*2.4.4.2 Gamma aminobutyric acid (GABA)*

GABA is an important neurotransmitter whose actions at the central level are mainly inhibitory. In 1984, GABA was reported to modulate the secretion of AP hormones secretion by acting at both the hypothalamic and pituitary levels [74]. Intraventricular administration of GABA in rats stimulated LH and GH release. This effect was attributed to the fact that GABA induced an elevation of hypothalamic NA and median eminence DA levels as well as AP DA levels. The authors concluded that GABA plays a physiological role in the control of AP hormone secretion, primarily

### *Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

through hypothalamic action [74]. These data supported a previous study in which a dual action of GABA was postulated in the control of the hormonal secretion of the AP gland: one mediated *via* the central nervous system and the other exerted directly at the level of the AP [75]. Inhibition of GABA degradation and blockade of GABA transmission as well as administration of GABA and GABA mimetic drugs have all been shown to affect GH secretion. However, there are many controversial findings. For example, ancient studies indicated that GABA inhibited the release of SS [76, 77]. The effects of GABA may depend on the site of action within the hypothalamicpituitary axis and the hormonal milieu. What is clear is that GABA participates in the regulation and actions of a very important GH secretagogue, such as ghrelin. In addition, long-term GHRH administration increases GABA levels in all brain regions [78], and GHRH activates GABA receptors in the cerebral cortex [79]. In all, these data suggest that GABA participates in the control of GH secretion but acts as a modulator of the actions of other neurotransmitters or peptides.

### *2.4.4.3 Nitric oxide (NO)*

The gaseous neurotransmitter nitric oxide (NO) is synthesized from arginine by the action of nitric oxide synthase (NOS). NO performs a number of very diverse actions in the body, both at the level of the central nervous system and in the periphery. NO plays a stimulatory role *in vivo* and *in vitro* on GH secretion in acromegalic patients [80]. In rats, NO stimulates secretion of GHRH therefore increasing secretion of GH; however, it was also found that GHRH, in turn, increases production of NO in somatotrophs blunting GH secretion [81]. In dogs, inhibition of NO blunts GHRHinduced GH secretion, suggesting that NO acts by decreasing hypothalamic SS release [82]. However, years before, Aguila [83] argued that the hypothalamic increase in NO induced by GHRH would act on SS neurons increasing both the synthesis and release of SS. More recent studies conducted in fetal human primary cell cultures indicated that NO stimulated GH secretion, probably *via* the cGMP pathway [84]. This is consistent with further data demonstrating that a strong GH secretagogue, such as ghrelin, requires the activation of the NOS/NO pathway, and its subsequent GC/ cGMP signal transduction pathway, as essential steps to induce GH secretion from somatotrophs [85].

In summary, it appears that NO plays a role in mediating the GH response to neuroendocrine factors, but its role appears to change depending on environmental conditions, including mitochondrial functioning, either in the hypothalamus or in the pituitary gland itself.

### *2.4.4.4 Endogenous opioids*

There are three major types of endogenous opioid peptides that can be considered as neurotransmitters: endorphins, enkephalins, and dynorphins, which are respectively derived from three different precursor proteins: pro-opiomelanocortin (POMC), preproenkephalin A and B [86], although this number increased after the discovery of endomorphin-1 and endomorphin-2 [87].

The endogenous opioid peptides play many different physiological and pharmacological effects in humans, including neuroendocrine actions. In the case of the control of GH secretion, it has been shown that β-endorphin, administered intravenously or directly into the rat hypothalamus, increases GH secretion, an effect mediated by α2-adrenergic pathways, *via* stimulating GHRH and inhibiting SS release, because

β-endorphin antiserum reduced the stimulatory effect of clonidine on GH release [88]. Treating rats with an antiserum against GHRH inhibits the GH stimulatory response to β-endorphin [89], and the inhibitory effect of SS on GH secretion is antagonized by endogenous opioids [90].

At this point, there is the need to remark that exogenous non-peptide opioids have different effects on GH secretion when administered to man. In general, whereas acute opioid administration increases GH secretion, the effects of chronic opioid administration are much more complex. For example, intrathecal administration of opioids in chronic pain patients inhibits GH secretion, but the response is affected by sex, body composition, and insulin resistance [91].

#### **2.5 Regulation of GH by metabolic substrates**

GH is a metabolic hormone that plays an important role in regulating carbohydrate, fat, and protein metabolism in humans. Therefore, it is logical that metabolic substrates play a modulator role in the control of GH secretion.

#### *2.5.1 Glucose and GH secretion*

Glucose is a nutrient that the brain uses almost exclusively to provide energy [92], so glycemic regulation is key to maintaining normal brain function.

Basically, acute hyperglycemia inhibits GH secretion, either in basal conditions or in response to a number of stimuli acting on the central nervous system. Conversely, the acute decrease in plasma glucose concentrations leads to a rapid GH discharge. GH is one of the four counter-regulatory hormones that oppose the hypoglycemic actions of insulin (the other three are catecholamines, glucagon, and cortisol).

The opposite effects of hypo- and hyperglycemia seem to take place in the hypothalamus in SS-producing neurons. Hypoglycemia leads to the inhibition of SS secretion, while hyperglycemia induces SS release as cholinergic agonism prevents hyperglycemic blockade of GHRH-induced GH secretion [53]. The increase in GH induced by hypoglycemia appears to be almost totally refractory to cholinergic blockade [53]. Since hypoglycemia strongly activates adrenergic transmission, an inhibition of SS release mediated by α2-adrenoceptors [54, 61] would favor GH secretion, also positively acting on the secretion of GHRH. The former is most likely dependent on increased glucose-induced SS release, as cholinergic agonism prevents hyperglycemic blockade of GHRH-induced GH secretion [53]. The latter, in turn, appears to be mainly due to by inhibition of SS secretion. The fact that the increase in GH induced by hypoglycemia appears to be almost totally refractory to cholinergic blockade [53] does not invalidate such a possibility. Since hypoglycemia strongly activates adrenergic transmission, an inhibition of SS release mediated by α2-adrenoceptors [54, 61] would favor GH secretion. Furthermore, as seen above, an enhancement of endogenous GHRH release would also take place. In fact, in mice, it has been seen that hypoglycemia activates GHRH neurons [93]. However, also in mice, it has been reported that hypoglycemia produces a decrease in GH release [94, 95], most likely mediated by specific activation of neurons producing Neuropeptide Y [96]. Therefore, it is not yet clear, at least in mice, what is the role of glucose detection in GHRH neurons in controlling GH release during hypoglycemia due to the possible involvement of other central systems, such as the neurons that produce NPY.

As it is logical, hyperglycemia has to act in the opposite way to hypoglycemia. Consequently, as described, hyperglycemia increases the hypothalamic release of SS [97] *Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

and GHRH-induced GH secretion drastically decreases [98]. Furthermore, GH content in the pituitary is decreased in diabetic rats.

However, GH responses to diabetes are different in rats and humans. In humans, type I diabetes presents with increased pulsatile GH secretion [99, 100], perhaps because there is a greater pituitary sensitivity to GHRH [101]; however, in type II diabetes, GH secretion is negatively affected [102–104]. These differences are most likely due to the different fat mass in both types of diabetes and its consequences on insulin secretion, suggesting that insulin may be a regulator of GH release in both types of diabetes.

#### *2.5.2 Free fatty acids (FFA) and GH secretion*

FFA can totally block GH responses to a number of stimuli, including that of exogenous GHRH [105]; therefore, it has been postulated that the inhibition of GH release induced by FFA is mediated by an increased secretion of hypothalamic SS, although the data obtained *in vitro* point to a direct pituitary inhibition also [106]. In humans, the GH response to combined administration of pyridostigmine plus GHRH is markedly altered when plasma FFAs are increased by administration of a lipid-heparin infusion [107]. This favors the possibility of a main inhibitory action of FFAs on somatotrophs, which could be exerted by modifying the bilayer structure of the membranes and, therefore, affect their ability to detect stimulating signals (e.g., GHRH) or activate subsequent secretory mechanisms (GHRH or increased intracellular Ca2+). Interestingly, the response of other pituitary hormones to their hypothalamic releasing factors appears to be unaffected by the increase in plasma FFA in humans. Therefore, the inhibitory action of FFAs on GH secretion appears to be very specific. This is curious, because an important metabolic effect of GH is to induce the release of FFA from adipose tissues [108, 109]. Obese subjects show increased plasma FFA levels, while GH secretion is practically abolished, and if we suppress circulating FFA with antilipolytic drugs, obese patients regain normal GH secretion [110, 111]. Elevated levels of FFA are associated with insulin resistance and insulin hypersecretion; therefore, the decrease in GH in obesity may be caused by altered insulin feedback, rather than being a consequence of elevated plasma FFA levels, although there is no doubt about the effect of FFAs on the pituitary response to GHRH and SS. The opposite situation is seen in fasting: GH secretion increases and stimulates FFA release [108, 109].

In total, these relationships between GH and FFA appear to be related to the need to maintain metabolic homeostasis.

#### *2.5.3 Amino acids and GH secretion*

Given the clear and important anabolic effects that GH exerts on protein synthesis and metabolism, it is at least surprising that only two basic amino acids, arginine and ornithine, exert a powerful effect on GH secretion in humans. Arginine clearly increases the maximal GH responsiveness to GHRH in humans [112, 113], even when an elevated SS tone seems to exist (e.g., in the elderly). Furthermore, pyridostigmine does not potentiate GH response to arginine [114]. Hence, the inhibition of hypothalamic SS release appears to be the mechanism involved in the GH-releasing effect of this amino acid. Furthermore, arginine proceeds from citrulline and can be transformed into NO by the action of NOS, which suggests that another mechanism of action of this amino acid in GH secretion may be secondary to the increase in NO synthesis at the central level.

#### **Figure 9.**

*Basic control of GH by metabolic substrates. Perhaps the main regulator is plasma glucose levels, given the effects of GH as a counterregulatory hormone. Hypoglycemia is detected by glucose-sensitive neurons and leads to the release of catecholamines (CA). These have a dual effect: they inhibit the release of SS (red arrow, −) and stimulate the release of GHRH (blue arrow, +). In somatotrophic cells, there are receptors for GHRH (GHRHR) and SS (SSR) that when stimulated induce respectively the synthesis and release of GH (GHRH, blue arrow, +) or the inhibition of the secretion of this hormone (SSR, red arrow, −), although in the case of SSR, it cannot be ruled out that they also act negatively on the synthesis of pituitary GH (?). The effects of FFA seem to be more varied, facilitating the release of SS, like hyperglycemia (blue arrow, +), and also acting at the level of somatotrophs, inhibiting the activation of GHRH and/or preventing the stimulating effect of GHRH on GH release (red arrow, −). Arginine appears to directly inhibit SS release (red arrow, −), allowing GHRH release. Although not depicted in the figure, the pituitary SS receptor is type 2, SSTR2.*

The effect of arginine on GH secretion is much more powerful than that of ornithine. The basic effects of metabolic substrates on GH secretion are schematically depicted in **Figure 9**.

#### **2.6 Regulation of GH by other hormones and peripheral and central peptides**

Endocrine products mainly related to GH control are those that play a general permissive role in the body, such as thyroid hormones and glucocorticoids, or those related to maturational changes, such as sex steroids, but also other hormones, such as Insulin and IGF-I play an important role in the control of GH synthesis and release. Furthermore, as with other pituitary hormones, GH secretion is self-regulated. In addition, as GH plays multiple important roles in practically all the tissues of the body, especially of a homeostatic metabolic regulatory nature, a series of central and peripheral peptides, some of which can be considered as authentic hormones, clearly participate in the modulation of GH synthesis and secretion, as we will analyze in this section.

#### *2.6.1 Thyroid hormones and GH secretion*

Thyroid hormones are permissive hormones needed for a physiological response to any other hormone. Hypothyroidism is known to be associated with impaired linear growth, which is easily normalized by the administration of replacement doses of

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

thyroid hormones. Consistent with it, there is a decrease in plasma levels of IGF-I, which are normalized after the treatment with thyroid hormones. Both spontaneous GH secretion and GH responses to classic stimuli are markedly reduced in clinical hypothyroidism in humans or in induced hypothyroidism in rats [115]. There are several explanations for these facts. First, the lack of thyroid hormones strongly affects the hypothalamic synthesis of GHRH [116]. Although this directly leads to impaired GH synthesis, additional effects of hypothyroidism may depend on the lack of both the positive modulation that thyroid hormone induces on the number of GHRH-receptor sites in somatotrophs and its facilitatory role on GHRH binding to its receptors. Furthermore, GH-secretory mechanisms are also impaired in hypothyroidism, not only because a decrease in the hypothalamic content of GHRH [116, 117] but also because SS appears to be increased, at least in the rat [117–119].

Here at this point, it is of interest to note that the administration of GH to hypothyroid children with GH deficiency can increase hypothyroidism, something that does not occur when GH is administered once the hypothyroidism has been corrected.

#### *2.6.2 Glucocorticoids and GH secretion*

The effects of glucocorticoids on GH synthesis and secretion are complex and reflect the pluripotential nature of these hormones. On the one hand, glucocorticoids are essential for the maintenance of GH secretion; thus, patients with adrenocortical insufficiency present GH deficiency that can be corrected by substitutive treatment with these steroids [120]. However, excess glucocorticoids decrease GH secretion and longitudinal growth [121, 122]. Acute administration of glucocorticoids leads to a GH-secreting response that lasts for a few hours, although after this there is a total blockade of the release of the hormone [123, 124]. This is probably all due to the fact that glucocorticoids stimulate transcription of the GH gene [125] and increase the stability of its mRNA [126], as well as they increase the expression rate of the GHRH receptor in somatotrophs.

The negative effect of excess glucocorticoids would depend on its induction of SS release, mediated by an increase in the responsiveness of SS neurons to β-adrenergic stimulation, as it happens in the periphery (**Figure 10**). It may also be that glucocorticoids directly modulate SS synthesis, since there is a glucocorticoid response element in the SS gene.

#### *2.6.3 Sex steroids and GH secretion*

The existence of a pattern of sexual dimorphism in GH secretion, first described in rat [127], has been a clearly well-established concept for many years [128]. Sexual differentiation occurs during fetal life; sexually different fetal sex steroid hormones regulate striking differences in the number of GHRH and SS neurons, their responses to sex steroids once the puberty begins, the adult hypothalamic synapse and its organization, and perhaps the number of somatotrophs and their responsiveness [129]. In humans of both sexes, spontaneous GH secretion is low during childhood, and it is gradually increasing until just before puberty, when the release of the hormone increases strongly [130, 131] and sexual dimorphism begins to manifest. Therefore, it appears that sex steroids explain this maturational change in the pattern of GH secretion. The question is: where does this action of sex steroids take place?

After puberty, GH secretion is greater in women than in men, although this can be modified depending on the phase of the menstrual cycle; the late follicular phase

#### **Figure 10.**

*Control of SS by glucocorticoids. Glucocorticoids (GC) stimulate the expression of β-adrenoceptors (β-R) in somatostatin neurons (SS neuron) and favor their response to low levels of catecholamines (1*′ *and 2), inducing the release of SS. Furthermore, GCs can stimulate the transcription of the SS gene (3). The result is an increased discharge of SS into the portal blood.*

leads to GH secretion greater than that seen in both the early follicular phase and the luteal phase.

A direct pituitary effect of sex steroids on GH secretion cannot be excluded; however, they most likely act primarily on the hypothalamic level, modulating the rhythmic interaction of GHRH-SS and, in particular, the availability of SS [61]. The difference between hypothalamic levels of free estradiol (fE2) in man and women can explain the differences in the secretory pattern of SS and, therefore, why GH release is sexually dimorphic.

In women, there is a large variation in plasma E2 levels throughout the menstrual cycle. We will have to bear in mind that only fE2 is biologically active, and that only in this form can it cross the blood-brain barrier. In fact, most of E2, but also of T, circulates bound to sex hormone-binding globulin (SHBG), although its affinity for this carrier is lower than that of T. However, plasma levels of SHBG are approximately two times higher in women than in men, because E2 increases the hepatic synthesis of this globulin. This, together with the fact that T circulates in levels of ng/ml, while E2 does so in pg/ml, suggests that free testosterone (fT) reaches higher levels in the hypothalamus than fE2, but this would be compensated by the hypothalamic aromatization from T to E2.

The data in rats clearly indicate that E2 replacement therapy rapidly reverses the decrease in SS mRNA observed after oophorectomy [132], although a direct effect of E2 on the SS gene is unlikely. E2 can affect both the biosynthesis and turnover of catecholamines [133] in hypothalamic areas involved in the control of SS, as well as the responsiveness of α2-adrenoceptors in SS neurons. This, together with our findings demonstrating the role of the adrenergic system in the regulation of SS [54, 61], allows to hypothesize that the action of E2 on SS neurons depends on its effects on adrenergic transmission to these neurons [4]. This is consistent with data indicating that, at the peripheral level, catecholamines appear to be integral signaling components for maintaining steroid sensitivity in some reproductive tissues [134].

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

In addition, E2 inhibits the hepatic synthesis of IGF-I, thus reducing the effects of this inhibitor of both SS and GH.

On the other hand, T seems to stimulate the release of SS, but it depends on its central aromatization to E2. Therefore, the different levels of free E2 fE2 related to gender could explain the dimorphism in GH secretion [4]. Indeed, in humans, plasma fE2 but not T, were shown to be strongly correlated with rates of total and pulsatile GH release [5]. Support for this approach is given by the fact that there is great aromatase activity in the hypothalamus, as well as by the fact that it is E2 and not T that modifies the activity of a series of enzymes involved in synthesis and turnover of catecholamines. This explains why in children with delayed puberty the administration of T produces an increase in the pituitary reserve of GH, which is not observed when a non-aromatizable androgen, such as oxandrolone, is administered. Furthermore, studies in pubertal boys and adult men given the antiestrogen tamoxifen abrogate the stimulatory effect of T on GH [135, 136].

In summary, higher levels of fE2 in the hypothalamus, in women, can cause decreased biosynthesis and/or release of SS. If fE2 exerts a positive effect on the production of catecholamines at the hypothalamic level, they will also act positively on the production of GHRH. In GHRH neurons, there is an estrogen receptor ERα whose deletion in mice delays female puberty, since in females a group of GHRH neurons changes their phenotype to start producing Kiss1 (a key peptide in gonadotropic regulation). Therefore, a direct action of estrogens on neurons with the dual GHRH/ Kiss1 phenotype modulates growth and puberty and may direct the sex differences in endocrine function observed during pubertal transition [137].

In women, episodic GH secretion is more frequent, and plasma GH values during trough periods are somewhat higher than in men. Therefore, in general, the amount of GH secreted in peak episodes is higher in women. But this can change throughout the menstrual cycle, as described before. Pulsatile GH release in women is doubled in the late follicular stage [138], and when estrogens are administered for superovulation, GH release is significantly increased [139]. Moreover, treatment of men with diethylstilbestrol (a potent estrogen) induces a change in the pulsatile pattern of GH release similar to that commonly seen in females [140]. It is important to remark that in humans, as in the rat [141], sex steroids would act mainly during the fetal and early neonatal stages, impregnating the hypothalamus so that from puberty there is an increase in the pulsatile secretion of GH, determined by an increase in the amplitude of the peaks rather than a change in secretory frequency [142]. In adults, the modification of the levels of sex steroids is not accompanied by significant modifications of the secretion of the hormone [4, 135].

The fact that estrogens produce an alteration in GH secretion may be due to the affectation of the feedback mechanisms through which IGF-I acts on GHRH and SS, in this case extraordinarily diminished by these steroids [143]. Alteration in GH output in response to estrogens is thought to occur as a consequence of altered negative feedback, wherein the actions of IGF-I on hypothalamic components of the GH-axis are greatly diminished [143]. This is something paradoxical [144], with evidence suggesting a complex relationship between IGF-I, estrogens, and GH output [129]. At low doses, estrogens increase GH output (while IGF-I production is high), while high doses of estrogens can decrease bioactive IGF-I levels, preventing the inhibitory effect of IGF-I on GH secretion. Furthermore, the route by which estrogens are administered clearly affects their effects on GH secretion. The pulsatile secretion of GH is very different when E2 is administered orally than when it is administered *via* transdermal patches, in both premenopausal and postmenopausal women. In two studies, oral administration of estradiol caused an increase in total daily GH secretion and its pulsatile release and a decrease in plasma IGF-I levels, whereas transdermal estrogen treatment increased circulating levels of IGF-I in post-menopausal to that seen in pre-menopausal women without altering the pulsatile release of GH [145, 146]. Therefore, it is likely that oral administration of E2 leads to a decrease in plasma IGF-I levels, consequently reducing its inhibitory effects, at the pituitary and hypothalamic levels, on GH secretion. Therefore, it is likely that the decrease in GH secretion observed at menopause is not due to the reduction in estrogen levels [146], since, furthermore, neither oral nor transdermal administration of E2 in menopausal women is able to reverse the decreased GH secretion associated with aging even when stimulated with GHRH. Moreover, the route of estrogen administration significantly alters the effect of estradiol on GH release. Assessment of pulsatile GH output in pre- and postmenopausal women following oral versus transdermal estradiol treatment revealed divergent effects on GH release. In two studies, oral administration of estradiol caused an increase in total daily GH secretion and pulsatility and a decrease in plasma IGF-I levels. Increased 24 h mean and pulsatile GH release and decreased circulating levels of IGF-I, whereas transdermal estrogen treatment increased circulating levels of IGF-I in post-menopausal to that seen in pre-menopausal women without altering the pulsatile release of GH [145, 146]. Thus, it is thought that oral administration of estrogen increases GH release through lowering circulating levels of IGF-I, thereby reducing negative feedback. These observations further indicate that

#### **Figure 11.**

*Control of SS by sex steroids. Depending on the sex, the testicles or the ovaries secrete sex steroids in the blood (1), testosterone (T) or estradiol (E2), respectively, but also low amounts of E2 (testicles) and T (ovaries). While T can cross the blood-brain barrier (BBB, 2), only free E2 (fE2, 2) can reach the brain. In the hypothalamus, T is aromatized to E2 (3), so the amount of fE2 is greater in women (4) than in men. fE2 modulates hypothalamic synthesis and catecholamine turnover (CA, 5), so the amount of catecholamines that reach SS neurons [5] is high enough to stimulate α2-adrenoceptors (6), inhibitors of the synthesis (7) and release of SS (8), but stimulators of GHRH synthesis (6) and release (9). E2 induces the hepatic synthesis of SHBG (10) and binds to it (11), as does T (11), but with lower affinity than T. Also in the liver, E2 decreases the synthesis of IGF-I (red arrow), a stimulator of SS release (12).*

### *Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

the reduced GH output following menopause is not due to a reduction in circulating levels estrogen [146]. To this extent, neither oral nor transdermal estrogen treatment in postmenopausal women can reverse age-related reductions in spontaneous or GHRH-stimulated GH and IGF-I release. However, a recent randomized, doubleblind, controlled study, performed in 60 healthy postmenopausal women conclude that while there was a E2 decrease when giving anastrozole (an inhibitor of aromatase activity), but no changes in E2 levels were observed when fulvestrant (a selective estrogen receptor-α antagonist) was administered. In spite of it, GH rebound after SS infusion declined markedly during both estrogen-deprivation schedules, suggesting a previously unrecognized dependence of hypothalamic-pituitary GH regulation on low levels of endogenous estrogen during menopause [147].

As we have seen throughout this section, although it is clear that sex steroids play a more than important role in the control of GH secretion, with E2 as the main actor, there are many controversies regarding both the mechanism of action and the effects they produce. Most likely this is a reflection of the great complexity of the world of GH and the number of factors involved in it.

In a study from our group carried out in young healthy volunteers of both sexes, it was concluded that testosterone acts on the release of GH at the supra-hypophyseal level and that its action is mediated by its aromatization to E2 [148]. Therefore, the association between reduced GH release at menopause and low circulating estrogen levels must be thoroughly analyzed in order to understand the mechanisms that may link these concepts.

Another point of interest is the fact that in women there is no cortico-adrenal production of estrogens and there is production of androgenic precursors that give rise to testosterone in the liver, as in men, which, although small, can help in GH secretory control.

A summary of these concepts is shown in **Figure 11**.

#### *2.6.4 Insulin and GH secretion*

Insulin and GH play some opposite actions at the metabolic level in the human body. Therefore, it is logical to think that insulin has effects on pituitary GH secretion.

As is known, chronic GH secretion counteracts insulin effects leading to peripheral insulin insensitivity, resulting in pancreatic hypersecretion of insulin that attempts to compensate for its loss of sensitivity [149]. On the contrary, as already defined, weight gain leads to poor GH secretion correlated with an increase in circulating insulin. On these basis, it appears that GH release is tightly controlled by prolonged changes in energy intake and supposed insulin release. There are many situations that support these inverse relationships in various metabolic conditions. For example, fasting, anorexia nervosa, and type-1 diabetes are associated with decreased insulin levels and increased GH secretion, while hyperphagia and obesity are associated with increased insulin secretion and decreased GH secretion.

A series of many evidences points to a direct relationship between hypoinsulinemia and excess increased secretion of GH. Intensive insulin treatment in type-1 diabetic patients reverses GH hypersecretion [99], whereas calorie restriction to reverse hyperinsulinemia [150] results in the recovery of GH secretion, presumably in response to a reduction in insulin levels [151].

Insulin inhibits the expression of the GH gene in isolated pituitary cells [152, 153], as well as acts selectively on its receptors in the pituitary gland [154] in such a way that it suppresses GH release from isolated somatotrophs [154, 155]. These indicate

that, although systemic insulin resistance exists [154], somatotrophs remain sensitive to insulin in obesity. Therefore, insulin can modulate GH release in relation to weight gain and promote continued suppression of GH release in obesity, independent of the inhibitory effect of plasma insulin. In contrast, *in vivo* deletion of somatotroph insulin receptors leads to increased pituitary GH content and release [155]. These effects occur independently of the development of systemic insulin resistance, suggesting that somatotrophs remain insulin sensitive in obesity. Thus, insulin can modulate GH release in relation to weight gain and promote sustained suppression of GH release in obesity, independent of systemic insulin resistance. In contrast, *in vivo* removal of insulin receptors from somatotrophs leads to an increase in the content and release of GH in the pituitary [155]. The suppressive actions of insulin on GH release occur independently of IGF-I, since the selective inactivation of the insulin receptor and IGF-I receptor (Insr and IgfIr) genes in mouse somatotrophs does not affect these effects of insulin [156], despite the fact that in the liver, insulin facilitates the expression of IGF-I, which suppresses the synthesis and secretion of GH. Therefore, it is likely that insulin can provide critical feedback to alter GH release in relation to longterm metabolic requirements. It should be noted that loss of somatotroph-specific insulin receptor expression does not completely reverse the suppression of GH release seen during diet-induced weight gain and thus leads to obesity [156].

The actions of insulin on GH release may not be restricted to the pituitary. Insulin receptors are expressed throughout the hypothalamus, including the arcuate and periventricular nuclei [157–160]; therefore, insulin can modulate GH release by acting through the hypothalamus, although this is not yet well known. As insulin does not predict ultradian GH release patterns, the actions of insulin at the hypothalamus level to mediate GH production may be limited to infradian regulation of maximal GH production [130].

#### *2.6.5 IGF-I and GH secretion*

IGF-I is the main mediator of the actions of GH in the body and shares many effects with it, although it also has specific effects of its own. IGF-I is produced in virtually any tissue, but plasma IGF-I is synthesized and released from the liver. There are specific relationships between IGF-I and GH, since IGF-I is part of a long feedback loop by which it inhibits GH secretion, acting both at the hypothalamic level, where it stimulates the release of SS, and at the pituitary level, inhibiting GH secretion directly. Even at the level of the median eminence, specific binding sites for IGF-I have been identified where it acts by inhibiting GHRH secretion [161, 162]. At the pituitary level, IGF-I inhibits the transcription of GH and Pit-1 genes, both under basal conditions and after stimulation with GHRH [41, 163].

#### *2.6.6 Ghrelin, Klotho, and GH secretion*

At the end of the past century, Bowers' group [164] tried to identify how peptides derived from endogenous opioids could stimulate GH secretion. They synthesized a Met-enkephalin derivative, without opioid activity, which they named GHRP-6 (growth hormone-releasing peptide-6) [165]; it exhibited GH-releasing activity *in vivo,* in humans, acting synergistically with GHRH without binding to any known receptor. After the synthesis of this GH secretagogue, many other were synthesized, until a specific receptor for these synthetic compounds was identified in 1996 [166]. This receptor was a GTP-binding protein (GHSR-1a) present in the pituitary and

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

arcuate and infundibular hypothalamus of several species, including humans [166], and its detection implied that a natural hormone had to exist. Indeed, this hormone was found in the stomach of rats and was called ghrelin [167].

Since we recently published an extensive review of the actions of ghrelin and klotho [168], only a few of the many important actions of these peptides will be discussed in this chapter.

#### *2.6.6.1 Ghrelin and GH secretion*

Ghrelin is a 28 amino acid peptide, which is in fact a true pleiotropic hormone. In spite of its peptide structure, ghrelin must bind to an octanoyl group on the third amino acid to acquire full biological activities [169].

The discovery of ghrelin and the fact that it acts synergistically with GHRH caused a change in the concept of basic regulation of GH; that is, in addition to the classical GHRH-SS interaction, GH secretion from the pituitary gland is also modulated by ghrelin produced by specific cells located in oxyntic cells in the stomach (**Figure 12A**). Ghrelin is an orexigenic hormone that appears in the blood during fasting and reaches the CNS to transmit a hunger signal. This explains why in anorexia nervosa there is an increase in plasma ghrelin concentrations, while in obese subjects it is reduced, and also why GH secretion is increased in anorexia nervosa patients and during fasting, while it is reduced or absent in obesity, but also in the elderly [170] (**Figure 12A**). Therefore, it seems that ghrelin appeared in evolution to stimulate eating behavior and optimize the use of digested food by promoting the secretion of an anabolic hormone, such as GH [171].

As indicated above, the active form of ghrelin that induces GH secretion, by stimulating GHRH secretion and inhibiting SS and IGF-I secretion (**Figure 12A**),

#### **Figure 12.**

*Control of GH secretion by ghrelin and GH secretion. A: In fasting situations, the empty stomach (1) produces ghrelin in P/DI cells (2), which is released to the blood (3, 4) from where it reaches the pituitary gland (5) and induces the synthesis and secretion of GH (6). Ghrelin also reaches the hypothalamus (7), where it stimulates (8) appetite neurons and GHRH and inhibits SS. Plasma ghrelin also inhibits IGF-I (9), therefore inhibiting its inhibitory effects on the pituitary synthesis and secretion of GH (10). In turn, IGF-I inhibits the increase in circulating ghrelin (11) produced by fasting. Blue arrows: stimulation. Red arrows: inhibition. Yellow arrow: synthesis and secretion of GH. Black arrow: hypothalamic effects of ghrelin. +: stimulation. −: inhibition. B: The active form of ghrelin is acyl ghrelin, obtained after an acylation performed by GOAT, which links after linking a fatty acid side chain to serine 3. Acyl ghrelin can then bind to its receptor GHSR-1a and activate it. This figure has been modified from reference 169, Front Endocrinol. 2021.*

and many other physiological functions, such as inhibiting cell death in cardiac and endothelial cells [172], is the acylated form (acyl ghrelin), produced by attaching an octanoyl group to amino acid 3 (serine) carried out by ghrelin O-acyl transferase (GOAT) (**Figure 12B**) [173, 174].

Ghrelin stimulates GH release: (1) directly on the pituitary somatotrophs and (2) antagonizing SS and inducing GHRH secretion. In addition, ghrelin decreases plasma levels of IGF-I, therefore inhibiting the negative effect of IGF-I on GH secretion.

In humans, the physiological nocturnal rise in GH was not inhibited by the infusion of a powerful analog of SS, such as octreotide [175, 176]. This indicates that the effect of ghrelin was not affected by SS. On the other hand, patients with inactive GHRH receptor, due to mutations, still had rhythmic GH secretion, suggesting that another factor, different to GHRH, was inducing pituitary GH secretion [177]. However, a non-sense mutation affecting ghrelin receptor in humans was found to be associated with short stature [178]. Furthermore, in rats, intravenous (iv) administration of ghrelin during a GH peak induced a marked increase in plasma GH [179], but the immunoneutralization of GHRH led to a virtual absence of ghrelin-induced GH secretion; however, when ghrelin was administered during a physiological trough period, the GH response was clearly diminished. Both effects indicate that ghrelin acts synergistically with GHRH while inhibits hypothalamic SS. In fact, ghrelin expression has been found in the hypothalamic arcuate nucleus [180].

Ghrelin and its receptor are also expressed in the pituitary [181, 182], so pituitary ghrelin may play an auto/paracrine role in the regulation of GH release. GHRH infusion increases pituitary ghrelin mRNA levels, suggesting that GHRH may be a regulator of pituitary ghrelin production [183]. Therefore, pituitary ghrelin can act physiologically on GH secretion, enhancing the response of somatotrophs to GHRH.

Ghrelin, as GHRH, acts through a G-protein-coupled receptor (GPCR), but in this case the activation of this receptor leads to the stimulation of the activity of phospholipase C (PLC) that induces the formation of IP3 and DAG (**Figure 13**); both IP3 and DAG induce an increase in cytosolic Ca2+ that facilitates the release of GH [179]. Ghrelin requires activation of the NOS/NO pathway and its subsequent guanylate cyclase (GC)/cGMP signal transduction pathway to induce GH release from the pituitary [85]. Chronic treatment with ghrelin produced an upregulation of GH transcription levels, as well as that of two isoforms of Na<sup>+</sup> channels, sensitive to blockade with tetrodotoxin (TTX), expressed in somatotrophs, such as NaV1.1 and NaV1.2. This indicates that ghrelin also regulates the expression of the Na+ channel gene in somatotrophs (**Figure 13**) [184].

Interestingly, the hypothalamic enzymatic complex AMP-activated protein kinase (AMPK), involved in the hypothalamic control of energy and metabolic homeostasis, is also activated by ghrelin. Therefore, AMPK also participates in the control of GH secretion, and as its blockade or its functional impairment inhibits ghrelin- or GHRHinduced GH secretion, most likely by increasing SS production tone [185] (**Figure 13**).

It is well known that aging is associated with a decrease in GH secretion from the second decade of life [186]. Something similar happens with ghrelin [170, 187], although the pituitary ghrelin receptor does not decline with aging and the GH response to ghrelin is still seen in the elderly and there is an age-related decline [188].

Therefore, what is the reason why the secretion of an orexigenic hormone, such as ghrelin, and also that of an anabolic hormone, such as GH, is lost with aging?

Gastric ghrelin synthesis and secretion increase during fasting and decrease during feeding [189]. This is the reason why chronic intake of high-calorie diets, prolonged ingestion of high fats, and obesity lead to a reduction in gastric ghrelin *Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

#### **Figure 13.**

*Ghrelin control of GH secretion. Mechanisms of action of ghrelin in the control of GH secretion. Hypothalamic ghrelin (coming from the circulation and/or synthetized in the hypothalamus) stimulates the release of GHRH into the portal blood from where GHRH reaches the somatotrophs (green arrow) and induces synthesis (blue arrow) and release (green arrow) of GH, but also that of pituitary ghrelin that also participates in the synthesis of GH (blue arrow). Hypothalamic ghrelin antagonizes SS by itself (red arrow) but also through a mechanism mediated by its stimulation of AMPK (blue arrow). At the pituitary level, ghrelin from the hypothalamus (green arrow) or from the systemic circulation stimulates the release of GH via activation of the PLC that activates the intracellular Ca2+ supply and the transcription of Na+ channels necessary for the release of GH. Likewise, ghrelin after the activation of its membrane receptor (GPCR) activates the NOS/NO pathway, also necessary for the release of GH. Taken from reference [169], Front Endocrinol. 2021.*

production and secretion [189, 190], while a low protein supply significantly increases plasma ghrelin [190].

Adrenergic hormones stimulate the release of gastric ghrelin by acting directly on the β1 receptors of ghrelin-producing cells, very rich in this type of adrenergic receptors [191]. Therefore, fasting acts on gastric ghrelin secreting cells through the sympathetic nervous system, something that appears to be logical given the relationships between this autonomic system and the hypothalamic production of GHRH and SS for controlling GH secretion. The administration of muscarinic agonists also increases plasma ghrelin concentrations [192], as does vagus nerve excitation in the gastric mucosa [193].

Furthermore, and given the relationships between ghrelin, SS, and GH secretion, it stands to reason that SS [194] and GH [195] inhibit gastric ghrelin secretion, although ghrelin also stimulates pancreatic SS production [196], to inhibit insulin release [197, 198] and contribute to glucose homeostasis [197].

Since, as we have seen, metabolic factors play an important role in the control of GH secretion, it is logical that they also participate, *via* hormonal modulation, in the control of gastric ghrelin secretion, as can be seen in detail in Ref. [169]. Perhaps the only hormones to be discussed in this section are leptin and IGF-I. Leptin, a hormone produced by adipocytes, which acts by decreasing food intake; that is, it is an

anorexigenic hormone, and it is logical that it acts by decreasing plasma ghrelin and inhibiting its actions at the central level [199].

Similarly, logical are the relationships between IGF-I and ghrelin. Plasma IGF-I concentration significantly determines plasma ghrelin concentrations, existing a negative correlation between them [200]. This has been found in children and adolescents [201, 202]. On this basis, the highest concentration of ghrelin was observed in GH-deficient children in whom there was low availability of free IGF-I [203], the bioactive form of IGF-I. That is, low plasma levels of IGF-I induce the synthesis and secretion of ghrelin, while in turn ghrelin decreases plasma levels of IGF-I.

#### *2.6.6.2 Klotho and GH secretion*

Although klotho was identified in 1997 as an antiaging agent, klotho-deficient mice exhibit growth retardation [204], implying that this transmembrane protein is also involved in the control of GH secretion. The kidneys are the main source of klotho, where it is stimulated by insulin [205] and IGF-I [206]. In contrast, klotho inhibits both receptor activation and intracellular signaling of these hormones [207], thus acting as a positive regulator of GH secretion.

Klotho induces GH secretion by activating the extracellular signal regulated kinase 1/2 (ERK1/2) pathway in AP somatotrophs [208]. Relationships between klotho and GH can be seen in untreated GH-deficient children and adults; in these patients, the plasmatic levels of klotho are low, while treatment with GH normalizes this klotho deficit, through a mechanism mediated by the activation of the Akt-mTOR pathway [206], a key pathway in GH signaling.

In addition to its production in the kidney, klotho is also produced in the somatotrophs, most likely to modulate auto/paracrine GH secretion. Interestingly, high production of klotho has been observed in pituitary adenomas that do not secrete GH, suggesting that klotho is also produced in other pituitary cells which are also a source of this still little known hormone that plays so many different roles in the body.

#### *2.6.7 Neuropeptide Y (NPY) and GH secretion*

NPY is a 36 amino-acid peptide belonging to the family of pancreatic polypeptides. It is expressed in the hypothalamus in neurons of the arcuate, paraventricular, and periventricular nuclei [209]. NPY exerts an important effect on the regulation of energy intake and expenditure, something that, as we have already analyzed, once again involves GH, so NPY has to play a role in regulating the secretion of this hormone, although this role is still little known. Ancient studies carried out in rodents showed that NPY inhibited the GH axis [210], most likely due to the fact that NPY neurons in the arcuate nucleus connect with periventricular SS neurons [211]. In fact, after 9 hours of fasting, it was observed, in mice, that the hypothalamic levels of NPY increased as did those of SS [95], which suggested that NPY would be the factor responsible for the inhibition of GH secretion, mediated by SS, associated with fasting. There are five receptors for NPY. Among them the Y1 receptor is mainly expressed at the post-synaptic level, while the Y2 receptor is expressed in NPY neurons [212, 213].

A study carried out in mice indicates that activation of Y1 receptor inhibits the secretion of GH during fasting, probably through induction of SS release, whereas Y2 receptor has no effect on GH secretion in fasting conditions, but maintains pulsatile GH release in mice fed *"ad libitum"* [96].

*Pituitary Growth Hormone Secretion and Cell Growth Hormone Production: Regulation of Their… DOI: http://dx.doi.org/10.5772/intechopen.108382*

All these data indicate that NPY neurons play a very important role in the control of energy intake and expenditure, as indicated above. This concept is reinforced by the fact that most of these neurons co-express NPY and Agouti-related peptide (AgRP, another important regulator of metabolic homeostasis), and more importantly, there is an insulin/NPY network that regulates energy homeostasis, so that the lack of insulin signaling in NPY neurons induces an increase in energy reserves and obesity and also produces an alteration of GH/IGF-I axis. This is a clear example of how GH control is key to proper homeostasis of metabolism and food consumption, and how these, in turn, control GH production.
