A molecular network map of orexin-orexin receptor signaling system

Abstract

Orexins are excitatory neuropeptides, which are predominantly associated with feeding behavior, sleep-wake cycle and energy homeostasis. The orexinergic system comprises of HCRTR1 and HCRTR2, G-protein-coupled receptors of rhodopsin family and the endogenous ligands processed from HCRT pro-hormone, Orexin A and Orexin B. These neuropeptides are biosynthesized by the orexin neurons present in the lateral hypothalamus area, with dense projections to other brain regions. The orexin-receptor signaling is implicated in various metabolic as well as neurological disorders, making it a promising target for pharmacological interventions. However, there is limited information available on the collective representation of the signal transduction pathways pertaining to the orexin-orexin receptor signaling system. Here, we depict a compendium of the Orexin A/B stimulated reactions in the form of a basic signaling pathway map. This map catalogs the reactions into five categories: molecular association, activation/inhibition, catalysis, transport, and gene regulation. A total of 318 downstream molecules were annotated adhering to the guidelines of NetPath curation. This pathway map can be utilized for further assessment of signaling events associated with orexin-mediated physiological functions and is freely available on WikiPathways, an open-source pathway database (https://www.wikipathways.org/index.php/Pathway:WP5094).

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12079-022-00700-3.

Introduction

Orexins are excitatory neuropeptides, which have a primary role in the regulation of feeding behavior, sleep or wakefulness and energy homeostasis (Yamanaka et al. 1999; Sutcliffe and de Lecea 2000; Willie et al. 2001). In 1998, the presence of orexins was independently identified by two groups; Sakurai et al. 1998 who named it “orexin” after the Greek word orexis, which means appetite and de Lecea et al. 1998 who called it “hypocretin” due to its expression in the hypothalamus as well as sequence homology with gut peptide hormone secretin. Orexins, also known as hypocretins (Hcrt), are produced by the neurons of the dorsolateral and perifornical area of the hypothalamus (de Lecea et al. 1998; Sakurai et al. 1998). While the cell bodies of orexinergic neurons are confined to the hypothalamus, dense projections are found all over the brain in regions such as the limbic system, thalamus and brain stem, implying complex functions (Peyron et al. 1998). Monoaminergic neurons are innervated by orexin neurons, they specifically activate noradrenergic neurons in the locus coeruleus, dopaminergic neurons in the ventral tegmental region, and histaminergic neurons in the tuberomammillary nucleus (Hagan et al. 1999; Yamanaka et al. 2002; Kohlmeier et al. 2008).

The HCRT gene, which has two exons and one intron encoding a 131-amino-acid precursor polypeptide, is located on chromosome 17q21-q24 (Sakurai et al. 1999). The two subtypes of orexin which are synthesized from this single pro-hormone preprohypocretin/prepro-orexin, include orexin A/hypocretin 1 and orexin B/hypocretin 2 (Ebrahim et al. 2003). Pre-prohypocretin is cleaved at unique sites to yield the two mature peptides upon removal of the N-terminal secretory signal sequence (Chieffi et al. 2017). Orexin A (Ox-A) is a 33 amino acid long peptide with two intrachain disulfide bonds, which has N-terminal proglutamyl residue and C-terminal amidation. Orexin B (Ox-B) is a 28 amino acid long peptide and the C-terminal is almost similar to Ox-A (Tsujino and Sakurai 2009). Orexins exert their function by binding to its cognate G-protein–coupled receptors (GPCRs), orexin receptor type 1 (OX1R, also named as Hcrtr-1) and type 2 (OX2R, or Hcrtr-2) that exhibit 64% amino acid identity (Sakurai 2005). OX1R has a higher affinity (~ 5–100-fold greater) towards Ox-A than Ox-B, whereas, OX2R has similar affinities to both Ox-A and Ox-B (Sakurai et al. 1998; Ammoun et al. 2003). The existence of two receptors and selective binding of agonists may be responsible for the cellular diversity of the orexin-orexin receptor functions.

The binding of orexins to the respective receptors activates at least three subtypes of G-proteins (G_q/11, G_i/0, and G_s) or other proteins such as β-arrestins. This subsequently controls the activation of downstream signaling cascades such as phospholipases, ion channels, and protein kinases (Leonard and Kukkonen 2014). As described in previous studies, the elevation of intracellular calcium (Ca²⁺) is a hallmark of orexin action in expression systems (Lund et al. 2000). Orexin receptors activate PKC in CNS neurons, which possibly necessitates PLC activity and may thus correlate with IP₃-triggered Ca²⁺ release (Ammoun et al. 2006; Johansson et al. 2008). OX1R couples to G_q/11 class of G-proteins, whereas, OX2R couples to G_q and G_i class of G-proteins. Additionally, stimulation of OX1R and OX2R is associated with the activation of the p38-MAPK signaling pathway and increases the level of phosphorylated ERK1/2 via G_q/PLC/PKC cascade, but not the PKA pathway (Milasta et al. 2005; Wenzel et al. 2009). The binding of orexins and the receptors also results in the stimulation of phospholipase D (PLD)/phosphatidic acid (PA), phospholipase A (PLA)/arachidonic acid (AA), PI3K/Akt and MTORC1 (ERK1/2-Akt-mediated) pathways (Johansson et al. 2008; Sokołowska et al. 2012; Turunen et al. 2012; Wang et al. 2014). Orexin signaling involves the activation of classical GPCR signaling as well as other intracellular signal transduction mechanisms.

Orexin signaling is critical in the regulation of feeding, energy metabolism (Haynes et al. 1999; Edwards et al. 1999; Moriguchi et al. 1999), gastrointestinal system (Kirchgessner and Liu 1999) and modulation of sense of pain (van den Pol 1999). They are also postulated to have roles in regulating neuroendocrine functions by altering the levels of corticotropin, gonadotropin, glucocorticoids and insulin (van den Pol et al. 1998; Ida et al. 2000; Sutcliffe and de Lecea 2000; Ziolkowska et al. 2005). Further, orexins also play an essential role in neuroprotection, regulation of apoptosis and inflammation (Butterick et al. 2012; Xiong et al. 2013; Sokołowska et al. 2014). Of the two ligands, Ox-A has potent neuroprotective and immuno-regulatory actions (Yuan et al. 2011; Duffy et al. 2019). The orexin/receptor system is ectopically expressed in a number of neurological disorders, implying that it plays a role in their occurrence and pathogenesis. Defects in the orexin/receptor system have been linked to human narcolepsy in multiple studies (Lin et al. 1999; Burgess et al. 2010; Hasegawa et al. 2014). It is also strongly correlated to drug addiction, especially to alcohol, nicotine, and cocaine (Smith et al. 2009; Moorman et al. 2017), Alzheimer’s disease (Fronczek et al. 2012; Liguori et al. 2016; Gabelle et al. 2017), schizophrenia (Nishino et al. 2002; Dalal et al. 2003; Huang et al. 2014) and depression (Salomon et al. 2003).

Orexins, with both neuroprotective and immunomodulatory properties, have emerged as a promising new class of biological agents for the treatment of immune-mediated CNS disorders such as narcolepsy, metabolic and neurological disorders. However, a comprehensive understanding of the signaling mechanisms underlying the orexin receptor is missing. The development of a consolidated map of orexinergic signaling could be appreciated for the definitive assignment of mechanisms underlying orexin/receptor-promoted functions. This will aid in the generation of orexin/receptor-targeted pharmacological therapies for the successful treatment of disorders.

Methodology

A literature search was executed in PubMed with key terms including ‘orexin, its aliases’ and ‘signaling’. Research articles excluding reviews were selected with information pertaining to the stimulation of orexin receptors. Following NetPath criteria, we have classified the annotated reactions into five categories; molecular association, (protein-protein interactions), catalysis (post-translational modifications, binding and cleavage), transport (translocation/transport of proteins between subcellular compartments), activation/inhibition, and, finally, gene regulation at the mRNA and/or protein level (both up and downregulation). The reactions pertaining to the orexin signaling pathway were filtered using NetPath criteria (Kandasamy et al. 2009, 2010) as described previously; Galanin receptor signaling (Gopalakrishnan et al. 2021), Serotonin (Sahu et al. 2018), oxytocin receptor (Chatterjee et al. 2016), Oncostatin M (Dey et al. 2013), AGE/RAGE signaling (Soman et al. 2013), MIF signaling (Subbannayya et al. 2015), IL33 signaling (Pinto et al. 2018), prolactin signaling (Radhakrishnan et al. 2012), FGF-1/FGFR (Raju et al. 2014), and VEGF-A/VEGFR2 (Sunitha et al. 2019) signaling pathways. The reactions were then represented in the form of a map with relevant information about orexin signaling using PathVisio, an open-source, free pathway depiction tool (van Iersel et al. 2008). The reactions were exported to WikiPathways, an open-source biological pathway database for the scientific community (Pico et al. 2008; Kelder et al. 2009). In addition, the pathway reactions and the map were subjected to manual review by internal curation experts and also by a pathway authority.

Results and discussion

We designed a literature search term to identify research articles that would provide information on orexin receptor signaling. The search term “OREXIN” OR “HYPOCRETIN” OR “HCRT” OR “OX” OR “NRCLP1” OR “PPOX” OR “HCRT1” OR “HCRT2” OR “HYPOCRETIN1” OR “HYPOCRETIN2” OR “OX1” OR “OX2” OR “OREXIN1” OR “OREXIN2” OR “OX1R” OR “OX2R” OR “HCRTR1” OR “HCRTR2” OR “OREXIN 1 RECEPTOR” OR “OREXIN 2 RECEPTOR” OR"HYPOCRETIN 1 RECEPTOR” OR “HYPOCRETIN 2 RECEPTOR” AND (“SIGNALING” OR “PATHWAY” OR “SIGNALLING”) yielded 5,548 PubMed articles, among which 125 articles were selected for manual curation of reactions based on the NetPath annotation criteria described in (Kandasamy et al. 2009, 2010). We identified a total of 318 downstream signaling reactions, which include, 23 transport, 31 catalysis events (post-translational modifications), 15 molecular associations, 23 activation/inhibition reactions and a total of 226 gene regulation events comprising 56 reactions reported at protein and 170 reactions at mRNA level. A compendium of these annotated reactions is represented as a systematic pathway map in Fig. 1. This pathway map was generated on the standalone free pathway drawing tool, PathVisio (van Iersel et al. 2008) and is made freely accessible on https://www.wikipathways.org/index.php/Pathway:WP5094. The data pertaining to the pathway is presented in a standard community exchange format, Biological PAthway eXchange (BioPAX) (Demir et al. 2010) Level 3 (OWL). The entire data can be downloaded in .png, .pdf and .svg image formats as well as in .txt format from the WikiPathways (Pico et al. 2008; Kelder et al. 2009). A supplementary table (Supplementary Table S1) provides all the annotated reactions with a brief description of each.

Fig. 1.

A schematic representation of the reactions induced by orexin-orexin receptor

The orexinergic neuropeptides (Ox-A and Ox-B) are known to be evolutionarily conserved across several mammalian species and are thus predicted to have important implications in several physiological conditions (Wong et al. 2011). The initiation of signaling by the orexin neuropeptides (Ox-A and Ox-B) ensues with the ligand binding to orexin receptors (OXRs) and the subsequent activation of G-protein subtypes. Initial studies point towards the strong association of OXRs with G_q (Sakurai et al. 1998; Smart et al. 1999) coupled with PLC-mediated calcium elevation (Kukkonen 2016) in the regulation of feeding behavior. However, over the years several studies have also indicated an orexin stimulated differential association of OXRs with other G-protein sub-types, such as G_i/0 and G_s in tissues other than the hypothalamus (Randeva et al. 2001; Holmqvist et al. 2005; Karteris et al. 2005). This differential G-protein activation downstream of OX1R and OX2R can also be attributed to the ligand used for receptor stimulation. OX1R stimulated by Ox-A/B and OX2R stimulated Ox-B activated a G_q mediated PLC/PKC cascade ultimately leading to phosphorylation-based activation of the ERK pathway. Whereas, Ox-A stimulated OX2R achieved the same through a G_s/AC mediated PKA cascade. The activation of the JNK or p38 pathways, however, was mediated via Gi-coupled OX1R or OX2R, respectively (Tang et al. 2008; Ramanjaneya et al. 2009).

The orexinergic signaling also comprises of the receptor association with GPCR kinases such as GRK2 and GRK5, and recruitment of the β-arrestin proteins, which aid not only in the receptor trafficking but also act as signaling scaffolds (Dalrymple et al. 2011; Kukkonen and Leonard 2014; Cai et al. 2020). Another established component of the OXR signaling is the elevation of intracellular Ca²⁺. Studies on neuronal as well as non-neuronal cells have indicated a surge of Ca²⁺ ions on OXR stimulation by either Ox-A or Ox-B. This elevation of Ca²⁺ levels can be attributed to both, an influx of extracellular Ca²⁺ through L-type or DAG mediated channels, as well as IP₃ mediated release from the intracellular Ca²⁺ stores in the sarcoplasmic reticulum (Ishibashi et al. 2005; Näsman et al. 2006; Xia et al. 2009; Peltonen et al. 2009; Wang et al. 2014). Interestingly, a study performed on Odora cells (derived from olfactory sensory neurons) revealed the contribution of both extracellular Ca²⁺ influx and intracellular Ca²⁺ release for the overall elevation of Ca²⁺ levels downstream of OX1R (Gorojankina et al. 2007). However, in the same cell line, the stimulation of OX2R led to an elevation of Ca²⁺ levels only through release from intracellular stores. Additionally, the stimulation of both the receptors in the rat arcuate nucleus in an agonist-dependent manner regulated the calcium signaling reciprocally to modulate feeding behavior (Muroya et al. 2004). Whilst a significant rise in Ca²⁺ levels was observed in rat NPY neurons in a G_q/PLC/IP₃/PKC dependent manner upon stimulation of OXIR by Ox-A, a G_i/0 mediated decrease in the intracellular Ca²⁺ levels was elicited by Ox-A/B stimulated OX2R in rat POMC neurons (Muroya et al. 2004). Another interesting study performed on rat INS-1E cells revealed a TRPV calcium channel-mediated Ca²⁺ influx on OX1R stimulation, leading to a rise in insulin secretion and cell proliferation (Skrzypski et al. 2016). Taken together, the calcium signaling downstream of OXRs is implicated in several physiological functions including regulation of feeding behavior, synthesis and release of hormones like catecholamines, nociception and integration of the neural systems involved in maintaining wakefulness, energy homeostasis, sensory processing and other autonomic functions (Xia et al. 2009; Ozcan et al. 2010; Nakamura et al. 2010; Nemoto et al. 2013).

Orexin neurons in the hypothalamus have a role in postsynaptic neurotransmitter release (Peltonen et al. 2009), which include the release of glutamate, GABA as well as acetylcholine (Kodama and Kimura 2002; Davis et al. 2003; Bernard et al. 2006; Dong et al. 2006). The release of glutamate and acetylcholine downstream of OXRs in rat locus coeruleus and somatosensory cortex, respectively, are implicated in the promotion of wakefulness or arousal thus affecting the sleep-wake cycle (Kodama and Kimura 2002; Dong et al. 2006). The release of these neurotransmitters from the pre-synaptic nerve terminal can also be mediated by a secondary metabolite, 2-arachidonoyl glycerol (2-AG), which facilitates retrograde paracrine signaling as an effect of OXR stimulation. The interaction between orexin and cannabinoid receptors (CB1), upon OX1R activation, is attributed to the release of secondary metabolites. For instance, a PLC or PLD-activated DAGL mediated 2-AG release followed by arachidonic acid (AA) release was observed in HEK293 and Neuro 2 A cells expressing the human OX1R gene (Turunen et al. 2012). Interestingly, a similar PLC/DAGL mediated increase in 2-AG leads to the generation of inhibitory inputs on dopaminergic neurons in rat brain regions such as the nucleus accumbens (NAc) and the ventral tegmental area (VTA). This rise in 2-AG in turn induces an association of the dopamine receptor D2 (DRD2) and β-arrestin 2 (ARRB2) leading to the desensitization of DRD2 resulting in the regulation of the mesolimbic dopaminergic circuitry (Tunisi et al. 2021). Additionally, the release of AA is also involved in providing feedback to Ca²⁺ influx under Ox-A stimulation of OX1R in CHO-hOX1 cells (Turunen et al. 2010) as depicted in the map (Fig. 1). This release of AA coincided with the release of other high potency lipid messengers in this cell system, suggesting a role of orexin in the regulation of the lipid signaling system.

Under OXR stimulation we observed the adenylyl cyclase (AC) mediated cyclic AMP (cAMP) cascade to be seemingly less prominent when compared with Ca²⁺ cascades. As depicted in the map we curated G_s-mediated increase in cAMP, as well as G_i-mediated decrease in cAMP downstream to OX2R. Also, a G_s-dependent increase in cAMP accumulation was observed under OX1R stimulation in the central nervous system (Gorojankina et al. 2007; Tang et al. 2008; Woldan-Tambor et al. 2011; Urbańska et al. 2012). Additionally, studies on human as well as rat adrenocortical cells have implicated the role of AC/PKA mediated cortisol secretion as well as autocrine-paracrine regulation of adrenal glucocorticoid release (Spinazzi et al. 2005; Ziolkowska et al. 2005). Apart from glucocorticoid and cortisol signaling, orexins also play an important role in the synthesis and secretion of several other endocrine hormones aiding the modulation of the hypothalamic-pituitary axis (HPA) (Kuru et al. 2000; Al-Barazanji et al. 2001; Xu et al. 2003; Ramanjaneya et al. 2009). The differential activation of AC downstream of OX1R resulted in an increase of cAMP accumulation in the adrenocortical cells led to a rise in cortisol and aldosterone secretion, however, the decrease in cAMP accumulation in the medullary cells was associated with an increase in epinephrine and norepinephrine release (Nanmoku et al. 2002). Some of the enzymes secreted downstream of OXRs include amylase and cholecystokinin (Harris et al. 2002; Larsson et al. 2003). Among other hormones, we curated and mapped the release of gonadotropin hormones (LH and FSH) (Cataldi et al. 2014), serotonin (Tao et al. 2006), and testosterone (Barreiro et al. 2004; Liguori et al. 2017), as well as a decrease in progesterone (Cataldi et al. 2012) under both the receptors. In addition, we identified an increase of leptin secretion (Pruszynska-Oszmalek et al. 2018) in porcine adipocytes and an increase in the release of growth hormone in ovine somatotrophs (Xu et al. 2003) under the stimulation of OX1R and OX2R, respectively. However, the exact mechanism involved in the secretion of these hormones is still poorly understood.

A very unique and intriguing phenomenon identified among the reactions curated under OXR stimulation is that of its dual role in regulating apoptosis. There is a PI3K-AKT mediated anti-apoptotic function induced through OX1R in INS1 cells and rat hepatocytes (Chen et al. 2013; Ju et al. 2014), nevertheless, in cancer cell types like HT29-D4 cells or rat C6 glioma OX1R stimulation are reported to induce apoptosis (Rouet-Benzineb et al. 2004; Biegańska et al. 2012). This dynamic modulation of the apoptotic pathway by the OX1R is arbitrated by the activation/inhibition of caspases such as CASP3 and CASP7 (Rouet-Benzineb et al. 2004; Liu et al. 2015; Wen et al. 2015). This facet of apoptotic signaling by the orexinergic systems needs to be explored further to enable the use of orexin in cancer therapeutics. Apart from its potential anti-tumorigenic role, orexinergic neuropeptides are also implicated in neuroprotection from hypoxic injury and anti-inflammatory actions. OXR signaling essentially exerts an anti-inflammatory effect by reducing the pro-inflammatory markers such as TNFα, IL-1β, IL-6, CXCL8, CXCL2, and NOX4 in a MAPK14 and NFκB dependent manner (Xiong et al. 2013; Zhang et al. 2018; Sun et al. 2018; Wang et al. 2019; Li et al. 2020), in cells/tissue insulted with either oxidative or ischemic stress. On the other hand, in normal rat paraventricular neurons stimulation of OX1R resulted in the upregulation of IL6, IL1B, TNF, FOSL1 and AVP mRNA leading to the development of hypertension (Huber et al. 2017; Fan et al. 2018). Thus, depending on the physiological state, the orexinergic system is seen to adapt its downstream signaling to either protect the cell or facilitate programmed cell death. Yet, the exact cascade or the signaling modalities remains unknown.

The regulation of glucose metabolism is a widely studied and well-illustrated physiological significance attributed to orexinergic signaling. In this regard, we curated two equally important cascades, both mediated by PI3K/AKT activation. One of these includes the AKT/MTOR mediated upregulation as well as activation of HIF1A protein which then instigates the upregulation of GLUT1 protein, which eventually leads to glucose uptake (Wan et al. 2017). This study performed on HepG2 cells also revealed the activation of PDP1, upregulation of PDHB and downregulation of LDHA and PDK1 thus, enabling the shift from glycolysis to TCA cycle of glucose metabolism. The other cascade, however, functions downstream of both receptors and proceeds via a series of phosphorylation-based activation of PI3K, PDK1, AKT, and AS-160, ultimately causing the translocation of GLUT4 receptor to the plasma membrane and subsequent rise in glucose uptake (Skrzypski et al. 2011). Another study performed on HEK293 cells and rat hypothalamic tissue revealed several other genes upregulated downstream of OX1R stimulated HIF1A upregulation (Sikder and Kodadek 2007). These include VEGFA, FLT4, SLC2A1, and NOS3.

Furthermore, we curated a predominantly upregulated set of genes associated with sleep deprivation, osteoblast differentiation, adipogenesis, mitochondrial biogenesis and modulation of excitatory transmission (Fig. 1) (Yamada et al. 2008; Sellayah et al. 2011; Koesema and Kodadek 2017; Han et al. 2018).

Conclusion

We attempted to consolidate the reactions relevant to the stimulation of orexin receptors by Ox-A and Ox-B, thus generate an extensive signaling map. With the relative representation of the Ox-A or Ox-B stimulation and their associations with the two orexin receptors for the signaling pathway reactions, this collective depiction of the signal transduction mechanisms and the signaling cascades associated with orexins will help scientists improve the current knowledge of the orexinergic system. This pathway illustration provides the downstream effectors and the varied physiological outcomes associated with differential stimulation of orexin receptors. Moreover, this would also serve as a reference for up-to-date inclusion of information on orexin signaling and thereby a tool for pathway enrichment analysis. Overall, this information could be further utilized for the advancement of orexin research in health and disease.

Electronic supplementary material

Below is the link to the electronic supplementary material.

List of abbreviations

CNS

Central Nervous System

HCRT

Hypocretin

GPCR

G protein-coupled receptor

HCRTR1

Hypocretin receptor 1

HCRTR2

Hypocretin receptor 2

OX1R

Orexin receptor 1

OX2R

Orexin receptor 2

Ox-A

Orexin A

Ox-B

Orexin B

OXR

Orexin receptors

PPIs

Protein-protein interactions

BioPAX

Biological Pathway Exchange

SBML

Systems Biology Markup Language

PKA

Protein kinase A

PKC

Protein kinase C

PLC

Phospholipase C

PLD

Phospholipase D

IP₃

Inositol-3-phosphate

MAPK

Mitogen activated protein kinases

GRK

G Protein-Coupled Receptor Kinase

Declarations

Conflict of interest

The author(s) declare no conflicts of interest.