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Acta Physiol 2010, 198, 361–371

REVIEW

Interactions of orexins/hypocretins with adrenocortical functions S. M. Kagerer and O. Jo¨hren Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lu¨beck, Lu¨beck, Germany

Received 2 April 2009, revision requested 4 June 2009, revision received 18 June 2009, accepted 14 August 2009 Correspondence: O. Jo¨hren, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lu¨beck, Ratzeburger Allee 160, D-23538 Lu¨beck, Germany. E-mail: [email protected]

Abstract The neuropeptides orexin A and B (hypocretin-1 and -2) are involved in numerous central regulation processes such as energy homeostasis, sleeping behaviour and addiction. The expression of orexins and orexin receptors in a variety of tissues outside the brain and the presence of orexin A in the circulation indicate the existence of an additional peripheral orexin system. Furthermore, it is well established that orexins exert an influence on the regulation of the hypothalamus–pituitary–adrenal axis, acting both on its central and peripheral branch. In rat and human adrenal cortices the expression of both orexin receptors has been verified with a predominance of OX2R. The local expression of orexin receptors was observed to be gender specific and to be modified by plasma glucose and insulin concentrations, nutritional status as well as gonadal steroids. Various studies consistently demonstrated orexin A to enhance glucocorticoid secretion of rat and human adrenal cortices, while orexin B was found to be either less potent or ineffective. On the contrary, the influence of orexins on adrenocortical aldosterone production and cell proliferation is still more controversial. Recent findings indicate that orexins stimulate adrenocortical steroidogenesis by augmenting transcription of selective steroidogenic enzymes and proteins such as steroidogenic acute regulatory protein. Both, Gq and Gs, signalling pathways with a downstream activation of MAP kinases appear to be involved in this regulation. Keywords adrenal cortex, glucocorticoids, hypocretin, orexin, OX1 receptor, OX2 receptor.

The neuropeptides orexin A (OxA) and orexin B (OxB), also referred to as hypocretin-1 and -2, were isolated as novel endogenous ligands for an orphan G-proteincoupled receptor (Sakurai et al. 1998). Both peptides originate from proteolytic cleavage of the common precursor prepro-orexin, whose cDNA is identical with that of the independently cloned prepro-hypocretin (Gautvik et al. 1996, de Lecea et al. 1998). The amino acid sequences of the 33-residue peptide OxA and the 28-residue peptide OxB are encoded by a single gene localized on human chromosome 17q21 and share 46% homology. While both peptides are amidated at their C terminals only OxA possesses an N-terminal pyroglut-

amyl residue and two intrachain disulfide bridges between Cys6–Cys12 and Cys7–Cys14, which are essential for its stability and binding characteristics (Sakurai et al. 1998). The receptors via which orexins were found to mediate their actions were termed orexin type 1 and type 2 receptors (OX1R and OX2R) and show 64% correspondence in their amino acid sequences. OxA binds with almost equal affinity to both receptors, whereas OxB seems to be selective for OX2R (Sakurai et al. 1998). Several studies report that orexin receptors interact with multiple G proteins, thus enabling the neurotransmitters to provoke finely adjustable tissue and cell-specific intracellular responses

2009 The Authors Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02034.x

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Orexins and adrenocortical functions

Æ S M Kagerer and O Jo¨hren

(Kukkonen et al. 2002, Holmqvist et al. 2005, Karteris et al. 2005). In the beginning, research on orexins largely focused on their central effects, as they were first discovered in the rat and human brains (de Lecea et al. 1998, Sakurai et al. 1998). The expression of prepro-orexin was found to be restricted to neurones of the lateral hypothalamic area, a region classically implicated in feeding behaviour (Gautvik et al. 1996). Accordingly, intracerebroventricular (ICV) administrations of OxA and OxB were shown to regulate food intake and energy homeostasis in rats (Lubkin & Stricker-Krongrad 1998, Sakurai et al. 1998). Yet, the projections of hypothalamic orexin-containing neurones to widespread areas of the central nervous system such as the locus coeruleus, spinal cord, arcuate nucleus and limbic system as well as the corresponding expression of orexin receptors suggested additional central effects (Peyron et al. 1998, Marcus et al. 2001). Further studies revealed a critical role for orexins in the mediation of autonomic and cardiovascular functions, sleep–wake regulation, central reward processes and drug addiction mechanisms (Matsuki & Sakurai 2008). Moreover, orexins interact with the neuroendocrine system and were found to alter plasma concentrations of luteinizing hormone, somatotropin, prolactin and thyroid-stimulating hormone after central administration (Pu et al. 1998, Hagan et al. 1999, Jones et al. 2001). Besides the demonstrated central effects, strong evidence of an extensive peripheral orexin system has accumulated over the past years. The expression of orexins and their receptors was identified using reversetranscription PCR and immunohistochemistry in numerous tissues including the pancreas, gonads, kidney, intestine, adrenal gland and adipose tissue (Kirchgessner & Liu 1999, Jo¨hren et al. 2001, Nanmoku et al. 2002, Nakabayashi et al. 2003, Digby et al. 2006, Silveyra et al. 2007b). Consequently, it has been confirmed, that orexins affect intestinal motility and secretion, adrenomedullary catecholamine release, testicular androgen production, ovulation, lipid metabolism in adipose cells as well as pancreatic insulin and glucagon secretion in vitro or when injected peripherally (Kirchgessner & Liu 1999, Nanmoku et al. 2002, Ouedraogo et al. 2003, Barreiro et al. 2004, Digby et al. 2006, Silveyra et al. 2007b). However, for many peripheral organs, the particular role of orexins remains to be elucidated. Furthermore, it still needs to be determined if any of the peripheral orexin expression sites might be a sufficient source for the concentrations of OxA detected in plasma, and whether orexins are capable of acting in an autocrine or paracrine fashion (Arihara et al. 2001, Jo¨hren et al. 2001, Ouedraogo et al. 2003). Alternatively, OxA might be released into circulation from OxA-immunoreactive fibres present in

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the median eminence and neurohypophysis, but plasma levels seem to be at least partially independent of central orexin production (Date et al. 2000, Dalal et al. 2001, Heinonen et al. 2008).

Central orexins and the hypothalamus– pituitary–adrenal axis Various studies report increased plasma concentrations of adrenocorticotropin (ACTH) and corticosterone after ICV injections of orexins, suggesting a central effect of the peptides on the activity of the hypothalamus–pituitary–adrenal (HPA) axis (Hagan et al. 1999, Ida et al. 2000, Jaszberenyi et al. 2000, Kuru et al. 2000, Al Barazanji et al. 2001, Russell et al. 2001, Brunton & Russell 2003, Samson et al. 2007). This effect is by all appearances mediated via the most potent endogenous stimulators of ACTH production, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), produced in the nucleus paraventricularis of the hypothalamus. Thus, orexins led to a rise in plasma levels of AVP and enhanced the release of CRH from hypothalamic explants but were unable to elevate basal ACTH release from cells of the anterior lobe of the pituitary in vitro despite the local expression of both orexin receptors (Date et al. 2000, Matsumura et al. 2001, Russell et al. 2001, Samson & Taylor 2001). Moreover, centrally injected OxA stimulated the expression of CRH, AVP and c-fos mRNA in the nucleus paraventricularis where orexin immunoreactive fibres and orexin receptors are present (Date et al. 1999, Kuru et al. 2000, Al Barazanji et al. 2001, Marcus et al. 2001). Lastly, the rise in plasma corticosterone after ICV administration of OxA or OxB was completely abolished by a CRH receptor antagonist (Jaszberenyi et al. 2000). These findings, together with the activation of orexincontaining neurones in the hypothalamus by cold exposure and immobilized stress but also by incubation with CRH indicate that orexins are interdependently involved in the central circuits of stress-induced HPA axis activation (Sakamoto et al. 2004, Winsky-Sommerer et al. 2004, Koob 2008).

Orexin receptor expression in the adrenal cortex The first investigations of orexin receptor expression in the rat adrenal gland utilizing RT-PCR and immunohistochemistry reported the presence of both receptors but located them exclusively in the adrenal medulla (Lopez et al. 1999) (Table 1). By contrast, subsequent studies have constantly demonstrated the simultaneous expression of OX1R and OX2R in adrenal cortex homogenates and freshly dispersed or cultured adreno-



Adrenocortical cells

ND

) + + + + ) + + ND +

+

)

)

) ) + + + ND +

Gs, Gq, cAMP, IP3 Gs, Gq, Gi, cAMP, IP3 ND ND ND ND cAMP, PKA ND cAMP, PKA

cAMP

ND ND ND ND ND ND Gs, Gq, Gi, Go, cAMP, PKA cAMP, PKA p42/44, p38 cAMP, PKA ND ND ND ND ND ND

ND

ND + + ND ND ND ND ND ND

+ ND + ) + + +

Signal transduction

+

ND + + ND ND ND ND ND ND

+ ND + ) + ) +

OX2R

ND ND ND ND C ›, A M C ›, PR › C ›, PR ›

ND

ND

ND

C ›, A ›

C ›, A M PR ›/fl C› C ›, A M C ›, A ›/fl CM C ›, A › C ›, A › C ›, PR ›/fl

ND CM ND ND ND ND ND

Effect

ISH, IHC ISH, IHC IHC RT-PCR, qPCR, WB RT-PCR, EI, HPLC, RIA HPLC, RIA, CPA RT-PCR, EI, HPLC, RIA

RT-PCR, WB, PLA, RIA

RT-PCR, WB, PLA, RIA

qPCR

RT-PCR, SB, EIA, AIA

HPLC, RIA, EI RT-PCR, EI,CPA RT-PCR, EI, HPLC, RIA s.c., HPLC, RIA s.c., HPLC, RIA i.p., FA s.c., HPLC, RIA s.c., HPLC, RIA s.c., HPLC, RIA, CPA

qPCR FA RT-PCR IHC RT-PCR ISH qPCR, WB, PLA, RIA

Methods

Karteris et al. (2001) Randeva et al. (2001) Blanco et al. (2002) Spinazzi et al. (2005a) Mazzocchi et al. (2001) Spinazzi et al. (2005a) Ziolkowska et al. (2005)

Randeva et al. (2001)

Karteris et al. (2001)

Zhang et al. (2005)

Nanmoku et al. (2002)

Malendowicz et al. (1999b) Spinazzi et al. (2005b) Ziolkowska et al. (2005) Malendowicz et al. (1999b) Malendowicz et al. (1999a) Jaszberenyi et al. (2000) Nowak et al. (2000) Malendowicz et al. (2001a) Malendowicz et al. (2001b)

Jo¨hren et al. (2001) Jaszberenyi et al. (2000) Lopez et al. (1999) Lopez et al. (1999) Malendowicz et al. (2001a,b) Jo¨hren et al. (2001, 2004) Karteris et al. (2005)

Reference

S M Kagerer and O Jo¨hren

Cortex dissections

Pigs Adrenocortical cells Sheep Whole adrenals Humans Whole adrenals

In vivo

Adrenocortical cells

Cortex dissections

Rats Whole adrenals

OX1R

Receptor subtype

Table 1 Receptor expression, signal transduction and effects of orexins in the adrenal cortex

Acta Physiol 2010, 198, 361–371

Æ Orexins and adrenocortical functions

363

364

Gs, Gq, Gi, p42/44, p38, PKA, PKC, Ca2+ + +

PKC, p42/44 ND

Æ S M Kagerer and O Jo¨hren +, detected; ), not detected; ND, no data given; IHC, immunohistochemistry; ISH, in situ hybridization, RT-PCR, reverse-transcriptase PCR; qPCR, quantitative PCR; WB, Western blot; C, corticosterone/cortisol; A, aldosterone; PR, cell proliferation; PLA, photoaffinity labelling assay; EI, enzyme inhibition assay; CPA, cell proliferation assay; DNGA, dominant-negative G protein assay; HPLC, high-performance liquid chromatography; RIA, radio immune assay; SB, Southern blot; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; AIA, automated immune assay; p42/44, p42/44 MAP kinase; p38, p38 MAP kinase; CA, calcium assay; s.c., subcutaneous; i.p., intraperitoneal; protein kinase A (PKA); protein kinase C, PKC; cAMP, cyclic AMP; IP3, inositol triphosphate.

Ramanjaneya et al. (2008, 2009) RT-PCR, qPCR, WB, EI, DNGA

Grabinski (2006) qPCR, EI, RIA

Jo¨hren et al. (2006b) qPCR, ELISA

C ›, HSD3B2 › CYP21 › HSD3B2 › CYP21 › StAR › ND +

ND

OX1R

+ NCI H295R cell line

Table 1 (Continued).

Receptor subtype

OX2R

Signal transduction

Effect

Methods

Reference


Acta Physiol 2010, 198, 361–371

cortical cells from rats using RT-PCR, quantitative realtime PCR or Western blotting (Malendowicz et al. 2001a,b, Karteris et al. 2005, Spinazzi et al. 2005b, Ziolkowska et al. 2005). Using in situ hybridization our group detected only OX2R mRNA in rat adrenal gland sections. It was distributed over all three layers of the adrenal cortex but undetectable in the medulla (Jo¨hren et al. 2001, 2004). Quantitative PCR verified high expression levels of OX2R mRNA and only low levels of OX1 receptor mRNA in adrenal dissections and expression of OX2R mRNA was found to be significantly higher in adrenal glands of male rats than in females (Jo¨hren et al. 2001, 2004). This apparent sex dimorphism may be due to different functional roles of the orexin system in males and females, as the expression of orexin receptors in the hypothalamus, pituitary, adrenals and ovaries has also been observed to be dependent on female sex hormone status (Jo¨hren et al. 2003, Silveyra et al. 2007a,b). In ovine and porcine adrenal glands only the detection of OX1R has been reported so far (Nanmoku et al. 2002, Zhang et al. 2005). Two of the earliest studies investigating orexin receptor expression in human adrenals found that the cortex was exclusively provided with OX2R using combined immunohistochemical methods, RT-PCR and Western blotting (Karteris et al. 2001, Randeva et al. 2001). Conversely, Blanco et al. (2002) detected immunoreactivity only for OX1R in the cortex of human adrenal gland sections. More recent studies evidenced the presence of both receptor subtypes in either freshly dispersed or cultured human adrenocortical cells, including the NCI H295R cell line, or adrenocortical homogenates using RT-PCR and Western blotting (Mazzocchi et al. 2001, Spinazzi et al. 2005a, Ziolkowska et al. 2005, Ramanjaneya et al. 2008). Both receptors seem to be distributed over all three functional zones of the adrenal cortex, but as in rat specimens quantitative real-time PCR demonstrated in NCI H295R cells overall mRNA levels of OX2R to be many times higher than that of OX1R (Mazzocchi et al. 2001, Randeva et al. 2001, Jo¨hren et al. 2006b, Ramanjaneya et al. 2008). Intriguingly, orexin receptors in the adrenal cortex have been observed to be differentially regulated by plasma glucose and insulin concentrations as well as by nutritional status (Karteris et al. 2005, Jo¨hren et al. 2006a). In cortisol-secreting adenomas, mRNA levels of orexin receptors were markedly elevated, indicating a possible parallel regulation of orexin receptors and cortisol release (Spinazzi et al. 2005a). Thus, orexin receptor expression might also increase in other situations of high glucocorticoid production, such as stressful conditions. These findings, together with the aforementioned sex-dependent expression of orexin


Acta Physiol 2010, 198, 361–371

receptors and potential yet unknown factors that may also regulate orexin receptor expression, might explain discrepancies between the illustrated results to a certain extent. Still, it is also possible that the presence and distribution of orexin receptors in the adrenal cortex differ between species. The fact that findings conflict, especially when histochemical techniques were applied, also casts doubts on the specificity of the methods used (Lopez et al. 1999, Karteris et al. 2001, Randeva et al. 2001, Blanco et al. 2002). Furthermore, results obtained using endpoint analysing RT-PCR at high cycle numbers should be interpreted with caution, as reliable results are only produced in the exponential phase of amplification (Raeymaekers 1995, Jo¨hren et al. 2001, 2004, Karteris et al. 2005, Ramanjaneya et al. 2008). Taking into account the results of all groups, the mechanisms of regulation, as well as the potential differences in specificity of detection methods, it seems most likely that both orexin receptor subtypes are expressed in rat and human adrenal cortex tissue with a predominance of OX2R.

Local effects of orexins on adrenocortical functions One of the first in vivo studies addressing the effect of orexins on adrenal function described an increase in both ACTH and corticosterone plasma levels 1–2 h after a single systemic administrations of OxA in rats (5 or 10 nmol kg)1), while OxB had no stimulating effect (Malendowicz et al. 1999a). In a comparable setting, Nowak et al. (2000) found both orexins (10 nmol kg)1) to elevate plasma corticosterone, but only OxA evoked an increase in ACTH plasma concentrations. These findings are in accordance with the notion that OxA is capable of rapidly crossing the blood–brain barrier, while OxB is quickly degraded in the circulation and the results might therefore at least partially be explained by a central activation of the HPA axis (Kastin & Akerstrom 1999). To date, though, compelling evidence clearly demonstrates an additional direct action of orexins on the adrenal cortex without the involvement of the HPA axis. Prolonged subcutaneous administrations of either peptide (20 ng kg)1) for 7 days were found to increase only corticosterone but not ACTH plasma levels in rats when measured 1 h after the last application (Malendowicz et al. 2001a). The enhancing effect of subcutaneously administered OxA (5 or 10 nmol kg)1) on adrenal corticosterone release after 1–4 h was confirmed by further in vivo studies with 20-day-old and adult rats (Malendowicz et al. 1999b, 2001b). Subsequent in vitro investigations consistently validated the direct secretagogue action of OxA (10)10–10)6 m) on the glucocorticoid release of rat, porcine and human



freshly dispersed or cultured adrenocortical cells after 1 or 24 h of incubation, while OxB was mostly observed to be ineffective (Malendowicz et al. 1999b, Mazzocchi et al. 2001, Nanmoku et al. 2002, Spinazzi et al. 2005a, Ziolkowska et al. 2005). In cells of the pluripotent human adrenocortical cell line NCI H295R, we observed a stimulating effect of OxA (10)6 m) on cortisol synthesis after 24 h of treatment (Jo¨hren et al. 2006b). This late rise in cortisol in comparison with the results of other groups might possibly be explained by biphasic regulation, as it has been reported for steroidogenesis before (Simpson & Waterman 1988). It has to be recalled, though, that cultured cells might not completely reflect the physiology of freshly dispersed cells or, of course, conditions in vivo. Yet, after time intervals of only 30 min no measurable effects of orexins (280 pmol) were detected either on corticosterone release of rat adrenal slices in vitro or on plasma glucocorticoid levels after single intraperitoneal injections in vivo (Jaszberenyi et al. 2000). The dose-dependent secretagogue effect of OxA on adrenocortical cells was evidenced to enhance only basal but not maximally ACTH-stimulated (10)9 m) glucocorticoid release and, accordingly, an ACTH receptor antagonist blunted the corticosterone response only to ACTH but not to orexin (Malendowicz et al. 1999b, Mazzocchi et al. 2001). Thus, orexins appear to exert their effect on the adrenal cortex independently of the local response to ACTH but might share the same intracellular transduction mechanisms with the ACTH receptor. The cortisol response to OxA was found to be even higher in cells derived from human adrenocortical adenomas than in normal adrenocortical cells, indicating the important role of orexins in adrenocortical cortisol production and perhaps also in cell proliferation (Spinazzi et al. 2005a). Several groups also noticed a stimulating effect of OxA or OxB on rat and porcine adrenal aldosterone production (Malendowicz et al. 1999a, 2001a, Nowak et al. 2000, Nanmoku et al. 2002). In contrast to the effect of orexins on glucocorticoid release, though, the effect on adrenal aldosterone production is still more controversial, as some groups failed to detect an enhancing effect of orexins on adrenal aldosterone release (Malendowicz et al. 1999a,b, Mazzocchi et al. 2001). In spite of the research on the effects of orexins on adrenocortical steroid secretion, little is known yet about the mechanisms involved therein. In a recent study, Ramanjaneya et al. (2008) described an upregulation of steroidogenic acute regulatory (StAR) protein, the first rate-limiting protein in steroidogenesis, in NCI H295R adrenocortical cells after incubation with OxA or OxB (10)7 m) for 4 or 24 h. To further characterize the processes involved in the effect of OxA


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on adrenal steroid production our group analysed the influence of OxA (10)6 m) on the gene expression of all essential steroidogenic enzymes in NCI H295R cells using quantitative real-time PCR. The mRNA levels of 3b-hydroxysteroid dehydrogenase type II (HSD3B2) and 21-hydroxylase (CYP21) were markedly increased after 12 h of incubation, while there was no difference in mRNA concentrations of the cholesterol side-chain cleavage cytochrome P450 (CYP11A) (Jo¨hren et al. 2006b). Preliminary results of our group indicate that this rise in HSD3B2 and CYP21 mRNA concentrations in OxA-treated H295R cells has to be attributed to an increased transcription rather than an alteration of mRNA stability (Kagerer SM & Jo¨hren O, unpublished data). These findings are consistent with the widely accepted conception that chronic regulation of steroidogenesis is controlled at the transcriptional level of steroidogenic genes (Simpson & Waterman 1988). The zone-specific regulation of steroidogenic enzyme expression presumably causes the functional zonation of the adrenal cortex, which is associated with adrenocortical cell proliferation and differentiation. This, together with the described upregulation of orexin receptors and detection of prepro-orexin mRNA in cortisol-secreting adenomas, suggests that orexins might also be involved in the regulation of proliferative activity of adrenocortical cells (Spinazzi et al. 2005a). Accordingly, both orexins (10 nmol kg)1) raised the metaphase index of zona glomerulosa cells of immature rats after three subcutaneous injections and either both orexins (10)8 m) or only OxA (10)10–10)6 m) was found to enhance the proliferation rate of cultured human adrenocortical cells (Malendowicz et al. 2001b, Spinazzi et al. 2005a,b). On the contrary, both orexins (10 nmol kg)1) lowered the metaphase index of regenerating rat adrenals at day 5 of regeneration, while there was no measurable effect on the metaphase index at day 8, and OxB (10)8–10)6 m) diminished the proliferation rate of cultured human adrenal cortex cells after treatment for 24 h (Malendowicz et al. 2001b, Spinazzi et al. 2005b). These results indicate that orexins, which have been detected in rat and human circulation, reach the adrenal gland through the bloodstream and exert their actions via the locally expressed orexin receptors. The receptor subtypes involved in the mentioned effects of orexins on adrenocortical functions and cell growth, however, remain to be determined. On the one hand, OX1R has been proposed by two studies to be the receptor subtype predominantly involved in the effect of OxA on glucocorticoid secretion or StAR expression (Ziolkowska et al. 2005, Ramanjaneya et al. 2008). On the other hand, various studies confirm expression of OX2R to be many times higher than that of OX1R in the adrenal cortex, thereby suggesting a predominance

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Acta Physiol 2010, 198, 361–371

of this receptor subtype in the mediation of local effects of orexins. Yet, OxB, which is selective for OX2R, was mostly found to have a lower potency than OxA on adrenal steroid production, although this might possibly be ascribed to its shorter metabolic half-life in comparison with OxA (Kastin & Akerstrom 1999) and receptor affinities to OxA or OxB may also vary between different species. Spinazzi et al. (2005b) reported the proliferogenic effect of OxA on human adrenocortical cells to be cancelled by OX1R blockade, while antiproliferogenic effects of both orexins were abolished by OX2R blockage. Intriguingly, the illustrated effects might also be mediated via a heterodimer of either OX1R/OX2R or a heterodimer of OX1R or OX2R with a yet undetermined third-party molecule, as a hypersensitizing crosstalk of OX1R with cannabinoid receptor CB1 has been demonstrated before (Hilairet et al. 2003). It remains to be ascertained whether such a heterodimerization of orexin receptors has any physiological relevance in the adrenal cortex and if any other molecules might have agonistic potency on such a heterologous receptor.

Signal transduction of orexin receptors in the adrenal cortex The bulk of evidence indicates that both OX1R and OX2R are capable of coupling to various G proteins, depending on the expression site and ligand concentrations (Kukkonen et al. 2002, Holmqvist et al. 2005). Regarding the coupling of orexin receptors in the adrenal cortex, differing results have been obtained so far. Several studies report the effect of OxA on glucocorticoid release of rat or human adrenocortical cells to be cancelled by either protein kinase (PK) A or adenylate cyclase (AC) inhibitors, suggesting a coupling of the involved receptor subtype to Gs (Malendowicz et al. 1999b, Mazzocchi et al. 2001, Ziolkowska et al. 2005). Accordingly, OxA (10)8–10)6 m) was found to evoke an increase in cyclic AMP (cAMP) production of cultured or freshly dispersed adrenal cortex cells within 30–60 min (Malendowicz et al. 1999b, Mazzocchi et al. 2001, Nanmoku et al. 2002, Ziolkowska et al. 2005). In a photoaffinity labelling assay for Ga subunits Karteris et al. (2001) revealed orexin receptors in human foetal adrenal membranes to be capable of coupling to Gs and Gi but not to Gq or Go after incubation with OxA (10)7 m). Applying the same technique in a subsequent study with adrenal cortices from adult rats, they found local orexin receptors to be capable of coupling to all of Gq, Gs, Gi and Go depending on the nutritional state of the animal. Subsequent second messenger studies confirmed a


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dose-dependent rise in cAMP and inositol triphosphate (IP3) in response to treatment with OxA (10)8–10)7 m) (Karteris et al. 2005). In adrenal membranes from human adult specimens Randeva et al. (2001) confirmed OxA (10)7 m) to increase the labelling of Gq, Gs and, to a lesser degree, Gi but not of Go also utilizing photoaffinity labelling techniques and also validated a consequent rise in cAMP and IP3. Correspondingly, the effect of OxA or OxB (10)7 m) on StAR expression in human NCI H295R adrenal cells was reduced by inhibition of either Gq, Gs or Gi in a dominant-negative G protein analysis and suppression of Gq showed the most diminishing capacity. Furthermore, both protein kinase A (PKA) and PKC inhibitors decreased the response of StAR expression to either OxA or OxB (10)7 m) in a subsequent signalling pathway study with the effect of PKC inhibition being more pronounced. Interestingly, both mitogen-activated protein (MAP) kinase isoforms p42/44 (also known as ERK1/2) and p38 were also found to be essential for the transduction of orexin-stimulated StAR expression (Ramanjaneya et al. 2008). Following investigations using Western blotting confirmed a dose-dependent rapid phosphorylation of p42/44 and p38 MAP kinases in NCI H295R cells in response to OxA or OxB. The activation of both MAP kinases was found to be mediated via Gq and Gs, while p38 phosphorylation also involved Gi-mediated signalling to a lower extent (Ramanjaneya et al. 2009). In collaboration with Ramanjaneya et al. (2009) our group detected an instantaneous rise in intracellular Ca2+ concentrations in response to orexins in the same cell line using the fluorescent Ca2+ indicator Fluo-4. Accordingly, we perceived the increase in HSD3B2 mRNA levels in response to OxA to be reduced by inhibition of PKC, while PKA inhibition had no effect on HSD3B2 expression nor did OxA stimulate cAMP production in NCI H295R cells. Moreover, the rise in HSD3B2 mRNA concentration was found to be prevented by inhibition of the p42/44, but not the p38 MAP kinase pathway (Grabinski 2006). The proliferogenic effect of OxA on adrenocortical cells via OX1R was reported to be mediated via p42/44 MAP kinases, while antiproliferogenic effects of both orexins via OX2R involved the p38 MAP kinase pathway (Spinazzi et al. 2005b). The outlined findings suggest that the orexin receptor subtype mediating the rise in glucocorticoid production in the adrenal cortex is capable of coupling to both Gq and Gs, while coupling to Gi or Go is less well established. Subsequent signal transduction studies confirmed the involvement of Ca2+, cAMP and IP3 as second messengers, but MAP kinase signalling pathways are likely to play an additional role, at least in the effects on proliferative activity. An involvement of p42/44 and p38 MAP kinases in the expression of HSD3B2 and StAR has also been reported



recently in response to angiotensin II in rat glomerulosa cells (Otis & Gallo-Payet 2007). However, coupling to a certain G protein might be related to a specific cell type and to ligand concentrations, as described for orexin receptors in other tissues. As coupling of orexin receptors in adrenal cortex cells was demonstrated to be dependent on the nutritional state, it might also be influenced by other metabolic or endocrine conditions (Karteris et al. 2005). Furthermore, a possible participation of orexins in adrenocortical cell proliferation and differentiation cannot be ruled out, wherefore the involved receptor subtype and its coupling might also differ in accordance with the differentiation status of the examined adrenal cortex tissue.

Model for orexin signalling in the adrenal cortex The outstanding roles of PKA and PKC in the regulation of adrenocortical steroidogenesis have long been known as they are considered the major signalling pathways for ACTH and angiotensin II receptors in the zonae glomerulosa and fasciculata (Foster 2004). These hormones function as the main stimulators of corticoid synthesis through an activation of transcriptional activity of steroidogenic genes (Payne & Hales 2004). Orexins appear to enhance adrenocortical steroidogenesis via an increased transcription of a subset of steroidogenic genes, most likely via locally expressed OX2R. Orexin receptors are capable of activating both PKA and PKC pathways through coupling to Gs or Gq and possibly also involving downstream p42/44 and

Figure 1 Model for orexin receptor signal transduction in adrenocortical cells, where OX2R is predominant. Orexin receptors are capable of coupling to both Gq and Gs G proteins and thereby induce PKC or PKA signalling pathways respectively. Orexins were observed to stimulate steroidogenesis via increased transcription of selective steroidogenic genes and to influence cell proliferation in adrenal cortex cells.


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p38 MAP kinases. These pathways have been reported to activate the expression of the described steroidogenic genes via some of their most important transcription factors, including SF-1, CREB/M, Nur77 and GATA-4. Thus, the Gs-mediated signal transduction in steroidogenic cells has not only been observed to lead to downstream activation of MAP kinases but also to involve all four mentioned transcription factors as intermediate elements (Tremblay et al. 2002, Hirakawa & Ascoli 2003, Val et al. 2003, Martin & Tremblay 2005, Kempna et al. 2007). The Gq-linked pathway, on the other hand, has also been proposed to lead to the activation of the Ras/Raf/MEK/MAP kinase system but only to involve Nur77 and SF-1 as transcription factors in the regulation of steroidogenic gene expression in steroidogenic cells (Leers-Sucheta et al. 1997, Tian et al. 1998, Nogueira et al. 2009). While CYP21, HSD3B2 and StAR have all been identified as target sites for SF-1 and Nur77, GATA-4 only enhances HSD3B2 and StAR expression and a CREB/M response element has been localized in the promoter of StAR (Chang & Chung 1995, Manna et al. 2002, Tremblay et al. 2002, Martin & Tremblay 2005, Martin et al. 2005, 2008). In conclusion, we propose a model for pathways linking orexin receptor response to increased transcriptional activities of steroidogenic genes in adrenocortical cells (Fig. 1). Future studies might clarify the nature of these signalling pathways further and evaluate the relevance of other well-known transcription factors of the depicted enzymes, such as STAT5, DAX-1 or Sp1, in this proposed signalling transmission.

Outlook Accumulating evidence substantiates an interaction of orexins with adrenocortical functions via locally expressed orexin receptors and suggests possible intracellular signal transduction pathways that may mediate these functions. Although this interaction of orexins with adrenocortical functions, especially with glucocorticoid production, is established now, its functional role in view of the entire orexin system and its physiological relevance remain uncertain. Recently, findings have accumulated that assign the adrenal cortex a key role in energy homeostasis and its pathophysiological conditions, associating adrenal steroid production with obesity, metabolic syndrome and polycystic ovarian syndrome (Marouliss & Triantafillidis 2006, Krug & Ehrhart-Bornstein 2008). Comparable with their interactions with the HPA axis, orexins seem to be involved in the control of energy homeostasis not only in the central nervous system, but also by direct interactions with peripheral organs. Thus, orexins modulate pancreatic insulin and glucagon release, motility and

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secretion of the gastrointestinal tract as well as lipolysis in adipocytes (Heinonen et al. 2008). Moreover, the functioning of the orexin system in the adrenal cortex has been evidenced to be modulated by metabolic conditions and OxA plasma concentrations are elevated in fasted rats (Ouedraogo et al. 2003, Karteris et al. 2005). Furthermore, food deprivation has been observed to induce HPA axis activity in rodents (Akana et al. 1994). A dual involvement in HPA axis functions and metabolic conditions is already established for other hypothalamic peptides, such as leptin and neuropeptide Y, which are considered major mediators in energy homeostasis (Rohner-Jeanrenaud et al. 1996, Nussdorfer & Gottardo 1998). Thus leptin, which reduces food intake and body weight, inhibits glucocorticoid secretion from the adrenal cortex (Glasow & Bornstein 2000). Conceivably, orexins, together with leptin, might comprise a counter-regulatory system that controls body weight and energy homeostasis through the regulation of adrenocortical steroid production.

Conflict of interest The authors declare no conflicts of interest. The authors thank Christine Eichholz for her excellent technical assistance.

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