GH-RELEASING HORMONE.

GHRH stimulates both GH synthesis and secretion. GHRH causes a prompt increase in blood GH levels, followed by a rapid return of GH to basal concentrations (Thorner et al, 1983). The pulsatile increases in GH primarily reflect the pulsatile secretion of GHRH from the hypothalamus (Thorner et al, 1998). Sustained infusions of GHRH over several hours result in an initial rise and then a progressive decrease in plasma GH concentrations, suggesting that GHRH depends on pulsatile secretion for its physiologic effect (Reichlin, 1998). The effects of GHRH are almost completely specific for GH, although a minimal increase in plasma prolactin may also occur.

SOMATOSTATIN.

Somatostatin denotes a family of peptides, including somatostatin-14, which is a cyclic peptide containing 14 amino acids, and the N-terminal-extended somatostatin-28, which is a cyclic peptide containing 28 amino acids. Somatostatin-14 is identical with the terminal 14 amino acids of somatostatin-28. Somatostatin-14 is found predominately in the central nervous system (CNS), including the hypothalamus, and somatostatin-28 is found predominately in the gastrointestinal tract, including the pancreas (Reichlin, 1998). As a pituitary regulator, somatostatin is a potent inhibitor of GH secretion. Somatostatin secretion is increased by elevated levels of GH and IGF-I. Somatostatin appears to determine the timing and amplitude of GH pulses but has no effect on GH synthesis (Thorner et al, 1998). Somatostatin inhibits secretion of thyroid-stimulating hormone (TSH) and, under certain conditions, secretion of prolactin and adrenocorticotropic hormone (ACTH). It also has nonpituitary roles, including activity as a neuromodulator in the central and peripheral nervous systems and as a gut and pancreatic regulatory peptide (Table 2-1; Reichlin, 1998). Somatostatin blocks hormone release in many endocrine-secreting tumors, including insulinomas, glucagonomas, vasoactive intestinal polypeptide-secreting tumors (VIPomas), carcinoid tumors, and some gastrinomas (see Chapters 14 and 15). In humans, long-acting analogs of somatostatin have been used therapeutically in the management of GH excess and functioning neuroendocrine tumors (Reichlin, 1998).

TABLE 2-1 BIOLOGIC ACTIONS OF SOMATOSTATIN OUTSIDE THE CENTRAL NERVOUS SYSTEM

STIMULI AFFECTING GH-REGULATING FACTORS.

The influence of GHRH and somatostatin on GH secretion is tightly regulated by an integrated system of neural, metabolic, and hormonal factors, including GH itself (Tables 2-2 and 2-3) (Buonomo and Baile, 1990). Most of these stimuli exert their effects on GH secretion by influencing hypothalamic secretion of GHRH or somatostatin. The predominant influence of the hypothalamus on GH secretion is stimulatory, as evidenced by the fact that damage to the hypothalamic-pituitary connection is followed by inhibition of both basal and induced GH release (Reichlin, 1998). One exception is ghrelin, a recently discovered hormone that is produced primarily in the stomach and to a lesser extent in the hypothalamus. Ghrelin is secreted after consumption of a meal; its primary actions are to decrease gastric emptying and promote satiety. Ghrelin also stimulates GH secretion, which in turn increases IGF-I concentrations. IGF-I promotes glucose utilization by binding to insulin receptors in tissues. Growth hormone also decreases IGFBP-1 concentrations, resulting in increased free IGF-I concentrations. As mentioned previously, the primary role of IGFBP-1 is the binding of IGF-I, thereby decreasing the circulating concentration of IGF-I.

TABLE 2-2 FACTORS THAT STIMULATE GH SECRETION IN PRIMATES

TABLE 2-3 FACTORS THAT INHIBIT GH SECRETION IN PRIMATES

Differences in the effect of stimuli on GH secretion appear to exist between dogs and humans. For example, moderate exercise stimulates GH secretion in humans but apparently not in dogs. Arginine, a potent stimulant of GH secretion in humans, has given inconsistent results when administered to dogs. The GH response to insulin-induced hypoglycemia has also been inconsistent in dogs, with some investigators reporting a clear-cut elevation and others reporting only a sluggish response. Likewise, hyperglycemia resulting from a glucose infusion inhibits GH secretion in humans but has a minimal effect in dogs. Because of this species variability, it seems inappropriate to assume that factors that affect GH secretion in humans automatically have a similar effect in dogs.

The reader is referred to the first edition of Canine and Feline Endocrinology and Reproduction for more information on the metabolic actions of GH and the factors that regulate GH secretion.

PITUITARY CYST.

Pituitary dwarfism may result from congenital cystic distention or persistence of the “intrasellar” portion of the craniopharyngeal duct (Rathke’s pouch). The intrasellar portion is found within the sella turcica, the region of the skull “encasing” the pituitary. During embryonic development, the anterior pituitary (adenohypophysis) arises from Rathke’s pouch, an evagination of the posterior wall of the pharynx. The portion of the pouch that comes in contact with the neurohypophysis differentiates into the pars intermedia of the anterior pituitary, and the anterior wall of the pouch differentiates into the pars distalis of the anterior pituitary. Lateral proliferations from the embryonic pouch extend around the infundibular stem to form the pars tuberalis (pituitary stalk). In the dog and cat, the distal and intermediate parts of the anterior pituitary are separated by the residual hypophyseal cleft, which is the residual lumen of Rathke’s pouch. The stalk of Rathke’s pouch is attenuated in the embryo to form the craniopharyngeal duct. This duct should disappear, although either or both ends of it commonly persist as vestiges in dogs. Persistence of the intrasellar portions of the duct is especially common in brachycephalic breeds. The degree of persistence and consequences varies. The remnants may become cystic; may be single or multiple, unilocular or multilocular; and frequently contain mucin produced by the epithelium that lines the cyst. The cysts are usually microscopic and clinically insignificant but sometimes are larger in volume than a normal pituitary gland (Fig. 2-3). Large cysts may cause pressure atrophy of pituitary cells.

FIGURE 2-3 CT scan of the pituitary region of a small 2-year-old female spayed Great Dane presenting for diagnostic evaluation of pituitary function. A large radiolucent cyst is evident in the pituitary region (arrow).

Pituitary cysts are relatively common in the dog, yet dwarfism is rare. Also, dwarfism is most often encountered in the German Shepherd dog, and German Shepherd dwarfs have been identified with only a very small pituitary cyst. For these reasons, the theory that cystic distention of the craniopharyngeal duct is the primary cause of destruction of the adenohypophysis has been questioned. An alternative hypothesis suggests a primary failure in the differentiation of the adenohypophyseal cells into normal tropic hormone-secreting cells (Kooistra et al, 2000b).

PITUITARY HYPOPLASIA.

The development of the anterior lobe of the pituitary gland is characterized by the expression of a series of homeodomain transcription factors, which are required for normal growth of the pituitary primordium (Treier et al, 1998; Watkins-Chow and Camper, 1998). After proliferation, different cell phenotypes arise in a distinct temporal fashion and undergo a highly selective determination and differentiation (Simmons et al, 1990; Voss and Rosenfeld, 1992). The corticotropic cell is the first distinct cell type that differentiates from the pituitary stem cell (Sheng et al, 1997). Ultimately, the mature pituitary anterior lobe is composed of five distinct endocrine cell types that are easily distinguished by the hormones they secrete. Any defect in the organogenesis of the pituitary gland may result in an isolated or combined pituitary hormone deficiency.

Pituitary dwarfism is encountered most often as a simple, autosomal recessive inherited abnormality in the German Shepherd dog (Kooistra et al, 2000b). Inherited pituitary dwarfism may be due to isolated GH deficiency or may be part of a combined anterior pituitary hormone deficiency syndrome. Concurrent deficiency in TSH and prolactin are most commonly identified in German Shepherd dwarfs; ACTH secretion is preserved (Fig. 2-4; Hamann et al, 1999; Kooistra et al, 2000b). Kooistra and colleagues (2000b) hypothesize that the disorder in German Shepherd dogs is caused by a mutation in a developmental transcription factor that precludes effective expansion of a pituitary stem cell after the differentiation of the corticotropic cells that produce ACTH. Mutations in the genes encoding for transcription factors Pit-1 or the Prophet of Pit-1 (Prop-1) cause combined pituitary anterior lobe hormone deficiencies in humans and mice (Pellegrini-Bouiller et al, 1996; Fofanova et al, 1998; Wu et al, 1998). Unfortunately, a mutation in the gene encoding transcription factors Pit-1 and Prop-1 was not identified in German Shepherd dwarfs, which indicates that another homeodomain gene is affected in dwarf German Shepherd dogs (Lantinga-van Leeuwen et al, 2000a and 2000b). Kooistra and colleagues (2000b) speculate that the underlying defect may involve a mutation that precludes effective expansion of a pituitary stem cell. Because ACTH secretion remains intact, a combined pituitary hormone deficiency in German Shepherd dwarfs would most likely be caused by a mutation in a developmental transcription factor that precludes effective expansion of a pituitary stem cell after the differentiation of the corticotropic cells. Such a factor would be analogous to the pituitary transcription factors Pit-1 and Prop-1 but would act at an earlier stage of pituitary gland development (Kooistra et al, 2000b).

FIGURE 2-4 Results of a combined pituitary anterior lobe function test (mean and SEM) in eight German Shepherd dogs with dwarfism (O) and in eight adult, healthy Beagle dogs (•). The bars indicating the SEM in the German Shepherd dwarfs are depicted only when they exceed the size of the symbols.

(Kooistra HS, et al: Dom Anim Endocr 19:177, 2000.)

The cystic changes involving the pituitary gland in dogs with congenital GH deficiency may be the result of primary failure of differentiation of the anterior lobe of the pituitary gland rather than the cause of the GH deficiency. Undifferentiated adenohypophyseal cells may secrete osmotically active proteinaceous material into the craniopharyngeal duct (Eigenmann, 1983). Analysis of cystic fluid obtained from rats has shown that the osmotically active proteinaceous material could attract water and lead to cystic changes, which is compatible with the observation that the cysts enlarge with time (Benjamin, 1981; Kooistra et al, 1998).

GH INSENSITIVITY SYNDROMES.

Pituitary dwarfism may develop from insensitivity to GH. The prototype of GH insensitivity is the syndrome of Laron-type dwarfism in human beings. Circulating levels of GH are increased in people with Laron-type dwarfism, but IGF-I levels are deficient. This disorder is due to inactivating mutations in the gene for the GH receptor, which results in absent or defective GH receptors and partial to complete GH insensitivity (Rosenfeld et al, 1994; Goddard et al, 1995). GH insensitivity can also result from an abnormality in the structure of GH (Takahashi et al, 1996), abnormalities of GH signal transduction (postreceptor defects), primary defects of synthesis of IGF-I, or lack of target tissue response to IGF-I (Lanes et al, 1980; Takahashi et al, 1996; Reiter and Rosenfeld, 1998).

Similar subcategories of dwarfism in dogs or cats have not yet been convincingly described. To date, all cases of pituitary dwarfism in the dog in which either GH or IGF-I concentrations were evaluated have revealed low to undetectable GH concentrations and low IGF-I concentrations. There has been no response of GH to stimulation tests (Rijnberk et al, 1993). Two dwarf German Shepherd dogs have been described with low IGF-I activity and histologically normal pituitary glands. Secondary GH deficiency (i.e., abnormal GH structure or nonresponsive target tissue) was proposed as the cause for the dwarfism, making the assumption that circulating GH levels were normal. Unfortunately, plasma GH concentrations were not evaluated to substantiate the claims. In another study in a German Shepherd dog, similar arguments were made for secondary GH deficiency as the cause of the dwarfism (Muller-Peddinghaus et al, 1980). In this study, basal GH concentrations rather than the GH secretory capacity of the pituitary were assessed. Unfortunately, basal GH concentrations in normal and hypopituitary dogs may be similar, rendering this parameter inadequate for assessing GH secretory capacity (Eigenmann and Eigenmann, 1981a).

Interestingly, two German Shepherd dogs have been described that had delayed growth, resulting in an initial stunted appearance, that eventually resolved by 1 year of age (Randolph et al, 1990). Both dogs had normal basal serum GH and IGF-I concentrations, normal GH secretory response to xylazine administration, and normal adrenal gland and thyroid gland function. These authors speculated that delayed growth was due to mild hypopituitarism or intermittent GH neurosecretory dysfunction. It seems logical that a spectrum of severity, from mild to severe, might exist with hypopituitarism and that the exhibited clinical signs would depend on the severity of impaired GH secretion.

BASAL GH CONCENTRATIONS.

Reported normal basal GH concentrations range from 1.5 ± 1.2 ng/ml to 4.3 ± 1.1 ng/ml in dogs (Eigenmann, 1983) and 1.2 ± 1.0 ng/ml to 3.2 ± 0.7 ng/ml in cats (Eigenmann et al, 1984d; Peterson et al, 1990). Unfortunately, basal GH concentrations in dogs and cats with hyposomatotropism (both congenital and acquired) may also be in this range, which makes it difficult to document hyposecretion when relying solely on basal GH levels. Two dogs with congenital GH deficiencies did have consistently undetectable serum concentrations (Rijnberk et al, 1993). Currently, assessment of random basal GH concentrations is inadequate for documentation of hyposomatotropism; assessment of GH secretory capacity after stimulation of the pituitary somatotrophs is recommended to establish the diagnosis.

GH STIMULATION TESTS.

Several stimulation tests have been developed to evaluate the GH secretory capabilities of the pituitary. The most commonly used stimulation tests in the dog are the clonidine, xylazine, and GHRH stimulation tests and a combined pituitary anterior lobe function test using four releasing hormones: GHRH, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and corticotropin-releasing hormone (CRH) (Table 2-6). Use of these tests to assess GH secretion in cats has not yet been reported.

TABLE 2-6 GROWTH HORMONE STIMULATION TESTING PROTOCOLS

* Currently preferred GH stimulation test.

† An abbreviated protocol in which plasma samples are obtained before and 20 and 30 minutes after stimulation can be done.

Interpretation of GH stimulation tests requires some knowledge of the conditions that may affect somatotroph responsiveness to various stimuli; this becomes especially important in the attempt to differentiate partial GH deficiency states from normal. A number of conditions may affect somatotroph responsiveness to stimuli in humans (Table 2-7) (Lazarus, 1984). Similar conditions have not been thoroughly evaluated in the dog, although hyperadrenocorticism, hypothyroidism, and some sex hormone imbalances may cause a reversible suppression of plasma GH concentrations (Peterson and Altszuler, 1981; Lothrop, 1988; Regnier and Garnier, 1995), and β-adrenergic antagonists potentiate the GH response to GHRH (Regnier et al, 1992).

TABLE 2-7 CONDITIONS THAT MAY AFFECT PITUITARY SOMATOTROPH RESPONSIVENESS TO GH STIMULATION TESTS IN HUMANS

Clonidine Stimulation Test.

Clonidine is an α-adrenergic agonist that stimulates secretion of GHRH, which in turn stimulates secretion of GH. Injection of clonidine increases circulating GH and glucose concentrations and decreases plasma insulin concentrations in the dog (Hampshire and Altszuler, 1981). These effects are dose dependent. The use of clonidine to provoke GH secretion appears to be more reproducible and results in more dramatic changes in plasma GH concentrations than do the other stimulation tests. Currently, either the clonidine stimulation test or the GHRH stimulation test is preferred for diagnosing hyposomatotropism.

The dose of clonidine used in the test is somewhat variable and depends on the protocol established for the individual laboratory. An increase in GH may be seen at dosages as low as 3 μg/kg body weight given intravenously (Hampshire and Altszuler, 1981), although dosages of 10 μg/kg (Eigenmann, 1983), 16.5 μg/kg (Roth et al, 1980), and 30 μg/kg have been reported. The usual dosage is 10 μg/kg given intravenously. It should be kept in mind that larger dosages of clonidine (i.e., 16.5 and 30 μg/kg) produce a more pronounced and prolonged hyperglycemia but have a greater incidence of adverse reactions, including sedation, bradycardia, hypotension, collapse, and aggressive behavior (Lothrop, 1988; Eigenmann and Eigenmann, 1981a). Adverse reactions may last 15 to 60 minutes. If necessary, atropine can be used to reverse bradycardia, and the α-adrenergic antagonists phentolamine or yohimbine can be used to antagonize the hypotensive effects of clonidine (and xylazine). Plasma samples for GH determination should be obtained prior to clonidine administration and 15, 30, 45, 60, and 120 minutes after stimulation. GH is relatively stable for prolonged periods if frozen and maintained at −20° C.

In the normal dog, plasma GH concentrations should increase after clonidine administration, reaching peak concentrations 15 to 30 minutes after initiation of the test (Fig. 2-7) (Hampshire and Altszuler, 1981; Eigenmann, 1983). In healthy dogs, GH concentrations should exceed 10 ng/ml after clonidine stimulation. The GH concentration should then decline rapidly toward basal levels. Pituitary dwarfs and dogs with adult-onset complete GH deficiency do not respond to clonidine (see Fig. 2-7). In these dogs, no increase in plasma GH concentrations follows clonidine administration. A partial GH deficiency may be suspected whenever subnormal results are obtained (Table 2-8) (Eigenmann and Patterson, 1984).

FIGURE 2-7 Results of a clonidine stimulation test in normal dogs and nine German Shepherd dogs with pituitary dwarfism. Clonidine, 10 μg/kg body weight, was given intravenously at time 0.

(Eigenmann JE, et al: Acta Endocrinol 105:289, 1984.)

TABLE 2-8 RESULTS OF A CLONIDINE STIMULATION TEST IN NINE ADULT DOGS WITH GH DEFICIENCY

Evaluation of plasma glucose concentrations during clonidine-induced GH secretion may be helpful in evaluating somatotroph function. In the normal dog, plasma glucose concentrations increase after clonidine administration, an effect that is dose dependent (Hampshire and Altszuler, 1981). The peak increase in the plasma glucose concentration occurs approximately 90 minutes after clonidine administration. In contrast, no increase in plasma glucose occurs in hypophysectomized dogs or in dogs with hyposomatotropism (Eigenmann, 1981). The exact mechanism of the hyperglycemia is not known, although it seems likely to be a result of GH-induced insulin antagonism and decreased glucose uptake by cells.

Growth Hormone-Releasing Hormone Administration Test.

The use of GHRH has been evaluated as a diagnostic test for canine congenital and adult-onset GH deficiency (Lothrop, 1986; Rijnberk et al, 1993; Kooistra et al, 2000b). Administration of human GHRH (1 μg/kg IV; Peninsula Laboratories, San Carlos, CA) results in a rapid increase in plasma GH, with peak concentrations occurring 10 to 30 minutes after GHRH administration (Fig. 2-8; Abribat et al, 1989; Meij et al, 1996). The mean (± SE) plasma GH concentration was 14.7 ± 3.7 ng/ml at 10 minutes after GHRH administration (Meij et al, 1996). In eight German Shepherd dwarfs, basal GH concentrations were low and there was no increase in the plasma GH concentration after GHRH administration (see Fig. 2-4; Kooistra et al, 2000b). In six dogs with suspected adult-onset GH deficiency, basal GH concentrations were either low or within normal limits and there was no increase in GH after GHRH stimulation (Rijnberk et al, 1993). Adverse reactions were not observed after administration of GHRH. For the GHRH stimulation test, plasma samples for GH measurement should be obtained prior to and 5, 10, 20, 30, 45, and 60 minutes after intravenous administration of 1 μg of GHRH/kg body weight. Availability and cost are obvious disadvantages of GHRH.

FIGURE 2-8 Plasma GH response (mean ± SE) in eight male Beagles after rapid (30 second) intravenous injection (arrow) of a combination of four hypothalamic releasing hormones (•) compared with single stimulation with 1 μg/kg GHRH (O). In the combined test, the releasing hormones were injected in the following order and doses: 1 μg of CRH/kg, 1 μg of GHRH/kg, 10 μg of GnRH/kg, and 10 μg of TRH/kg. The area under the curve (AUC) and the increment (peak basal level at 0 minutes) in the plasma GH concentration were not significantly different between combined and single stimulation tests. CRH, Corticotropin-releasing hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; TRH, thyrotropin releasing hormone.

(Meij BP, et al: Dom Anim Endocr 13:161, 1996.)

BASAL IGF-I CONCENTRATION.

IGF-I concentrations are low in dogs with pituitary dwarfism. In one study involving nine German Shepherd dwarfs, the mean plasma IGF-I concentration in normal adults was 280 ± 23 ng/ml; in immature normal animals, 345 ± 50 ng/ml; and in the dwarf dogs, 11 ± 2 ng/ml (Eigenmann et al, 1984c). Low plasma IGF-I concentrations have also been documented in three additional studies involving nine pituitary dwarfs (Rijnberk et al, 1993; Kooistra et al, 1998; Kooistra et al, 2000b). These findings support the theory of GH control over plasma IGF-I concentrations (see Fig. 2-2) and imply the important role that IGF-I plays in the regulation of growth. Radioimmunoassays for measurement of IGF-I in humans have been validated in dogs (Randolph et al, 1990) and cats (Church et al, 1994) and are commercially available. Measurement of serum IGF-I concentrations by radioimmunoassay provides a way to gain further evidence of GH deficiency as the cause of poor growth in dogs and cats when measurement of GH is not available. However, interpretation of results in dogs must take into consideration the size of the breed (i.e., smaller breeds have lower normal IGF-I concentrations and larger breeds have higher normal concentrations) (Eigenmann et al, 1984b).

THYROID FUNCTION.

Evaluation of thyroid gland function is imperative in any animal with suspected hyposomatotropism, because the clinical signs of hypothyroidism and hyposomatotropism are similar, making hypothyroidism an important differential diagnosis; also, TSH deficiency is the most common concurrent hormone deficiency in pituitary dwarfs with pituitary hypoplasia and combined hormonal deficiencies. In theory, the basal serum thyroxine, free thyroxine, and endogenous TSH concentrations should be low or undetectable in pituitary dwarfs with concurrent hypothyroidism. Unfortunately, an undetectable endogenous TSH concentration does not confirm pituitary TSH deficiency because the lower limit of the normal range using the TSH assay currently available extends below the sensitivity of the assay. In addition, low serum thyroxine or free thyroxine concentrations may be caused by extraneous factors affecting the thyroid gland function (e.g., euthyroid sick syndrome) rather than by pituitary TSH deficiency. The serum thyroxine concentration may also be within the reference range in a pituitary dwarf with impaired TSH secretion (Kooistra et al, 1998). For these reasons, evaluation of pituitary and thyroid gland function using thyrotropin-releasing hormone (TRH) stimulation testing is recommended (see Chapter 3, page 120). The results of TRH stimulation testing should be normal in dogs and cats with isolated GH deficiency. In dogs and cats with congenital hypothyroidism or pituitary dwarfism caused by combined anterior pituitary hormone deficiency, the baseline TSH, thyroxine, and free thyroxine concentrations should be low and there should be minimal to no increase in the concentrations of these hormones after administration of TRH (see Fig. 2-4). The reader is referred to Chapter 3 for more information on thyroid gland function tests.