Exogenous orexin-A Research Articles

Therapeutic effects of orexin-A in sepsis-associated encephalopathy in mice

BackgroundSepsis-associated encephalopathy (SAE) causes acute and long-term cognitive deficits. However, information on the prevention and treatment of cognitive dysfunction after sepsis is limited. The neuropeptide orexin-A (OXA) has been shown to play a protective role against neurological diseases by modulating the inflammatory response through the activation of OXR1 and OXR2 receptors. However, the role of OXA in mediating the neuroprotective effects of SAE has not yet been reported.MethodsA mouse model of SAE was induced using cecal ligation perforation (CLP) and treated via intranasal administration of exogenous OXA after surgery. Mouse survival, in addition to cognitive and anxiety behaviors, were assessed. Changes in neurons, cerebral edema, blood-brain barrier (BBB) permeability, and brain ultrastructure were monitored. Levels of pro-inflammatory factors (IL-1β, TNF-α) and microglial activation were also measured. The underlying molecular mechanisms were investigated by proteomics analysis and western blotting.ResultsIntranasal OXA treatment reduced mortality, ameliorated cognitive and emotional deficits, and attenuated cerebral edema, BBB disruption, and ultrastructural brain damage in mice. In addition, OXA significantly reduced the expression of the pro-inflammatory factors IL-1β and TNF-α, and inhibited microglial activation. In addition, OXA downregulated the expression of the Rras and RAS proteins, and reduced the phosphorylation of P-38 and JNK, thus inhibiting activation of the MAPK pathway. JNJ-10,397,049 (an OXR2 blocker) reversed the effect of OXA, whereas SB-334,867 (an OXR1 blocker) did not.ConclusionThis study demonstrated that the intranasal administration of moderate amounts of OXA protects the BBB and inhibits the activation of the OXR2/RAS/MAPK pathway to attenuate the outcome of SAE, suggesting that OXA may be a promising therapeutic approach for the management of SAE.

Orexin A alleviates LPS-induced acute lung injury by inhibiting macrophage activation through JNK-mediated autophagy

Crosstalk between the central nervous system and immune system by the neuroendocrine and autonomic nervous systems is critical during the inflammatory response. Exposure to endotoxin alters the activity of hypothalamic homeostatic systems, resulting in changed transmitter release within the brain. This study investigated the effects and cellular molecular mechanisms of neurogenic and exogenous orexin-A (OXA) in LPS-induced acute lung injury (ALI). We found the production of OXA in the hypothalamus and lungs was both decreased following LPS infection. LPS-induced lung injury including the destruction of the structure, inflammatory cell infiltration, and pro-inflammatory cytokines generation was aggravated in mice in which orexin neurons were lesioned with the neurotoxin orexin-saporin (orexin-SAP). Administration of exogenous OXA greatly improved lung pathology and reduced inflammatory response. Orexin receptors were found in cultured mouse bone marrow-derived macrophages (BMDMs) and lung macrophages (LMs), adoptive transfer of OXA-treated macrophages showed alleviative lung injury compared to adoptive transfer of macrophages without OXA treatment. Mechanistically, it is the induction of autophagy via JNK activation that is responsible for OXA to suppress macrophage-derived pro-inflammatory cytokine production. These findings highlight the importance of neuro-immune crosstalk and indicate that OXA may be a potential therapeutic agent in the treatment of ALI.

Orexin-A aggravates cognitive deficits in 3xTg-AD mice by exacerbating synaptic plasticity impairment and affecting amyloid β metabolism

Dementia is the main clinical feature of Alzheimer's disease (AD). Orexin has recently been linked to AD pathogenesis, and exogenous orexin-A (OXA) aggravates spatial memory impairment in APP/PS1 mice. However, the effects of OXA on other types of cognitive deficits, especially in 3xTg-AD mice exhibiting both plaque and tangle pathologies, have not been reported. Furthermore, the potential electrophysiological mechanism by which OXA affects cognitive deficits and the molecular mechanism by which OXA increases amyloid β (Aβ) levels are unknown. In the present study, the effects of OXA on cognitive functions, synaptic plasticity, Aβ levels, tau hyperphosphorylation, BACE1 and NEP expression, and circadian locomotor rhythm were evaluated. The results showed that OXA aggravated memory impairments and circadian rhythm disturbance, exacerbated hippocampal LTP depression, and increased Aβ and tau pathologies in 3xTg-AD mice by affecting BACE1 and NEP expression. These results indicated that OXA aggravates cognitive deficits and hippocampal synaptic plasticity impairment in 3xTg-AD mice by increasing Aβ production and decreasing Aβ clearance through disruption of the circadian rhythm and sleep-wake cycle.

Exogenous orexin-A downregulates luteinizing hormone secretory activity in prepubertal female rats.

Orexin-A is a neuropeptide synthesized in the lateral hypothalamus. Orexin-A immunoreactive fibres overlap distribution with GnRH neurons. In adult rats, orexin A is known to affect LH secretion via GnRH release modulation. Because data concerning the impact of orexin-A on the hypothalamo-pituitary axis activity are limited, we focused on the involvement of orexin-A and receptors of NPY in the modulation of LH release and LH subunit b (Lhb) mRNA expression in prepubertal female rats. Forty immature female Wistar rats were divided into 4 groups and received 2 intracerebroventricular (icv) microinjections of: 1 - artificial cerebrospinal fluid (CSF) (controls); 2 - CSF followed by orexin A; 3 - selective NPY receptor antagonist (BIBP) followed by CSF; 4 - BIBP followed by orexin A. One hour after the last microinjection, all rats were decapitated. Trunk blood was collected, and serum was stored at -20°C for the LH RIA examination. The adenohypophysis was immediately excised, flash-frozen, and kept at -80°C for RNA extraction. Real-time PCR amplification was carried out, and relative Lhb gene expression was calculated. In comparison to the CSF-treated controls with a mean LH serum concentration of 0.40 ± 0.02 ng/mL, the mean LH serum level was diminished both after orexin-A (0.27 ± 0.01 ng/mL) and after BIBP (0.30 ± 0.02 ng/mL) icv microinjections. In the presence of BIBP, orexin-A more effectively inhibited LH release (0.20 ± 0.01 ng/mL) when compared to the BIBP-treated group. Orexin-A and BIBP exerted a consistent inhibitory effect on Lhb mRNA expression levels in the anterior pituitary gland. In comparison to the CSF-treated controls, orexin-A, and BIBP-treated females responded with, respectively, 35% and 40% reduction of Lhb mRNA expression. Orexin-A and BIBP co-administration evoked a further reduction of Lhb gene transcriptional activity. Orexin-A exerts a down-regulatory effect on LH synthesis and release in immature female rats. Considering that Y1R-oriented down-regulation of endogenous NPY activity did not reverse the suppressive effect of exogenous orexin-A, it might be suggested that NPY and orexin A systems can operate independently to affect gonadotropin activity in the anterior pituitary of the immature female rats.

Common carp (Cyprinus carpio) orexin: Molecular identification, tissue expression, and the role of Orexin-A in glucose metabolism

Orexins are neuropeptides involved in energy homeostasis. In fish, the function of orexin-A in glucose metabolism is less reported. The present study aims to identify the potential role of orexin-A in the glucose absorption and metabolism in common carp. The full length of orexin cDNA was cloned using RT-PCR and RACE method. The prepro-orexin cDNA was 513 bp containing an open reading frame of 474 bp and encoded 157 amino acids which including 47 amino acids of orexin-A and 28 amino acids of orexin-B. The tissue distribution results showed that orexin was expressed in multiple tissues, especially with high-level expression in hypothalamus. The exogenous orexin-A protein was chemical synthesized. Intraperitoneal injection of orexin-A alleviated the increase of plasma glucose level at 1 h and 2 h after OGTT (Oral glucose tolerance test). Orexin-A also decreased glucose uptake activity and the expression of sglt1 and glut2 mRNA in intestine. However, orexin-A significantly increased the mRNA expression of glut2 in liver and glut4 in muscle. Moreover, the gluconeogenesis-related gene fbpase mRNA was significantly down-regulated, while glycolysis-related genes gk and hk were significantly up-regulated after treatment with orexin-A in vivo and vitro. These results implied that hyperglycemia could be alleviated in common carp after the injection of orexin-A and suggest a role of this peptide as a plasma glucose regulator by inhibition glucose absorption in intestine, promotion glucose transport to liver and muscle. Furthermore, hepatic gluconeogenesis was inhibited. Taken together, this study helped to understand the characterization of prepro-orexin and the function of orexin-A in fish glucose homeostasis.

Exogenous Orexin-A Microinjected Into Central Nucleus of the Amygdala Modulates Feeding and Gastric Motility in Rats.

Orexin-A is a circulating neuropeptide and neurotransmitter that regulates food intake and gastric motility. The central nucleus of the amygdala (CeA), which regulates feeding behavior and gastric function, expresses the orexin-1 receptor. The aim of this study was to evaluate the effects of microinjection of exogenous orexin-A into the CeA, on food intake and gastric motility, and to explore the mechanisms of these effects. Normal chow and high fat food (HFF) intake were measured, gastric motility and gastric emptying were evaluated, extracellular single unit firing was recorded, and c-fos expression was determined. The results showed that microinjection of orexin-A into the CeA resulted in increased HFF intake but did not affect normal chow intake. This effect was blocked by an orexin-1 receptor antagonist-SB-334867 and was partially blocked by a dopamine D1 receptor antagonist-SCH-23390. Gastric motility and gastric emptying were enhanced by orexin-A, and the former effect was abolished by subdiaphragmatic vagotomy. The firing frequency of gastric distention-related neurons was regulated by orexin-A via the orexin-1 receptor. Furthermore, c-fos expression was increased in the ventral tegmental area (VTA) and the nucleus accumbens (NAc), the lateral hypothalamus (LHA), and the dorsal motor nucleus of the vagus (DMV) in response to microinjection of orexin-A into the CeA. These findings showed that orexin-A regulated palatable food intake and gastric motility via the CeA. The LHA, the VTA, and the NAc may participate in palatable food intake and the CeA-DMV-vagus-stomach pathway may be involved in regulating gastric motility through the regulation of neuronal activity in the CeA.

Orexin A-induced inhibition of leptin expression and secretion in adipocytes reducing plasma leptin levels and hypothalamic leptin resistance.

Orexin A (OXA) is a neuropeptide associated with plasma insulin and leptin levels involved in body weight and appetite regulation. However, little is known about the effect of OXA on leptin secretion in adipocytes and its physiological roles. Leptin secretion and expression were analysed in 3T3-L1 adipocytes. Plasma leptin, adiponectin and insulin levels were measured by ELISA assay. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the hypothalamus were evaluated by western blotting. OXA dose-dependently suppressed leptin secretion from 3T3-L1 adipocytes by inhibiting its gene expression while facilitating adiponectin secretion. The leptin inhibition by OXA was mediated via orexin receptors (OXR1 and OXR2). In addition to the pathway via extracellular signal-regulated kinases, OXA triggered adenylyl cyclase-induced cAMP elevation, which results in protein kinase A-mediated activation of cAMP response element-binding proteins (CREB). Accordingly, CREB inhibition restored the OXA-induced downregulation of leptin gene expression and secretion. Exogenous OXA for 4 weeks decreased fasting plasma leptin levels and increased hypothalamic pSTAT3 levels in high-fat diet-fed mice, regardless of increase in body weight and food intake. These results suggest that high dose of OXA directly inhibits leptin mRNA expression and thus secretion in adipocytes, which may be a peripheral mechanism of OXA for its role in appetite drive during fasting. It may be also critical for lowering basal plasma leptin levels and thus maintaining postprandial hypothalamic leptin sensitivity.

Arcuate Nucleus Orexin-A Signaling Alleviates Cisplatin-Induced Nausea and Vomiting Through the Paraventricular Nucleus of the Hypothalamus in Rats.

The most common side effects of cisplatin chemotherapy are nausea and vomiting, and the overwhelming majority of research studies on the mechanism of cisplatin-induced nausea have been focused on the “vomiting center.” As a modulatory center of gastric motility, the roles of the hypothalamus in nausea and vomiting remain unclear. In the present study, we investigated the effects of exogenous orexin-A injected into the arcuate nucleus (ARC) on cisplatin-induced nausea and vomiting, and the possible underlying mechanism. Kaolin intake was calculated daily in cisplatin-treated and saline-treated rats. Gastric motility recording, injections into the ARC, and lesions of the paraventricular nucleus (PVN) were used to study the effects of orexin-A and the hypothalamic nucleus on disorders of gastrointestinal function in cisplatin-treated rats. The pathway from the ARC to the PVN was observed through Fluoro-Gold retrograde tracing. Furthermore, an NPY Y1 receptor antagonist was administered to explore the possible mechanisms involved in the effects of orexin-A in the ARC. We illustrated that exogenous orexin-A injected into the ARC reduced kaolin intake and promoted gastric motility in cisplatin-treated rats, and these effects could have been blocked by an ipsilateral PVN lesion or co-injected antagonist of orexin-A-SB334867. Additional results showed that orexin-A-activated neurons in the ARC communicated directly with other neurons in the PVN that express neuropeptide Y (NPY). Furthermore, activation of the downstream NPY pathway was required for the observed effects of orexin in the ARC on cisplatin-induced nausea and vomiting. These findings reveal a novel neurobiological circuit from the ARC to the PVN that might provide a potential target for the prevention and treatment of cisplatin-induced nausea and vomiting.

Ectopic expression of OX1R in ulcerative colitis mediates anti-inflammatory effect of orexin-A

Orexins (orexin-A and orexin-B) are hypothalamic peptides that are produced by the same precursor and are involved in sleep/wake control, which is mediated by two G protein-coupled receptor subtypes, OX1R and OX2R. Ulcerative colitis (UC) is an inflammatory bowel disease, (IBD) which is characterized by long-lasting inflammation and ulcers that affect the colon and rectum mucosa and is known to be a significant risk factor for colon cancer development. Based on our recent studies showing that OX1R is aberrantly expressed in colon cancer, we wondered whether orexin-A could play a role in UC. Immunohistochemistry studies revealed that OX1R is highly expressed in the affected colonic epithelium of most UC patients, but not in the non-affected colonic mucosa. Injection of exogenous orexin-A specifically improved the inflammatory symptoms in the two colitis murine models. Conversely, injection of inactive orexin-A analog, OxB7–28 or OX1R specific antagonist SB-408124 did not have anti-inflammatory effect. Moreover, treatment with orexin-A in DSS-colitis induced OX1R−/− knockout mice did not have any protective effect. The orexin-A anti-inflammatory effect was due to the decreased expression of pro-inflammatory cytokines in immune cells and specifically in T-cells isolated from colonic mucosa. Moreover, orexin-A inhibited canonical NFκB activation in an immune cell line and in intestinal epithelial cell line. These results suggest that orexin-A might represent a promising alternative to current UC therapies.

Effect of orexin-A in the arcuate nucleus on cisplatin-induced gastric side effects in rats

The most common side effects of the cancer chemotherapy drug cisplatin are nausea and vomiting. These effects are heavily influenced by orexigenic and anorexigenic peptides. We explored the effects of orexin-A on the cisplatin-treated rats and a possible mechanism for its effects on cisplatin-induced side effects. Quantitative real-time PCR was used to measure the change of prepro-orexin mRNA in the hypothalamus following cisplatin treatment. The effect of orexin-A and cisplatin on the firing rate of arcuate nucleus neurons was recorded. The effect of administration of orexin-A and a neuropeptide Y1 receptor antagonist to the arcuate nucleus on food intake, pica, and gastric motility on cisplatin treated rats were also measured. The relative expression of prepro-orexin mRNA in the hypothalamus was reduced by cisplatin. Exogenous orexin-A altered cisplatin-induced changes to the neuronal firing of gastric distension-responsive neurons, alleviated the cisplatin-induced anorexia, pica and improves the weakened gastric motility in the arcuate nucleus of rats. These effects could be partially blocked by intracerebroventricular injection (i.c.v.) of a neuropeptide Y1 receptor antagonist. These results suggest that orexin-A signaling ameliorates the gastric disorder induced by cisplatin in rats, and may act through neuropeptide Y neurons in the arcuate nucleus.

Effects of orexins on 17β-estradiol synthesis and P450 aromatase modulation in the testis of alpaca (Vicugna pacos)

The steroidogenic enzyme P450 aromatase (ARO) has a key role in the conversion of testosterone (T) into estrogens (E), expressed as 17β-estradiol. The presence and localization of this key enzyme have not been described before in the South American camelid alpaca (Vicugna pacos). In our previous studies of the expression and biological effects of orexin A (OxA) and OxB on the alpaca testis demonstrated that OxA, via its specific receptor 1 (OX1R), stimulated T synthesis. In order to extend these findings, we presently explored the presence and localization of ARO in the alpaca male gonad, and the possible correlation between ARO and the orexinergic complex. Western blotting and immunohistochemistry demonstrated the presence of ARO in tissue homogenates and its localization in the tubular and interstitial compartments of the alpaca testis, respectively. The addition of OxA to fresh testicular slices decreased the 17β-estradiol E levels. This effect was annulled by the sequential addition of the selective OX1R antagonist, SB-408124. OxB incubation did not have any effect on the biosynthesis of E. Furthermore, the OxA-mediated down-regulation of E secretion could be ascribed to ARO inhibition by exogenous OxA, as indicated by measurement of ARO activity in tissue slices incubated with OxA. Overall, our findings suggest that locally secreted OxA interacting with OX1R could indirectly inhibit ARO activity, disabling the conversion of T to E, and consequently lowering E biosynthesis and increasing the production of T in mammalian testis.

Orexin A regulates plasma insulin and leptin levels in a time-dependent manner following a glucose load in mice.

Orexin A (OXA) is a neuropeptide implicated in the regulation of arousal status and energy metabolism. Orexin receptors are expressed not only in the central nervous system but also in the pancreas and adipose tissue. However, little is known about the physiological function of orexins. This study investigated the role of exogenous OXA in blood glucose control after glucose load in mice. In addition, the effect of OXA on insulin secretion was also identified in mouse pancreatic beta cells. Insulin secretion and intracellular Ca(2+) levels were measured in perifused mouse islets. To investigate the effects of exogenous OXA on blood glucose levels in vivo, intraperitoneal glucose tolerance tests were performed after a subcutaneous injection of OXA in normal and high-fat diet-induced diabetic mice. OXA significantly potentiated glucose-stimulated insulin secretion in vitro, which increased intracellular Ca(2+) levels, mainly through adenylate cyclase and ryanodine receptor activation. This Ca(2+)-dependent insulinotropic effect of OXA was blocked in Epac2 (Rapgef4)-deficient beta cells. After a glucose load in mice, exogenous OXA decreased blood glucose levels, compared with the control, by enhancing plasma insulin and decreasing plasma glucagon levels. Additionally, OXA caused a delayed increase in plasma leptin levels, resulting in lower plasma insulin levels when blood glucose levels fell to baseline. These results suggest that OXA might be a critical regulator of insulin, glucagon and leptin secretion in response to glucose. Thus, exogenous OXA might have therapeutic potential in improving blood glucose control in patients with type 2 diabetes.

The Effect of Orexin-A on Cardiac Dysfunction Mediated by NADPH Oxidase-Derived Superoxide Anion in Ventrolateral Medulla

Hypocretin/orexin-producing neurons, located in the perifornical region of the lateral hypothalamus area (LHA) and projecting to the brain sites of rostral ventrolateral medulla (RVLM), involve in the increase of sympathetic activity, thereby regulating cardiovascular function. The current study was designed to test the hypothesis that the central orexin-A (OXA) could be involved in the cardiovascular dysfunction of acute myocardial infarction (AMI) by releasing NAD(P)H oxidase-derived superoxide anion (O2 −) generation in RVLM, AMI rat model established by ligating the left anterior descending (LAD) coronary artery to induce manifestation of cardiac dysfunction, monitored by the indicators as heart rate (HR), heart rate variability (HRV), mean arterial pressure (MAP) and left intraventricular pressure. The results showed that the expressions of OXA in LHA and orexin 1 receptor (OX1R) increased in RVLM of AMI rats. The double immunofluorescent staining indicated that OX1R positive cells and NAD(P)H oxidative subunit gp91phox or p47phox-immunoreactive (IR) cells were co-localized in RVLM. Microinjection of OXA into the cerebral ventricle significantly increased O2 − production and mRNA expression of NAD(P)H oxidase subunits when compared with aCSF-treated ones. Exogenous OXA administration in RVLM produced pressor and tachycardiac effects. Furthermore, the antagonist of OX1R and OX2R (SB-408124 and TCS OX2 29, respectively) or apocynin (APO), an inhibitor of NAD(P)H oxidase, partly abolished those cardiovascular responses of OXA. HRV power spectral analysis showed that exogenous OXA led to decreased HF component of HRV and increased LF/HF ratio in comparison with aCSF, which suggested that OXA might be related to sympathovagal imbalance. As indicated by the results, OXA might participate in the central regulation of cardiovascular activities by disturbing the sympathovagal balance in AMI, which could be explained by the possibility that OXR and NAD(P)H-derived O2 − in RVLM mediates OXA-induced cardiovascular responses.

Aging-related alterations in orexin/hypocretin modulation of septo-hippocampal amino acid neurotransmission.

GABAergic neurons of the medial septum of the basal forebrain make up a substantial portion of the septo-hippocampal pathway fibers, and are known to modulate hippocampal amino acid neurotransmission and support cognitive function. Importantly, these neurons are also implicated in age-related cognitive decline. Hypothalamic orexin/hypocretin neurons innervate and modulate the activity of these basal forebrain neurons and also provide direct inputs to the hippocampus. However, the precise role of orexin inputs in modulating hippocampal amino acid neurotransmission--as well as how these interactions are altered in aging--has not been defined. Here, orexin A (OxA) was administered to CA1 and the medial septum of young (3-4 months) and aged (27-29 months) Fisher 344 Brown Norway rats, and hippocampal GABA and glutamate efflux was analyzed by in vivo microdialysis. Following CA1 infusion of OxA, extracellular GABA and glutamate efflux was increased, but the magnitude of orexin-mediated efflux was not altered as a function of age. However, medial septum infusion of OxA did not impact hippocampal efflux in young rats, while aged rats exhibited a significant enhancement in GABA and glutamate efflux compared to young counterparts. Furthermore, immunohistochemical characterization of the medial septum revealed a significant decrease in parvalbumin (PV)-positive cell bodies in aged animals, and a significant reduction in orexin fiber innervation to the remaining GABAergic cells within the septum, while orexin innervation to the hippocampus was unaltered by the aging process. These findings indicate that: (1) OxA directly modulates hippocampal amino acid neurotransmission in young animals, (2) Aged animals show enhanced responsivity to exogenous OxA activation of the septo-hippocampal pathway, and (3) Aged animals undergo an intrinsic reduction in medial septum PV-immunoreactivity and a decrease in orexin innervation to remaining septal PV neurons. Alterations in orexin regulation of septo-hippocampal activity may contribute to age-related dysfunctions in arousal, learning, and memory.

Neuroprotective Effect of Orexin-A Is Mediated by an Increase of Hypoxia-inducible Factor-1 Activity in Rat

Recent studies suggest that the novel neuropeptide orexin-A may play an essential role during neuronal damage. However, the function of orexin-A during brain ischemia remains unclear. Recently, hypoxia-inducible factor-1α (HIF-1α) was shown to be activated by orexin-A. The aim of the current study is to test the hypothesis that administration of exogenous orexin-A can attenuate ischemia-reperfusion injury through the facilitation of HIF-1α expression. Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion for 120 min. Rats were treated with different doses of orexin-A or vehicle before the ischemia and at the onset of reperfusion. To investigate the action of HIF-1α in the neuroprotective effects of orexin-A, the HIF-1α inhibitor YC-1 was used alone or combined with orexin-A. Neurologic deficit scores and infarct volume were assessed. Brains were harvested for immunohistochemical staining and western blot analysis. Orexin-A significantly ameliorated neurologic deficit scores and reduced infarct volume after cerebral ischemia reperfusion. Administration of 30 μg/kg orexin-A showed optimal neuroprotective effects. This effect was still present 7 days after reperfusion. Furthermore, orexin-A decreased the number of apoptotic cells and significantly enhanced HIF-1α expression after cerebral ischemia reperfusion. Moreover, the facilitation of HIF-1α expression was accompanied with inhibition of von Hippel-Lindau expression. Administration of HIF-1α inhibitor suppressed the increase of HIF-1α and reversed the neuroprotective effects of orexin-A. Orexin-A has a neuroprotective effect against cerebral ischemia-reperfusion injury. These effects may be mediated through the HIF-1α pathway.

Intra-Periaqueductal Gray Matter Microinjection of Orexin-A Decreases Formalin-Induced Nociceptive Behaviors in Adult Male Rats

Intracerebroventricular injection of orexin-A (hypocretin-1) has been shown to elicit the analgesic responses. However, the locations of central sites that may mediate these effects have not been clearly elucidated. This study was performed using male Sprague Dawley rats to investigate the antinociceptive effects of intra-periaqueductal gray matter (PAG) administration of orexin-A, 5 minutes prior to formalin (50 μL of 2%) injection. Orexin-A had no effect on tail-flick test as thermal and acute model. In the formalin test, intra-PAG injection of orexin-A (10 nM) decreased the formalin-induced nociceptive behaviors in the interphase and phase 2, but not in phase 1, indicating an antinociceptive role of exogenous orexin-A in the PAG. While Orexin-A failed to produce a dose-dependent decrease in formalin-evoked behaviors in phase 1, it may have induced a dose-dependent decrease in formalin-evoked behaviors in early phase 2. Control injections of orexin-A into the sites near the PAG resulted in less or no reduction in pain, indicating that the analgesia observed is probably due to a site of action within the PAG rather than at surrounding neural structures. The antinociceptive effect of orexin-A was compared with intra-PAG administration of morphine (.5 μL of 20 mM, 5 minutes before the formalin injection). Morphine decreased the formalin-induced nociceptive behaviors in all phases. To investigate whether the orexin has a special action on the early part of the second phase, or its delayed effects are related to its pharmacokinetics, the orexin-A was injected into the PAG, 10 minutes before the formalin injection. No difference was observed between 5 and 10 minutes injection of orexin-A prior to formalin injection. The antinociceptive effect of orexin was blocked by intra-PAG injection of SB-334867, a putative type 1 orexin receptor antagonist, suggesting the involvement of orexin receptor type 1 in antinociception produced with intra-PAG injection of orexin-A. The results showed that the orexin-A plays an antinociceptive role in PAG in the interphase and the late phase of formalin test through type 1 orexin receptor dependent mechanism. Orexin is produced exclusively in the lateral hypothalamus, where it is known to modulate the pain processing through PAG. The antinociceptive effect of orexin in PAG may provide a role for this neurotransmitter in the up-down modulating pain system and further support the development of orexin-1 agonists for pain treatment.

A Major Role for Perifornical Orexin Neurons in the Control of Glucose Metabolism in Rats

OBJECTIVEThe hypothalamic neuropeptide orexin influences (feeding) behavior as well as energy metabolism. Administration of exogenous orexin-A into the brain has been shown to increase both food intake and blood glucose levels. In the present study, we investigated the role of endogenous hypothalamic orexin release in glucose homeostasis in rats.RESEARCH DESIGN AND METHODSWe investigated the effects of the hypothalamic orexin system on basal endogenous glucose production (EGP) as well as on hepatic and peripheral insulin sensitivity by changing orexinergic activity in the hypothalamus combined with hepatic sympathetic or parasympathetic denervation, two-step hyperinsulinemic-euglycemic clamps, immunohistochemistry, and RT-PCR studies.RESULTSHypothalamic disinhibition of neuronal activity by the γ-aminobutyric acid receptor antagonist bicuculline (BIC) increased basal EGP, especially when BIC was administered in the perifornical area where orexin-containing neurons but not melanocortin-concentrating hormone–containing neurons were activated. The increased BIC-induced EGP was largely prevented by intracerebroventricular pretreatment with the orexin-1 receptor antagonist. Intracerebroventricular administration of orexin-A itself caused an increase in plasma glucose and prevented the daytime decrease of EGP. The stimulatory effect of intracerebroventricular orexin-A on EGP was prevented by hepatic sympathetic denervation. Plasma insulin clamped at two or six times the basal levels did not counteract the stimulatory effect of perifornical BIC on EGP, indicating hepatic insulin resistance. RT-PCR showed that stimulation of orexin neurons increased the expression of hepatic glucoregulatory enzymes.CONCLUSIONSHypothalamic orexin plays an important role in EGP, most likely by changing the hypothalamic output to the autonomic nervous system. Disturbance of this pathway may result in unbalanced glucose homeostasis.

Orexin A affects ascending contraction depending on downstream cholinergic neurons and descending relaxation through independent pathways in mouse jejunum

The involvement of orexin in neural pathways for peristalsis was examined in mouse jejunal segments. Localized distension of the segments using a small balloon resulted in ascending contraction and descending relaxation. Ascending contraction was abolished by atropine and tetrodotoxin. Desensitization to orexin A (OXA) and SB-334867-A, an orexin-1 receptor antagonist, significantly inhibited ascending contraction. Hexamethonium also produced a significant inhibition. Exogenous administration of either OXA or nicotine induced a transient contraction that was completely inhibited by atropine and tetrodotoxin. The OXA-induced contraction was significantly inhibited by hexamethonium and SB-334867-A, whereas the nicotine-induced contraction was not inhibited by SB-334867-A. Descending relaxation was either partially or completely inhibited by l-nitroarginine and tetrodotoxin, respectively. Both SB-334867-A and hexamethonium partially inhibited descending relaxation. A combination of SB-334867-A and hexamethonium had an additive inhibitory effect on descending relaxation. Exogenous OXA, in the presence of atropine, induced a relaxation that was significantly inhibited by both l-nitroarginine and SB-334867-A, but not by hexamethonium. Nicotine in the presence of atropine relaxed the jejunal segment. SB-334867-A, unlike hexamethonium, did not affect nicotine-induced relaxation. These results suggest that OXA plays an important role in the ascending and descending neural reflexes in the mouse jejunum.

Stimulatory effect of endogenous orexin A on gastric emptying and acid secretion independent of gastrin

Orexin A (OXA) increases food intake and inhibits fasting small bowel motility in rats. The aim of this study was to examine the effect of exogenous OXA and endogenous OXA on gastric emptying, acid secretion, glucose metabolism and distribution of orexin immunoreactivity in the stomach. Rats equipped with a gastric fistula were subjected to intravenous (IV) infusion of OXA or the selective orexin-1 receptor (OX1R) antagonist SB-334867-A during saline or pentagastrin infusion. Gastric emptying was studied with a liquid non-nutrient or nutrient, using 51Cr as radioactive marker. Gastric retention was measured after a 20-min infusion of OXA or SB-334867-A. Plasma concentrations of OXA, insulin, glucagon, glucose and gastrin were studied. Immunohistochemistry against OXA, OX1R and gastrin in gastric tissue was performed. OXA alone had no effect on either acid secretion or gastric emptying. SB-334867-A inhibited both basal and pentagastrin-induced gastric acid secretion and increased gastric retention of the liquid nutrient, but not PEG 4000. Plasma gastrin levels were unchanged by IV OXA or SB-334867-A. Plasma OXA levels decreased after intake of the nutrient meal and infusion of the OX1R antagonist. Only weak effects were seen on plasma glucose and insulin by OXA. Immunoreactivity to OXA and OX1R were found in the mucosa, myenteric cells bodies and varicose nerve fibers in ganglia and circular muscle of the stomach. In conclusion, endogenous OXA influences gastric emptying of a nutrient liquid and gastric acid secretion independent of gastrin. This indicates a role for endogenous OXA, not only in metabolic homeostasis, but also in the pre-absorptive processing of nutrients in the gut.