pituitary adenomas³³ and in patients who have undergone pituitary irradiation. Thus GH replacement therapy is an important clinical intervention in GH-deficient adults and children and is considered the standard of care.

GH insufficiency can be a consequence of (1) hypothalamic disease, (2) disruption of the portal system between hypothalamic nuclei and the anterior pituitary, (3) GHRHR loss-of-function mutations, or (4) somatotroph disease. GH deficiency can occur in isolation [isolated GH deficiency] or together with other pituitary deficiencies [multiple pituitary hormone deficiencies (MPHD) or combined pituitary hormone deficiencies (CPHD)]. Patients with isolated GH deficiency should be followed clinically for the development of other pituitary hormone deficiencies because MPHD can evolve over time. Biochemical testing is necessary to establish the diagnosis of GH deficiency, growth hormone resistance, or MPHD. In most affected children, the cause of GH deficiency is unknown [idiopathic GH deficiency].¹⁵⁹ Approximately one in four children with proven GH deficiency has an organic origin for their GH deficiency; half of these children will be diagnosed with a CNS tumor.

Any type of hypothalamic disease or dysfunction can lead to GHRH deficiency, including (1) tumor, (2) inflammation, (3) previous infection, (4) trauma (including previous surgery),¹²⁴ (5) bleeding, (6) irradiation, and (7) malformation [septo-optic dysplasia (SOD)].¹¹⁶ Low-dose irradiation of the hypothalamus/pituitary can cause idiopathic GH deficiency, whereas higher doses of irradiation can cause MPHD. SOD is defined by the triad of (1) midline brain defects such as agenesis of the septum pellucidum and/or corpus callosum, (2) hypoplasia of the optic nerve, and (3) anterior and/or posterior pituitary hormone abnormalities. A small number of cases of SOD are explained by mutations in HESX1, SOX2, and SOX3, all of which are transcription factors. HEXS1 (chromosome 3p14.3) is a paired-like homeobox gene, SOX2 (chromosome 3q26.3) is the SRY (sex-determining region on the Y chromosome) box 2 gene, and SOX3 (chromosome Xq27.1) is the SRY box 3 gene. Midline brain tumors, such as meningiomas, gliomas, germinomas, third ventricle colloid cysts, ependymomas, and optic nerve gliomas, also affect the hypothalamus. Disruptions in the hypothalamic-pituitary portal system can result from tumor, inflammation, previous infection, trauma (including previous surgery), and irradiation.

Congenital GH deficiency from pituitary disease has many causes, including GHRHR gene mutations (idiopathic GH deficiency type IB), GH1 mutations (idiopathic GH deficiency types IA, IB, II, and III, and bioinactive GH), and transcription factor mutations (usually causing MPHD: LHX3, LHX4, PROP1, Pit-1, RIEG) and malformations (anencephaly and holoprosencephaly) (Table 53-5).¹⁶⁸ Homozygous LHX3 mutations have caused panhypopituitarism, with the exception that ACTH was not affected. Heterozygous LHX4 mutations have caused deficiencies of GH, TSH, and ACTH. PROP1 (name derived from “PROphet of Pit-1”; POU1F1; Pit-1 stands for “paired-like homeodomain transcription factor”)¹⁵⁷ mutations causes deficiencies of GH, prolactin, TSH, and gonadotropins. ACTH deficiency has also occurred in some families. PROP1 gene mutations are inherited as autosomal recessive traits. Pit-1 gene mutations cause GH, prolactin, and variable degrees of TSH deficiency. These transcription factor mutations may be inherited as autosomal recessive or dominant traits. Heterozygous RIEG gene mutations (PITX2, a paired-like homeobox gene) are the cause of Rieger syndrome, which may include GH deficiency. Features of Rieger syndrome encompass developmental abnormalities of teeth, the anterior chamber of the eye, and the umbilicus. Mutations in GLI1, GLI2, Shh, ZIC2, SIX3, tgif, PATCHED1, DGF1, and FAST1 have variously been cited as causes of holoprosencephaly.⁸⁸ Children with midline facial clefts (cleft lip, cleft palate, or combined) or a single central incisor can exhibit GH deficiency. Two GH1 gene mutations have been identified to cause bioinactive GH.¹⁸⁹ In these mutations, GH does not have full biological activity but retains normal immunoreactivity. Therefore, in contrast to other forms of pituitary or hypothalamic disease, the GH concentration is not deficient. At least one reference laboratory provides GHRHR, GH1, and GHR gene sequencing services (Athena Diagnostics, Worcester, Mass).^7,62,207

Acquired causes of pituitary disease include (1) tumor (craniopharyngioma, Rathke cleft cyst, arachnoid cyst, or anterior pituitary adenoma), (2) infiltrative disease—amyloidosis, (3) inflammation (autoimmune hypophysitis and granulomatous inflammation), (4) infection, (5) trauma (including surgery), (6) bleeding, (7) irradiation, (8) infarction (pituitary apoplexy from Sheehan syndrome), and (9) metabolic derangement (hemochromatosis, iron overload from long-term transfusion therapy, or certain anemias, such as sideroblastic anemia).⁵ Hemochromatosis does not cause hypopituitarism until many decades have passed.³⁵ Some investigators believe that all patients with “idiopathic” hypopituitarism should be screened for hemochromatosis.¹⁴¹

A staged approach to the evaluation of GH secretion is advised.¹⁶⁶ Initial screening can involve one of the following tests: (1) measurement of IGF-I (with or without IGFBP-3), (2) GH measurement following exercise, or (3) pharmacologic GH screening. All forms of GH testing should be performed after the subject has fasted overnight. If a GH screening test is abnormal, definitive testing should be pursued.²⁰¹ If GH adequacy is demonstrated by GH screening tests, definitive GH testing need not be performed unless there is a very high index of suspicion for GH deficiency. In children, the likelihood of a falsely abnormal GH screening test can be reduced by pretreatment of both boys and girls with a short course of sex steroids [ethinylestradiol: for body weights <50 lb, 20 µg/dose 18 hours, 12 hours, and 1 hour before testing; for body weights ≥50 lb: 50 µg/dose 18, 12, and 1 hour before testing, or 100 µg/d for 3 days; Premarin: for body weights <30 lb: 2.5 mg twice daily for 3 days; for body weights ≥30 lb, 5 mg twice daily for 3 days; diethylstilbestrol (DES): 5 mg/d for 3 days; or, exclusively in boys, testosterone enanthate 50 mg injected intramuscularly 2 or 3 weeks before testing]. The mechanism is unclear; however, sex steroids appear to play a major role in increasing the response of IGF-I to GH at the time of puberty. From clinical experience, GH deficiency is overdiagnosed in some peripubertal children who are tested without the benefit of sex hormone priming.

Exercise physiologically enhances GH release.⁷² Typically, in the fasting state, the subject exercises vigorously for ≈20 minutes (e.g., running up and down stairs, running on a treadmill). At the completion of the exercise, when the subject is tachycardic and sweating, a sample is collected for GH measurement (Box 53-2). A baseline GH measurement is not required. A GH concentration may also be obtained 40 minutes after exercise, in case of delayed GH release. Finally, screening for GH deficiency can be performed by measuring GH 60 to 90 minutes following clonidine, glucagon, or L-dopa administration. (Note: L-dopa is not currently available in the United States.) Doses of clonidine, glucagon, and L-dopa are identical to doses used in formal GH testing scenarios (see later).

It has been argued that measuring GH during sleep is a physiologic assessment of the hypothalamic-somatotroph-GH axis. Some authorities suggest electroencephalographic (EEG) monitoring with GH measured during deep sleep (e.g., stage III, stage IV). More simply, GH could be measured 1 hour after the onset of sleep. However, in reality these types of sleep studies are cumbersome because they require hospitalization or overnight boarding in a sleep laboratory. In practice, this is rarely done. Furthermore, the high cost of hospitalization or the sleep laboratory suggests that an exercise tolerance test or a simple pharmacologic stimulus would be a more cost-effective approach to initial GH testing.

Reference intervals for IGF-I and IGFBP-3 are age and gender dependent.⁷³ If IGF-I is within its reference interval for age and gender in children, GH deficiency is excluded. If IGF-I is low, definitive GH testing is required. Because IGF-I concentrations can be depressed in states of (1) malnutrition, (2) malabsorption, (3) chronic disease, (4) hypothyroidism, and (5) sex hormone deficiency, a low IGF-I concentration does not confirm GH deficiency. IGFBP-3 is less dependent on good nutrition to achieve normal concentrations, so it may be a superior marker of GH deficiency compared with IGF-I. However, one study failed to demonstrate that measuring IGFBP-3 alone or together with free IGF-I was superior to measuring IGF-I alone as a screening test for GH deficiency.⁴⁸ In another study, only ≈50% of GH-deficient children had a low IGFBP-3 concentration; this finding calls into question its value as a sensitive screening test.¹⁹⁸

Analytical advantages of IGFBP-3 over IGF-I include the following: (1) IGF-I must be separated from its binding proteins to be measured, but IGFBP-3 does not require a dissociation step; (2) IGFBP-3 is present in higher concentration than IGF-I; and (3) less age dependency is seen for IGFBP-3 compared with IGF-I.⁴⁷ Because of their stability over the course of a day, IGF-I and IGFBP-3 measurements may be obtained randomly. However, some researchers have concluded that IGFBP-3 measurements are too nonspecific to be used for the evaluation of GH deficiency.⁴⁵ Furthermore, reports indicate that IGF-I and IGFBP-3 exhibit imperfect sensitivity and specificity for the diagnosis of GH deficiency.¹⁶⁹

IGF-I measurements in adults often are not diagnostically helpful.¹³⁰ For reasons that are unclear, IGF-I concentrations can be normal in GH-deficient adults. Therefore, a normal IGF-I does not rule out adult GH deficiency. If the IGF-I concentration is very low and suspicion for GH deficiency is high (MPHD or childhood-onset severe GH deficiency), some experts would diagnose GH deficiency in the absence of GH testing.⁸⁷

GH responses to insulin-induced hypoglycemia [insulin tolerance test (ITT); Box 53-3] and GH responses to centrally acting pharmacologic or biological agents (Box 53-4) are considered definitive tests. The stimuli can be sequential or administered on different days. The classical diagnosis of pediatric GH deficiency requires that GH responses to two different stimuli (Table 53-6) be deficient. In research settings, GHRH and GHRP-6 have been used to stimulate GH release. However, because these agents are not available for clinical use, they are not included in Table 53-6. Note that many variations of these protocols are available because endocrinologists often customize these tests. Diazepam and pentagastrin have been studied as GH secretogogues; however, experience with these agents is limited, and they are not included in Table 53-6.

Of children with appropriate stature for age, approximately 80% will have normal GH responses to one stimulus, and at least 95% will have normal GH responses to at least one of two stimuli. This is why two stimuli are generally recommended—to avoid overdiagnosis of GH deficiency. However, the GH Research Society advises that a single definitive abnormal test is adequate to diagnose GH deficiency if the child has (1) confirmed CNS pathology, (2) a history of CNS irradiation, (3) multiple pituitary hormone deficiencies, or (4) a genetic defect.¹

A history of childhood GH deficiency, CNS disease, trauma, or irradiation is an indication to test adults for GH deficiency.39,190 Retesting of adults with the diagnosis of childhood GH deficiency is necessary because not all adults with childhood GH deficiency remain deficient as adults. In adults, a single abnormal GH response to a stimulus is diagnostic of GH deficiency if the deficiency is congenital or genetic, or if multiple pituitary hormone deficiencies are due to organic disease. Guidelines published in 2009 recommended that a low IGF-I concentration in a patient with three or more pituitary hormone deficiencies is sufficient to diagnose GH deficiency.⁸ Unfortunately GH testing is not very reproducible.¹⁰³

Insulin-induced hypoglycemia (ITT) is often considered to be the “gold standard” stimulus when hypoglycemia is indeed achieved (glucose <40 to 45 mg/dL).¹¹² The risk associated with this type of test is that untreated severe hypoglycemia can be life threatening. Venous access for infusion of glucose is very important during the ITT. Should vascular access be lost during the ITT, glucagon should be readily available for IM injection (the dose of glucagon is 1 mg). If intravenous access cannot be ensured, stimuli other than insulin should be considered. In general, GH stimulation tests are not conducted by laboratory personnel because of the risks and complexities of testing. Arginine infusion presents the danger of acidosis and even death.¹⁸³

Stimulated GH concentration less than 7 to 10 µg/L defines GH deficiency in children.²⁶ In adults, GH deficiency is present when stimulated GH is less than 5 µg/L. GH deficiency in adults can be parsed according to the stimulus. For ITTs, a deficient peak GH response is less than 3 µg/L. For GHRH plus arginine, a deficient peak GH response is less than 11 µg/L when the body mass index (BMI) is less than 25 kg/m². However if the BMI is 25 to 30 kg/m², deficient is defined as less than 8 µg/L, and for BMI greater than 30 kg/m², deficient is identified at less than 4 µg/L. Because GHRH is not commercially available, and clonidine and arginine alone are not helpful in defining GH deficiency in adults, the ITT remains the best test of GH secretion in adults.

Controversy continues as to what constitutes a “normal” GH response to stimuli, because insulin-induced hypoglycemia is considered by some to be a nonphysiologic stimulus. Discordance between normal stimulated GH concentration and a deficient spontaneous rise in GH concentration has been described as neurosecretory GH deficiency.⁵³ Neurosecretory GH deficiency can result from CNS or hypothalamic disease. The diagnosis of neurosecretory GH deficiency requires overnight, every 20 minute blood sampling with GH measurements—a protocol that ordinarily requires hospitalization. The combined costs of GH assays, physician fees, and inpatient services would exceed several thousand dollars.

The definition of partial GH deficiency is especially problematic because the definition of simple GH deficiency is itself controversial.91,110 Eliminating GH stimulation testing has been proposed, with the diagnosis of GH deficiency based on growth parameter, IGF-I and IGFBP-3 measurements, neuroradiologic investigation, and genetic considerations.^14,137,211

Growth Hormone Resistance

In children with short stature and low growth velocity, if IGF-I is (1) below the reference interval for the child’s bone age and gender, (2) if the GH concentration is normal or elevated, and (3) if non–GH-dependent causes of IGF-I deficiency (malnutrition, malabsorption, chronic disease, hypothyroidism, and sex hormone deficiency compared with the patient’s bone age) have been excluded, GH resistance should be considered.¹⁷¹ As uncommon as GH deficiency is in the general pediatric population (1 in 10,000 children), GH resistance as a primary problem is far less frequent.

GH resistance can be congenital, resulting from loss-of-function GHR mutations or GHR signaling defects (STAT5b mutations),123,215 or from defects in the production of IGF-I itself. Most GHR mutations involve the extracellular domain that involves GH binding to the GHR. Some GHR mutations affect homodimerization. Loss of the intracellular GHR domain can result from splice-site mutations. GHR is not expressed on the cell surface if the transmembrane domain is defective. Recall that circulating GHBP is derived from the extracellular domain of the GHR. Most cases of GHR deficiency display low or absent concentrations of GHBP. STAT5b is necessary for normal GH-GHR signaling to the cell nucleus. These are autosomal recessive disorders. Size at birth is normal because in utero IGF-I production is independent of GH.

IGF-I gene inactivating mutations or deletions are rare, and only two such mutations have been described. In contrast to GH receptor and signaling defects, when IGF-I itself cannot be produced because of intrinsic IGF-I gene mutations, intrauterine growth retardation will result, in addition to extrauterine growth failure. Other consequences of IGF-I gene mutations include severe mental retardation, deafness, and micrognathia (mandibular hypoplasia).206,216

IGF-I and IGFBP-3 concentrations are very low in rare cases of ALS deficiency, while baseline and poststimulation GH concentrations are normal.⁶⁷ Although adolescence was delayed, adult stature was nearly normal in persons with ALS deficiency.

Acquired GH resistance is far more common than congenital GH resistance. In cases of acquired GH resistance, the IGF-I is low (despite sufficient GH secretion) because of malnutrition, malabsorption, chronic disease, hypothyroidism, or sex hormone deficiency. An acquired form of GH resistance is also observed in patients with idiopathic GH deficiency type I, who develop GH-inhibitory antibodies when treated with exogenous GH. Because GH is absent in this form of congenital GH deficiency, exogenous GH is seen by the immune system as foreign. Apparently, the resulting antibodies directed against exogenous GH bind to, and inactivate, the GH. This is reminiscent of the development of factor VIII antibodies in some boys with severe hemophilia A, who are treated with exogenous factor VIII.

A number of criteria for GH resistance have been proposed, including (1) height more than 3 standard deviations below the mean for age; (2) basal GH greater than 2.5 ng/mL; (3) basal IGF-I less than 50 ng/mL; (4) basal IGFBP-3 more than 2 standard deviations below the mean for age; (5) an increase in IGF-I of less than 15 ng/mL after 4 days of GH treatment (0.05 mg/kg/d); and (6) increase in IGFBP-3 of less than 0.4 µg/mL after GH treatment. The largest concentrations of subjects with GH resistance are found in Israel and southern Ecuador.¹⁷⁰