Endocrine Actions Of Hypocretins

The presence of abundant immunoreactive hypocretin fibres and hypocretin receptors in hypothalamic nuclei such as the arcuate nucleus (ARC), the paraventricular nucleus (PVN), the periventricular nucleus (PeN) and the preoptic area (PO) suggests a neuroendocrine regulatory role for hypocretins.14 Moreover, the presence of both hypocretin receptors in rat and human pituitary65'68 could indicate direct actions of hypocretins at this level. In this section we will review the endocrine and neuroendocrine actions of hypocretins.

4.1. Hypocretins and Adrenal Axis

Anatomical and physiological data demonstrate that the lateral hypothalamus (LHA) is involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and there is substantial evidence to support this contention. There are neuronal connections between the LHA and the corticotropin-releasing hormone (CRH) neurons in the parvocellular part of the paraventricular hypothalamic nucleus (PVN),2 CRH is expressed in the LHA94 and in rats with selective destruction of the LHA a disruption of the normal circadian corticosterone rhythm is observed.95

The HPA axis is the endocrine axis in which hypocretins play their most extensive role; in fact the rat adrenal gland was the first peripheral organ in which Hcrtrs were detected.70 Additional papers have demonstrated the existence of hypocretin receptors at all levels of the HPA. Thus, immunohistochemical studies have revealed the anatomical contact between hypocretin terminals and corticotropin-releasing hormone (CRH) cells in the parvocellular part of the paraventricular hypothalamic nucleus,38'57 which express Hcrtrl.61 Moreover, in the human pituitary there is abundant expression of Hcrtr2 in corticotrope cells,68 suggesting direct hypocretin actions, and in the rat adenohypophysis all the corticotrope cells present Hcrt2 immunoreactivity,67 suggesting a paracrine role of hypocretins in these cells.

In addition to this anatomical evidence, physiological studies have revealed the existence of a functional link between hypocretins and the HPA axis. It has been shown that intracerebroventricular administration of both hypocretins results in the induction of c-fos activity and a depolarization of CRH-synthesizing parvocellular neurons of the paraventricular hypothalamic nucleus.96-99 Furthermore, central administration of Hcrtl increased mRNA levels of CRH and arginine vasopressin (AVP) in this neuronal population100 (our unpublished data). Central administration of Hcrtl also raised the circulating levels of adrenocorticotropin hormone (ACTH) and corticosterone.98101102

The hypothalamic interaction between hypocretins and the adrenal axis is mediated by neuropeptide Y (NPY). This is demonstrated by the blockade of central NPY function using antagonists or antibodies. Blocking NPY activity inhibits the hypocretin-induced corticosterone release.103 In this sense, it is interesting to note that Hcrt1 stimulates the expression of NPY in the rat, both in vitro104 and in vivo9 and in the goldfish (Carassius auratus)}05 The participation of NPY arcuate neurons in the hypothalamic actions of hypocretins is not exclusive of the adrenal axis. In fact, the hypocretin-NPY neuronal circuit3961106 plays a fundamental role in hypocretin actions in the gonadal axis,107108 prolactin release,109 regulation of body temperature,110111 drinking behaviour,106 orexigenic actions of Hcrt1 and Hcrt29112-115 and probably the GH -axis (see below, Hypocretins and Growth Hormone Axis).

In addition to their hypothalamic and pituitary actions on the HPA axis, hypocretins also play an important role in the development and proliferation116 and secretory activity of rat adrenal cortex, acting on mineralocorticoid and glucocorticoid secretion. In this sense, peripheral administration of Hcrt1 and Hcrt2 to rats enhances the blood levels of both aldosterone and corticosterone73 and in vitro studies have reported stimulatory effects of both hypocretins on cortisol secretion.75,117

The physiological relevance of hypocretins in the HPA axis remains unclear. Some evidence suggests that some behavioural effects of hypocretins, such as grooming and face washing, are mediated through CRH and glucocorticoids.118 Moreover, the ability of hypocretins to modulate the response to stress is supported by neuroendocrine96,101,102,118,119 and cardiovascular studies.120-122 In this sense, it is very interesting to note that during late pregnancy in the rat, the responsiveness of paraventricular CRH neurons to Hcrt1, and hence the pituitary-adrenal axis, is markedly reduced,123 suggesting that hypocretin system plays a role in the attenuation of the HPA response to stressors observed in the pregnancy.123

The relevance of the hypocretin-HPA axis in humans remains unclear. Nevertheless, regulatory actions of hypocretins on pituitary-adrenal axis are supported by data obtained in narcoleptic humans. These patients exhibit normal cortisol levels despite the fact that ACTH secretion is markedly decreased, suggesting alterations in the interaction between the HPA hormones.124,125 In this sense, a recent study in rat has reported that the expression of the enzymes involved in the steroid metabolism is altered during sleep.126 Thus, it is tempting to speculate that changes in the hypocretin tone during sleep could act directly on adrenocortical metabolism, explaining then the lack of association between ACTH and cortisol levels.

On the other hand, it is possible that hypocretins play a role in the regulation of adrenal axis during the sleep cycle in normal healthy humans. In this sense, in humans deep sleep has an inhibitory effect on the HPA axis and sleep deprivation has a stimulatory effect on it.127-129 Whether these changes are mediated by the hypocretinergic tone in humans merits further investigation.

Despite all the evidence linking hypocretins and the HPA axis, the actions of glucocorticoids on hypocretin expression are not very well established. It has been demonstrated that bilateral adrenalectomy induces a marked reduction in prepro-Hcrt mRNA levels in the LHA, which is reversed by peripheral dexamethasone treatment130 but not reversed by corticosterone administration.131 Additionally, treating normal rats with dexamethasone does not cause any change in prepro-Hcrt expression11 (our unpublished data). Further work is required to understand the precise relation between hypocretins and glucocorticoids.

Finally, the role of hypocretins in the adrenal medulla is still unclear, although high expression of both hypocretin receptors has been detected.70,75 In vitro studies have provided contradictory results. Using human adrenal slices, no effect of hypocretins on catecholamine release has been demonstrated.75 However, in porcine adrenal cultures74 and in the rat pheochromocytoma cell line PC 12,132 hypocretins decreased basal catecholamine secretion. In vivo studies, using conscious rabbits, have demonstrated that central administration of Hcrt1 increases plasma adrenaline.133 These results suggest that Hcrt1 activates sympathoadrenal outflow and then it could influence cardiovascular physiology.120-122,134,135

4.2. Hypocretins and Growth Hormone Axis

Although the lateral hypothalamic area (LHA) does not belong to the hypophysiotropic area (HTA) of the hypothalamus, there are data to suggest that it is implicated in the regulation of the growth hormone (GH) axis. Both growth hormone-

releasing hormone (GHRH)136"139 and somatostatin (SST)140 have been found in the LHA. Moreover, the LHA contains glucorreceptors that are implicated in the hypoglycaemia-induced GH secretion.141 Finally, LHA destruction in rats results in linear growth reduction possibly related to GH deficiency.5

The relationship between hypocretins and the GH axis is well supported by anatomical and functional evidence. Hypocretin-containing neurons project into the periventricular (PeN), paraventricular (PVN) and arcuate (ARC) hypothalamic nuclei.35,36 Hypocretin receptors are present in all these three nuclei.60,62,64 Furthermore, it has been demonstrated that somatostatin neurons in the PeN express Hcrtr161 and that Hcrt1 stimulates SST release from hypothalamic explants.142 Finally, Hcrt1 has been shown to decrease endogenous rat plasma GH levels101 and to inhibit pulsatile GH secretion in the rat143 (Fig. 2). Intriguingly, in primary culture of sheep somatotropes, it has been found that GH secretion is stimulated by Hcrt1144 and Hcrt2.145,146 These discrepancies, between in vivo and in vitro data, suggest that hypocretin actions on growth hormone release are mainly exerted at hypothalamic level.

Figure 2. Effect of i.c.v. Hcrt1 (3 nmol) treatment on GH plasma levels. Representative plasma GH profiles in normally fed male rats that received either vehicle or Hcrt1.

Recent data suggest that Hcrt1 actions on GH secretion in the rat are mediated by a hypothalamic mechanism involving GHRH and SST neurons. First of all, Hcrt1 and Hcrt2 do not modify GH secretion in rat pituitary cultures119,143 suggesting a lack of a direct effect of hypocretins on pituitary GH release. Secondly, in keeping with this, Hcrt1 failed to modify in vivo GH responses to GHRH, although it markedly blunted GH responses to ghrelin.143 And thirdly, central Hcrt1 treatment induces a decrease in the GHRH mRNA levels in the parvocellular part of the PVN (Fig. 3A) and a GH-dependent stimulatory effect on somatostatin mRNA content in the PeN.147 However, GHRH cells in the ARC, the classically implicated GHRH cell group controlling GH secretion,148,149 is

not regulated by Hcrt1 (Fig. 3B). The implication of PVN GHRH neurons in GH regulation is not well established; however it is supported by anatomical studies, showing that GHRH containing neurons in the PVN project their axons to the fenestrated capillaries of portal vessels in the median eminence.150 Curiously, in spite of the abundant presence of Hcrtrs in the parvocellular part of the PVN and in the ventrolateral ARC, and the presence of GHRH neurons in the PVN and ARC, no study has so far addressed show whether Hcrtrs are express by GHRH neurons. However, these data suggest that Hcrtrs could be expressed in those cells.

NPY plays a role in the regulation of the somatotropic axis by inhibiting GH synthesis and secretion.151152 Since that Hcrt1 treatment increases NPY mRNA expression9 and release104 it is possible that NPY could mediate the inhibitory effects of hypocretins on the GH axis. In relation to this, it is interesting to note that in models with GH deficiency, namely hypophysectomized rats and dwarf rats, Hcrt1 does not affect the mRNA levels of NPY (our unpublished data) suggesting that GH tone could regulate this interaction. Additional work is required to address this issue.

Figure 3. Effect of i.c.v. Hcrtl (3 nmol) treatment on GHRH mRNA levels in the rat hypothalamus. A.

mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus of the described experimental groups. **: P<0.01 vs. vehicle 2 hours. B. GHRH mRNA levels in the arcuate nucleus of the hypothalamus (ARC) of the described experimental groups. The data (mean +/- SEM) are expressed as the percentage of change in relation to the control at each time (vehicle treated animals = 100%).

Figure 3. Effect of i.c.v. Hcrtl (3 nmol) treatment on GHRH mRNA levels in the rat hypothalamus. A.

mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus of the described experimental groups. **: P<0.01 vs. vehicle 2 hours. B. GHRH mRNA levels in the arcuate nucleus of the hypothalamus (ARC) of the described experimental groups. The data (mean +/- SEM) are expressed as the percentage of change in relation to the control at each time (vehicle treated animals = 100%).

Although the physiological relevance of the hypocretin-GH axis interaction is still unclear, several options should be considered. Firstly, it is possible that hypocretins serve as a signal linking metabolic status and GH secretion. It is known that the levels of prepro-Hcrt are markedly influenced by nutritional status, being up-regulated upon fasting.7'8'153 This is particularly interesting, since food-deprivation in the rat is associated with a marked decrease in spontaneous GH secretion.30 Thus, it is possible that a fasting-induced increase in hypocretin-gene expression inhibits GH secretion, constituting an additional mechanism for adaptation of the hypothalamus to nutritional status.

Furthermore, the interaction between hypocretins and GH axis could be important as well in terms of sleep regulation. As it was described above, the hypocretins11 and hypocretin receptors154 are important regulators of the sleep-wake cycle. On the other hand, a growing body of evidence has linked GH secretion to sleep. It is well established that a major burst of GH secretion occurs during non-rapid eyes movement (NREM)

sleep in humans and in several animal species.29,30,149 Furthermore, functional alteration in the somatotropic axis can lead to marked changes in the regulation of sleep-wake activity. Thus, GH-deficiency in children is often associated with decreases in REMs.29,155,156 Also, data obtained in genetic strains of GH-deficient rats, e.g. dwarf Lewis rats157 or in transgenic models,158 linked to an impairment in GH secretion, showed the presence of sleep alterations. Although the nature of the hypothalamic mechanisms through which the different components of GH-axis influences sleep is poorly understood, several possibilities have been put forward. Therefore, GH155,157,159 SST160,161 and GHRH157,162 have been implicated in the regulation of the sleep-wake cycle in humans and rodents. Interestingly, GHRH neurons in the PVN play a major role in sleep control, stimulating REM sleep patterns.163,164 The central inhibitory action of Hcrt1 on REM sleep101,165 could be mediated, at least in part, by decreasing GHRH mRNA expression in the PVN.147

Data in narcoleptic humans link hypocretins and the somatotrope axis. GH secretion in narcolepsy shows alterations, such as abnormal circadian pattern and the decrease of plasma GH peak associated with slow wave sleep at the nocturnal sleep onset.166-168 These alterations are likely mediated by changes in the expression and levels of GHRH in the hypothalamus.147 In this sense, it has been recently demonstrated that hypocretin deficiency disrupts the circadian release of GHRH in narcoleptic patients causing abnormal circadian GH release and promoting sleep alterations.168 All this evidence suggests that the relationship between hypocretins and the somatotropic axis is playing an important role in the regulation of sleep in physiological and pathological states.

Although the actions of hypocretins on GH and GHRH are well established, neither GH deficiency nor GHRH central treatment affect hypocretin gene expression.147 These data indicate that the sleep alterations associated with GH deficiency do not appear to be mediated by changes in hypocretin gene expression and that the central effects of GHRH are mediated through a hypocretin-independent mechanism. Finally, prepro-Hcrt mRNA content in the LHA was unchanged after treatment with ghrelin169 indicating that hypocretins are not involved in the hypothalamic action of this hormone on the GH

4.3. Hypocretins and Gonadal Axis

The role of the lateral hypothalamus (LHA) in the regulation of the reproductive axis was discovered in experiments involving LHA lesions. Animals with LHA lesions show behavioural and feeding patterns very close to those induced by estrogen.170-174 Moreover, electrical stimulation of the LHA in rats affect ovarian steroidogenesis175 and sexual behaviour.176 On the other hand, the LHA has been implicated in the cholinergic blockade of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH).177

Anatomical data suggest that hypocretins play important roles in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis and sexual behaviour. The hypocretin neurons in the LHA project to CNS sites involved in the regulation of the HPG axis. In rat and sheep hypothalamus, Hcrt1 immunoreactive fibers project to the septal (SPO), medial preoptic area (mPO) and the arcuate nucleus-median eminence (ARC-ME) region.38,50,57 Luteinizing hormone-releasing hormone (LHRH) neuronal bodies and their axons terminals are located in all three nuclei (SPO, MPO and ARC-ME).178 Furthermore, hypocretin fibers make contact with LHRH neurons, and Hcrtr1 is expressed in the septal and medial preoptic area of the rat58,60,62 on LHRH neurons.

Curiously, LHRH nerve terminals in the median eminence do not express Hcrtr1.63 Outside the hypothalamus Hcrt1 immunoreactive fibers project to the medulla, the midbrain dorsal raphe, the pontine locus coeruleus, the amygdala, the olfactory bulbs and the central gray matter, all of them important areas in the control of the HPG axis and sexual behaviour.3857 Moreover, Hcrtr1 is expressed at high levels in the monoaminergic locus coeruleus and dorsal/median raphe.58-60

These morphological data have been supported by functional studies showing that central administration of both Hcrt1 and Hcrt2 stimulate or suppress, depending on the presence or the absence of ovarian steroids respectively, the pulsatile secretion of luteinizing hormone (LH) in ovariectomized rats.107 179-181 Such a bimodal mode of action is apparently linked with site-specific effects of Hcrt1 within different hypothalamic nuclei; Hcrt1 being stimulatory at the rostral preoptic area (rPO), and inhibitory at the medial PO or the arcuate nucleus-median eminence.182 It has also been demonstrated that immunoneutralization of Hcrt1 in the brain abolished the LH surge,180 whereas the central administration of the Hcrtr1 antagonist SB 334867-A attenuates this LH surge.182 Moreover, the central administration of Hcrt1 leads to a dose-dependent recovery of fasting-suppressed LH surge.180 These data are important in terms of food intake regulation because they support the idea that in fasting there is a depletion of Hcrt1/Hcrt2 in several hypothalamic areas183 despite the fact that prepro-Hcrt mRNA levels are strongly increased after fasting.78153

The effects of hypocretins on the gonadal axis appear to be entirely mediated through the stimulation of LHRH secretion108 179 182 because either Hcrt1 or Hcrt2 failed to inhibit LH secretion from primary cultures of rat pituitary.119 However, Hcrt1 inhibited LHRH-stimulated LH release in dispersed pituitaries from proestrous female (but not from male) rats.108 The stimulatory action of hypocretins on LHRH is mediated by an NPY-dependent mechanism, as evidenced by the treatment with selective NPY Y1-receptor antagonist that abolishes the stimulatory effect of Hcrt1 on LHRH release in vitro.108 On the other hand, recent evidence suggests that NPY regulates hypocretin neurons through the NPY Y4 receptor in a postsynaptic fashion.63 106 All this evidence suggests that the NPY-hypocretin circuit plays a major role in the hypothalamic regulation of the gonadal axis.

Notably, hypocretins exert their endocrine action at testicular level as well. Both prepro-Hcrt and the Hcrtr1 are expressed in rat testis7,66,70,78 and Hcrt1 stimulates testosterone secretion in vitro and in vivo, further suggesting a role for the hypocretin system in the control of the male reproductive axis78 (Fig. 4). In addition, considering the predominant location of Hcrtr1 in the tubular compartment within the rat testis, we are currently investigating additional biological actions of Hcrt1 in the direct control of the seminiferous epithelium.

The physiological relevance of the hypocretin system in reproductive function could be related to the adaptation of the gonadal axis to changes in the metabolic state. It is well known that variations in the metabolical status of the animal are linked to changes in reproductive function.178 Hypocretin neurons in the lateral hypothalamic area express the long form of the leptin receptor in rat184 185 and sheep,186 as well as the NPY Y4 receptor in the rat.63 106 On the other hand, hypocretin neurons sense the nutritional status of the animal, being regulated by leptin,8 187 188 glucose,153 189-192 pancreatic polypeptide (PP)106 and visceral signals,153 192 Taken together, these data suggest that hypocretin neurons provide an important link between energy balance and reproductive function by integrating central (NPY) and peripheral signals (glucose, leptin, PP).

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