Orexin-A attenuated motion sickness through modulating neural activity in hypothalamus nuclei.

BACKGROUND AND PURPOSE: We evaluated the hypothesis that central orexin application could counteract motion sickness responses through regulating neural activity in target brain areas. EXPERIMENTAL APPROACH: Thec effects of intracerebroventricular (i.c.v.) injection of orexin-A and SB-334867 (OX1 antagonist) on motion sickness-induced anorexia, nausea-like behaviour (conditioned gaping), hypoactivity and hypothermia were investigated in rats subjected to Ferris wheel-like rotation. Orexin-A responsive brain areas were identified using Fos immunolabelling and were verified via motion sickness responses after intranucleus injection of orexin-A, SB-334867 and TCS-OX2-29 (OX2 antagonist). The efficacy of intranasal application of orexin-A versus scopolamine on motion sickness symptoms in cats was also investigated. KEY RESULTS: Orexin-A (i.c.v.) dose-dependently attenuated motion sickness-related behavioural responses and hypothermia. Fos expression was inhibited in the ventral part of the dorsomedial hypothalamus (DMV) and the paraventricular nucleus (PVN), but was enhanced in the ventral part of the premammillary nucleus ventral part (PMV) by orexin-A (20 µg) in rotated animals. Motion sickness responses were differentially inhibited by orexin-A injection into the DMV (anorexia and hypoactivity), the PVN (conditioned gaping) and the PMV (hypothermia). SB-334867 and TCS-OX2-29 (i.c.v. and intranucleus injection) inhibited behavioural and thermal effects of orexin-A. Orexin-A (60 µg·kg-1) and scopolamine inhibited rotation-induced emesis and non-retching/vomiting symptoms, while orexin-A also attenuated anorexia with mild salivation in motion sickness cats. CONCLUSION AND IMPLICATIONS: Orexin-A might relieve motion sickness through acting on OX1 and OX2 receptors in various hypothalamus nuclei. Intranasal orexin-A could be a potential strategy against motion sickness.

Chaihu-Longgu-Muli decoction improves sleep disorders by restoring orexin-A function in CKD mice.

Introduction: Chaihu-Longgu-Muli decoction (CLMD) is a well-used ancient formula originally recorded in the "Treatise on Febrile Diseases" written by the founding theorist of Traditional Chinese Medicine, Doctor Zhang Zhongjing. While it has been used extensively as a therapeutic treatment for neuropsychiatric disorders, such as insomnia, anxiety and dementia, its mechanisms remain unclear. Methods: In order to analyze the therapeutic mechanism of CLMD in chronic renal failure and insomnia, An adenine diet-induced chronic kidney disease (CKD) model was established in mice, Furthermore, we analyzed the impact of CLMD on sleep behavior and cognitive function in CKD mice, as well as the production of insomnia related regulatory proteins and inflammatory factors. Results: CLMD significantly improved circadian rhythm and sleep disturbance in CKD mice. The insomnia related regulatory proteins, Orexin, Orexin R1, and Orexin R2 in the hypothalamus of CKD mice decreased significantly, while Orexin and its receptors increased remarkably after CLMD intervention. Following administration of CLMD, reduced neuron loss and improved learning as well as memory ability were observed in CKD mice. And CLMD intervention effectively improved the chronic inflflammatory state of CKD mice. Discussion: Our results showed that CLMD could improve sleep and cognitive levels in CKD mice. The mechanism may be related to the up-regulation of Orexin-A and increased phosphorylation level of CaMKK2/AMPK, which further inhibits NF-κB downstream signaling pathways, thereby improving the disordered inflammatory state in the central and peripheral system. However, More research is required to confirm the clinical significance of the study.

Nutritional efficacy of Astaxanthin in modulating orexin peptides and fatty acid level during adult life of rats exposed to perinatal undernutrition stress.

Perinatal undernutrition stress predisposes several disorders in adult life, which could be programed using nutraceuticals. However, the effect of perinatal undernutrition stress on orexin peptides, brain lipids, and its amelioration by a potent antioxidant (Astaxanthin) needs exploration. The present study focussed on the effect of perinatal undernutrition stress on brain fatty acid levels, Orexin peptides A and B, and its amelioration by Astaxanthin.Twenty-four male Wistar rats (Rattus norvegicus) were allocated to four groups (n = 6) as Normal, Perinatally Undernourished (UN), Astaxanthin treated (AsX, 12mg/kg), and perinatally Undernourished-but-Astaxanthin treated (UNA), and are allowed to grow for 1, 6 and 12 months. The fatty acid and orexin peptides A & B at different brain parts were measured and compared. Orexin peptides were assessed using an ELISA kit. Fatty acid levels were estimated using HP 5890 gas chromatograph. Data were analyzed by ANOVA followed by Tukey's posthoc test. P < 0.05 was considered significant.The hair cortisol, Orexin-A, and B were significantly increased (p < 0.001) in the UN group compared to normal and were modulated significantly by AsX in the UNA group. Undernutrition stress during the perinatal period altered the lipid profile, Total SFA, Total MUFA, Total n-3 PUFA, Total n-6 PUFA, n-3: n-6 PUFA, which Astaxanthin effectively modulated at 6 and 12 months of postnatal life. There was no difference between DHA and AA ratio. These results indicate that nutritional enrichment with Astaxanthin during the perinatal period positively contributes to adult health. Further, the mechanism of regulation of brain chemistry by Astaxanthin is warranted.

Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis.

AIM: Modified Suanzaoren Decoction (MSZRD) is obtained by improving Suanzaoren Decoction (SZRT), a traditional Chinese herbal prescription that has been used to treat insomnia for more than thousands of years. Our previous study showed that MSZRD can improve the gastrointestinal discomfort related insomnia by regulating Orexin-A. This study is the first study to evaluate the effects and possible mechanisms of MSZRD in mice with insomnia caused by p-chlorophenylalanine (PCPA) combined with multifactor random stimulation. METHODS: After 14 days of multifactor stimulation to ICR mice, a PCPA suspension (30 mg/mL) was injected intraperitoneally for two consecutive days to establish an insomnia model. Three different doses of MSZRD (3.6, 7.2, and 14.4 g/kg/day) were given to ICR mice for 24 days. The food intake and back temperature were measured, and behavioral tests and pentobarbital sodium-induced sleep tests were conducted. The levels of Orexin-A, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and adrenocortical hormones (CORT) in the serum and 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in hypothalamus were measured using enzyme-linked immunosorbent assay (ELISA) kits. The levels of γ-aminobutyric acid (GABA) and glutamic acid (Glu) were measured by high-performance liquid chromatography (HPLC). The expression of 5HT1A receptor (5-HTRIA) and orexin receptor 2 antibody (OX2R) was measured by Western blot (WB) and immunohistochemical staining (ICH). Hematoxylin and eosin (H&E) staining and Nissl staining were used to assess the histological changes in hypothalamus tissue. RESULTS: Of note, MSZRD can shorten the sleep latency of insomnia mice (P < 0.05, 0.01), prolonged the sleep duration of mice (P < 0.05, 0.01), and improve the circadian rhythm disorder relative to placebo-treated animals. Furthermore, MSZRD effectively increased the content of 5-HT and 5-HTR1A protein in the hypothalamus of insomnia mice (P < 0.05, 0.01), while downregulated the content of DA and NE (P < 0.05, 0.01). Importantly, serum GABA concentration was increased by treatment with MSZRD (P < 0.05), as reflected by a decreased Glu/GABA ratio (P < 0.05). Moreover, MSZRD decreased the levels of CORT, ACTH, and CRH related hormones in HPA axis (P < 0.05, 0.01). At the same time, MSZRD significantly downregulated the serum Orexin-A content in insomnia mice (P < 0.05), as well as hypothalamic OX2R expression (P < 0.05). In addition, MSZRD also improved the histopathological changes in hypothalamus in insomnia mice. CONCLUSION: MSZRD has sleep-improvement effect in mice model of insomnia. The mechanism may be that regulating the expression of Orexin-A affects the homeostasis of HPA axis and the release of related neurotransmitters in mice with insomnia.

Expression of orexin-A (hypocretin-A) in the hypothalamus after traumatic brain injury: A postmortem evaluation.

Traumatic brain injury (TBI) is one of the leading causes of mortality and morbidity. The key component of TBI pathophysiology is traumatic axonal injury (TAI), commonly referred to as diffuse axonal injury (DAI). Coma is a serious complication which can occur following traumatic brain injury (TBI). Recently, studies have shown that the central orexinergic/ hypocretinergic system exhibit prominent arousal promoting actions. Therefore, the purpose of this study is to investigate by immunohistochemistry the expression of beta-amyloid precursor protein (ß-APP) in white matter of parasagittal region, corpus callosum and brainstem and the expression of orexin-A (ORXA) in the hypothalamus after traumatic brain injury. RESULTS: DAI was found in 26 (53.06%) cases, assessed with ß-APP immunohistochemical staining in parasagittal white matter, corpus callosum and brainstem. Orexin-A immunoreactivity in hypothalamus was completely absent in 5 (10.2%) of the cases; moderate reduction of ORXA was observed in 9 (18.4%) of the cases; and severe reduction was observed in 7 (14.3%) of the cases. A statistically significant correlation was found between ß-APP immunostaining in white matter, corpus callosum and brainstem in relation to survival time (p < 0.002, p < 0.003 and p < 0.005 respectively). A statistically positive correlation was noted between ORX-A immunoreactivity in hypothalamus to survival time (p < 0.003). An inverse correlation was noted between the expression of ß-APP in the regions of brain studied to the expression of ORX-A in the hypothalamus of the cases studied (p < 0.005). CONCLUSIONS: The present study demonstrated by immunohistochemistry that reduction of orexin-A neurons in the hypothalamus, involved in coma status and arousal, enhanced the immunoexpression of ß-APP in parasagital white matter, corpus callosum and brainstem.

The Important Role of Adiponectin and Orexin-A, Two Key Proteins Improving Healthy Status: Focus on Physical Activity.

Exercise represents the most important integrative therapy in metabolic, immunologic and chronic diseases; it represents a valid strategy in the non-pharmacological intervention of lifestyle linked diseases. A large body of evidence indicates physical exercise as an effective measure against chronic non-communicable diseases. The worldwide general evidence for health benefits are both for all ages and skill levels. In a dysregulated lifestyle such as in the obesity, there is an imbalance in the production of different cytokines. In particular, we focused on Adiponectin, an adipokine producted by adipose tissue, and on Orexin-A, a neuropeptide synthesized in the lateral hypothalamus. The production of both Adiponectin and Orexin-A increases following regular and structured physical activity and both these hormones have similar actions. Indeed, they improve energy and glucose metabolism, and also modulate energy expenditure and thermogenesis. In addition, a relevant biological role of Adiponectin and Orexin A has been recently highlighted in the immune system, where they function as immune-suppressor factors. The strong connection between these two cytokines and healthy status is mediated by physical activity and candidates these hormones as potential biomarkers of the beneficial effects induced by physical activity. For these reasons, this review aims to underly the interconnections among Adiponectin, Orexin-A, physical activity and healthy status. Furthermore, it is analyzed the involvement of Adiponectin and Orexin-A in physical activity as physiological factors improving healthy status through physical exercise.

Associations of plasma hypocretin-1 with metabolic and reproductive health: Two systematic reviews of clinical studies.

The hypocretin system consists of two peptides hypocretin-1 and hypocretin-2 (HCRT1 and HCRT2). Hypocretin-containing neurons are located in the posterior and lateral hypothalamus, and have widespread projections throughout the brain and spinal cord. In addition to its presence in the cerebrospinal fluid (CSF), peripheral HCRT1 has been detected in plasma. Robust experimental evidence demonstrates functions of hypothalamic-originated HCRT1 in regulation of multiple biological systems related to sleep-wake states, energy homeostasis and endocrine function. In contrast, HCRT1 studies with human participants are limited by the necessarily invasive assessment of CSF HCRT1 to patients with underlying morbidity. Regulation by HCRT1 of energy homeostasis and reproduction in animals suggests similar regulation in humans and prompts these two systematic reviews. These reviews translate prior experimental findings from animal studies to humans and examine associations between HCRT1 and: 1) metabolic risk factors; 2) reproductive function in men, women and children. A total of 21 studies and six studies met the inclusion criteria for the two searches, respectively. Research question, study design, study population, assessments of HCRT1, reproductive, cardiometabolic data and main findings were extracted. Associations between HCRT1, metabolic and reproductive function are inconsistent. Limitations of studies and future research directions are outlined.

Xiaoyaosan regulates depression-related behaviors with physical symptoms by modulating Orexin A/OxR1 in the hypothalamus.

The functional regulation of the orexin system in the central nervous system is closely related to the occurrence and development of psychotic disorders. Abnormal changes in the lateral region of the hypothalamus are associated with the comorbidity of depression and physical symptoms, and how the traditional Chinese formula Xiaoyaosan regulates these changes may reveal aspects of the pathogenesis of depression. This study aimed to establish a rat model of depression in order to examine changes in Orexin A/OxR1 expression in the lateral region of the hypothalamus and the effects of Xiaoyaosan. Sixty specific pathogen-free (SPF) male healthy Sprague-Dawley (SD) rats were used in the experiment and randomly divided into the control group, the model group, the Xiaoyaosan group and the fluoxetine group. The depression model was established by 21-day chronic immobilization stress (CIS). Food intake and body weight were recorded, and the sucrose preference test (SPT) and open field test (OFT) were used to evaluate the model. Then, the expression of Orexin A/OxR1 in the hypothalamus was measured by ELISA, Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression of Orexin A and OxR1 in the lateral hypothalamic area was significantly down regulated in the model group, compared with the control group. Xiaoyaosan significantly reversed these changes with obvious curative effects. Abnormal changes in Orexin A/OxR1 in the lateral hypothalamic area of rats with depression caused by chronic stress are closely related to the pathogenesis of depression accompanied by physical symptoms. Xiaoyaosan can improve depression accompanied by physical symptoms by regulating Orexin A/OxR1.

Chronic Consumption of Fructose Induces Behavioral Alterations by Increasing Orexin and Dopamine Levels in the Rat Brain.

It has been widely described that chronic intake of fructose causes metabolic alterations which can be associated with brain function impairment. In this study, we evaluated the effects of fructose intake on the sleep⁻wake cycle, locomotion, and neurochemical parameters in Wistar rats. The experimental group was fed with 10% fructose in drinking water for five weeks. After treatment, metabolic indicators were quantified in blood. Electroencephalographic recordings were used to evaluate the sleep architecture and the spectral power of frequency bands. Likewise, the locomotor activity and the concentrations of orexin A and monoamines were estimated. Our results show that fructose diet significantly increased the blood levels of glucose, cholesterol, and triglycerides. Fructose modified the sleep⁻wake cycle of rats, increasing the waking duration and conversely decreasing the non-rapid eye movement sleep. Furthermore, these effects were accompanied by increases of the spectral power at different frequency bands. Chronic consumption of fructose caused a slight increase in the locomotor activity as well as an increase of orexin A and dopamine levels in the hypothalamus and brainstem. Specifically, immunoreactivity for orexin A was increased in the ventral tegmental area after the intake of fructose. Our study suggests that fructose induces metabolic changes and stimulates the activity of orexinergic and dopaminergic neurons, which may be responsible for alterations of the sleep⁻wake cycle.

Yokukansan (Kampo medicinal formula) prevents the development of morphine tolerance by inhibiting the secretion of orexin A.

BACKGROUND: Yokukansan (YKS), a traditional herbal (Kampo) medicine consisting of seven herbs, is effective in the treatment of pain disorders, such as headache, postherpetic neuralgia, fibromyalgia, and trigeminal neuralgia, and we have previously shown it to be effective against morphine analgesic tolerance in rats. It has been reported that orexin receptor antagonists prevent the development of morphine tolerance and that YKS inhibits the secretion of orexin A in the hypothalamus. This study examined whether the inhibition of the secretion of orexin A by YKS is one mechanism underlying its effect against morphine analgesic tolerance. METHODS: Male Wistar rats were administered a subcutaneous injection of morphine hydrochloride (10 mg/kg/day) for 5 days. One group was preadministered YKS, starting 3 days before the morphine. The withdrawal latency following thermal stimulation was measured daily using a hot plate test. On day 5, the levels of orexin A in the plasma and the midbrain were measured, and the appearance of activated astrocytes in the midbrain was examined by immunofluorescence staining. RESULTS: The preadministration of YKS prevented the development of morphine tolerance. The repeated administration of morphine significantly increased the plasma and midbrain levels of orexin A and the activation of astrocytes. These increases were significantly inhibited by the preadministration of YKS. CONCLUSION: These results suggest that the preadministration of YKS attenuated the development of antinociceptive morphine tolerance and that the inhibition of orexin A secretion may be one mechanism underlying this phenomenon.

The hypothalamic neuropeptide orexin A- a possible regulator in glucose homeostasis and germ cell kinetics in adult mice testes.

Orexin A (OXA), a hypothalamic neuropeptide, regulates food intake, sleep-wake cycle and energy balance by binding to its receptor (OX1R). Apart from brain, OXA and OX1R are also present in peripheral organs including reproductive tissues. Mammalian reproduction depends on uptake and proper utilization of glucose in the testes. This study, therefore, examined role of OXA/OX1R system in regulation of glucose homeostasis in adult mouse testis under in vivo and ex vivo conditions. Binding of OXA to OX1R was blocked using an OX1R antagonist, SB-334867. Mice were given a single bilateral intratesticular injection of the antagonist at doses of 4 and 12µg/mouse and sacrificed 24 h post-injection. In order to understand the direct role of OXA in testes of adult mice, an ex vivo experiment was performed where binding of OXA to OX1R in the testis was blocked by using the same OX1R antagonist. The antagonist treatment affected testicular glucose and lactate concentration with concomitant down-regulation in the expression of glucose transporters 3 and 8. A decreased activity in lactate dehydrogenase enzyme and imbalance between germ cell survival and proliferation were also noted in testes in treated mice. The results of ex vivo study supported the results obtained from in vivo study. The findings thus suggest involvement of OXA/OX1R system in regulation of testicular glucose homeostasis and germ cell kinetics in adult mice.

Effects of central RVD-hemopressin(α) administration on anxiety, feeding behavior and hypothalamic neuromodulators in the rat.

BACKGROUND: The endocannabinoid (eCB) system is strongly involved in the regulation of anxiety and feeding behavior. RVD-hemopressin(α) [RVD-hp(α)], a N-terminally extended form of hemopressin, is a negative allosteric modulator of the cannabinoid (CB) 1 receptor and a positive allosteric modulator of CB2 receptor which has been recently reported to exert anxiolytic/antidepressant and anorexigenic effects after peripheral administration in rats. Pharmacological evidences reported a possible link between brain hypocretin/orexin, monoamine and eCB systems, as regards appetite and emotional behavior control. Considering this, the aim of our work was to investigated the effects of RVD-hp(α) on anxiety like behavior and food intake after central administration and related it to monoamine levels and orexin-A gene expression, in the hypothalamus. METHODS: We have studied the effects of central RVD-hp(α) (10nmol) injection on anxiety-like behavior and feeding using different behavioral tests. Hypothalamic levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and gene expression of orexin-A and proopiomelanocortin (POMC) were measured by high performance liquid chromatography (HPLC) and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis, respectively. RESULTS: Central RVD-hp(α) administration decreased locomotion activity and stereotypies. Moreover, RVD-hp(α) treatment inhibited anxiogenic-like behavior and food intake, NE levels and orexin-A gene expression, in the hypothalamus. CONCLUSION: Concluding, in the present study we demonstrated that central RVD-hp(α) induced anxiolytic and anorexigenic effects possibly related to reduced NE and orexin-A and POMC signaling, in the hypothalamus. These findings further support the central role of the peptide in rat brain thus representing an innovative pharmacological approach for designing new anorexigenic drugs targeting eCB system.

Relationship between cerebrospinal fluid concentrations of orexin A/hypocretin-1 and body composition in humans.

The hypothalamic neuropeptide orexin A (hypocretin-1) is a key signal in sleep/wake regulation and promotes food intake. We investigated the relationship between cerebrospinal fluid orexin A concentrations and body composition in non-narcoleptic human subjects with a wide range of body weight to gain insight into the role of orexin A in human metabolism. We collected cerebrospinal fluid and blood samples and measured body composition by bioelectric impedance analysis in 36 subjects (16 women and 20 men) with body mass indices between 16.24 and 38.10 kg/m2 and an age range of 19-80 years. Bivariate Pearson correlations and stepwise multiple regressions were calculated to determine associations between orexin A and body composition as well as biometric variables. Concentrations of orexin A in cerebrospinal fluid averaged 315.6 ±â€¯6.0 pg/ml, were comparable between sexes (p > 0.15) and unrelated to age (p > 0.66); they appeared slightly reduced in overweight/obese compared to normal-weight subjects (p = .07). Orexin A concentrations decreased with body weight (r = -0.38, p = .0229) and fat-free mass (r = -0.39, p = .0173) but were not linked to body fat mass (p > 0.24). They were inversely related to total body water (r = -0.39, p = .0174) as well as intracellular (r = -0.41, p = .0139) and extracellular water (r = -0.35, p = .0341). Intracellular water was the only factor independently associated with cerebrospinal fluid orexin A concentrations (p = .0139). We conclude that cerebrospinal fluid orexin A concentrations do not display associations with body adiposity, but are inversely related to intracellular water content. These cross-sectional findings suggest a link between orexin A signaling and the regulation of water homeostasis in humans.

[Impacts of the repetitive transcranial acupuncture stimulation on the content of serum orexin A in patients with post-stroke insomnia].

OBJECTIVE: To compare the effect on post-stroke insomnia between the repetitive transcranial acupuncture stimulation (rTAS) and the conventional western medication in the patients and to explore the mechanism. METHODS: Ninety patients of post-stroke insomnia were randomized into a rTAS group, a medication group and a placebo group, 30 cases in each one. In the rTAS group, patients were intervened by rTAS. The acupoints were Baihui (GV 20), Ningshen (Extra), emotion area, Wangu (GB 12), Taiyang (EX-HN 5), Neiguan (PC 6), Shenmen (HT 7), Sanyinjiao (SP 6), Zhaohai (KI 6), Zusanli (ST 36) and Taichong (LR 3). Fast twist with small amplitude was used at Baihui (GV 20) and emotion area for 2-3 min, 200-300 r/min, once 15 min. Electroacupuncture (EA) was applied at Baihui (GV 20) and Ningshen (Extra), bilateral Wangu (GB 12), Sanyinjiao (SP 6) and Zhaohai (KI 6) on the same side, 10 Hz, 0.5-1 mA. The treatment was given for 40 min in the rTAS group, once a day. Diazepam was prescribed orally in the medication group before sleep, 2.5 mg a day. Starch capsule was used in the placebo group before sleep, once a day. All the treatment was given for continuous 1 month. The level of serum orexin A was observed before and after treatment. The effects were compared. The recurrence rate was recorded 3 months after treatment. RESULTS: The total effective rates in the rTAS group and the medication group were 86.7% (26/30) and 90.0% (27/30) repectively after treatment, which were better than 20.0% (6/30) in the placebo group (both P<0.01). After treatment, the levels of serum orexin A in the rTAS group and the medication group were lower than those before treatment (both P<0.01), which were lower than that in the placebo group after treatment (both P<0.01), without statistical significance between the rTAS group and the medication group after treatment (P>0.05). The total effective rates in the rTAS group and the medication group were 86.7% (26/30) and 86.7% (26/30) at follow-up repectively, which were better than 16.7% (5/30) in the placebo group (both P<0.01). CONCLUSION: The rTAS is safe and effective for post-stroke insomnia, which is similar to oral medication of diazepam. The decreasing serum orexin A may be one of the mechanisms.

Impacts of the repetitive transcranial acupuncture stimulation on the content of serum orexin A in patients with post-stroke insomnia / 中国针灸

OBJECTIVE@#To compare the effect on post-stroke insomnia between the repetitive transcranial acupuncture stimulation (rTAS) and the conventional western medication in the patients and to explore the mechanism.@*METHODS@#Ninety patients of post-stroke insomnia were randomized into a rTAS group, a medication group and a placebo group, 30 cases in each one. In the rTAS group, patients were intervened by rTAS. The acupoints were Baihui (GV 20), Ningshen (Extra), emotion area, Wangu (GB 12), Taiyang (EX-HN 5), Neiguan (PC 6), Shenmen (HT 7), Sanyinjiao (SP 6), Zhaohai (KI 6), Zusanli (ST 36) and Taichong (LR 3). Fast twist with small amplitude was used at Baihui (GV 20) and emotion area for 2-3 min, 200-300 r/min, once 15 min. Electroacupuncture (EA) was applied at Baihui (GV 20) and Ningshen (Extra), bilateral Wangu (GB 12), Sanyinjiao (SP 6) and Zhaohai (KI 6) on the same side, 10 Hz, 0.5-1 mA. The treatment was given for 40 min in the rTAS group, once a day. Diazepam was prescribed orally in the medication group before sleep, 2.5 mg a day. Starch capsule was used in the placebo group before sleep, once a day. All the treatment was given for continuous 1 month. The level of serum orexin A was observed before and after treatment. The effects were compared. The recurrence rate was recorded 3 months after treatment.@*RESULTS@#The total effective rates in the rTAS group and the medication group were 86.7% (26/30) and 90.0% (27/30) repectively after treatment, which were better than 20.0% (6/30) in the placebo group (both 0.05). The total effective rates in the rTAS group and the medication group were 86.7% (26/30) and 86.7% (26/30) at follow-up repectively, which were better than 16.7% (5/30) in the placebo group (both <0.01).@*CONCLUSION@#The rTAS is safe and effective for post-stroke insomnia, which is similar to oral medication of diazepam. The decreasing serum orexin A may be one of the mechanisms.

Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A.

Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy.

Increased plasma orexin-A levels in patients with insomnia disorder are not associated with prepro-orexin or orexin receptor gene polymorphisms.

Orexins, also known as hypocretins, play a regulatory role in the sleep-wake cycle by activating orexin receptors. Previous animal studies have shown that sleep deprivation can elevate orexinergic peptide levels. However, the relationship between insomnia disorder and orexin-A levels in humans has not been explored. In the current study, we examined plasma orexin-A levels in patients with insomnia disorder and in normal sleepers. We also studied the possible mechanisms underlying changes in orexin-A levels between the study groups; this included investigations of prepro-orexin and orexin receptor gene polymorphisms as well as exploration of other variables. We measured plasma orexin-A levels in 228 patients with insomnia disorder and 282 normal sleepers. The results indicated that the patients with insomnia disorder had significantly higher orexin-A levels than normal sleepers (63.42±37.56 vs. 54.84±23.95pg/ml). A positive relationship was detected between orexin-A level and age in patients with insomnia disorder. Orexin-A levels were elevated in relation to course of insomnia, as well as in relation to increased Insomnia Severity Index score. None of the evaluated prepro-orexin gene single nucleotide polymorphisms were informative between the two study populations. After sequencing all orexin receptor exons, one variation (rs2271933) in the OX1R gene and one variation (rs2653349) in the OX2R gene were found. However, no significant differences were found in either genotypic or allelic frequency distributions between the two study groups. It is suggested that the increased plasma orexin-A levels in patients with insomnia disorder are associated with the course and severity of insomnia, but not with prepro-orexin and orexin receptor gene polymorphisms.

Central inhibitory effects on feeding induced by the adipo-myokine irisin.

Irisin, the soluble secreted form of fibronectin type III domain containing 5 (FNDC5)-cleaved product, is a recently identified adipo-myokine that has been indicated as a possible link between physical exercise and energetic homeostasis. The co-localization of irisin with neuropeptide Y in hypothalamic sections of paraventricular nucleus, which receives NPY/AgRP projections from the arcuate nucleus, suggests a possible role of irisin in the central regulation of energy balance. In this context, in the present work we studied the effects of intra-hypothalamic irisin (1µl, 50-200nmol/l) administration on feeding and orexigenic [agouti-related peptide (AgRP), neuropeptide Y (NPY) and orexin-A] and anorexigenic [cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)] peptides in male Sprague-Dawley rats. Furthermore, we evaluated the effects of irisin on hypothalamic dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) concentrations and plasma NE levels. Compared to vehicle, irisin injected rats showed decreased food intake, possibly mediated by stimulated CART and POMC and inhibited DA, NE and orexin-A, in the hypothalamus. We also found increased plasma NE levels, supporting a role for sympathetic nervous system stimulation in mediating increased oxygen consumption by irisin.

Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation.

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3µM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3µM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1µM)+ACEA (0.1µM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2µM), or OX-A (0.1µM)+ACEA (0.1µM), but not after ACEA (0.2µM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2µM) or OX-A (0.1µM)+ACEA (0.1µM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2µM), was induced by incubation with OX-A (0.1µM)+ACEA (0.1µM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Localization, expression and role of Orexin A and its receptor in testes of neonatal mice.

Orexin A (OXA), a hypothalamic neuropeptide, and its receptor (OX1R) are primarily expressed in lateral hypothalamus and are involved in the control of various biological functions. Expressions of OXA and OX1R have also been reported in peripheral organs like gastrointestinal and genital tracts. In the present study, expressions of OXA and OX1R have been observed in the testis of Parkes strain neonatal mice by semi-quantitative RT-PCR and western blot analyses. Immunohistochemical study also revealed their presence on spermatogonia, Sertoli cells and in the interstitium of the testis. In order to understand the role of OXA and OX1R in testicular development, an in vitro study was also performed. For this, binding of OXA to OX1R was blocked using OX1R specific antagonist, SB-334867. Eighteen mice were sacrificed and their testes were cultured in complete media containing vehicle and two doses (0.1 and 4.0µg/ml media) of SB-334867 for 72h in CO2 incubator at 37°C. At the end of culture period, testes were used for western blot and RT-PCR analyses to study the expression of various markers of gonadal development, such as steroidogenic factor 1 (SF-1), Wilms' tumor 1 (Wt1), Mullerian inhibiting substance (MIS) and stem cell factor (SCF). Further, expressions of OXA, OX1R and glucose transporter 3 (GLUT 3) were also studied. A marked increase in the expression of SF-1 and a decrease in the expression of Wt1 at both transcript and protein levels were noted, while there was a decrease in the expression of SCF and MIS at transcript level at both doses of the antagonist; this suggests that blockage of OXA binding to OX1R by SB-334867 affects testicular development. The decrease in expressions of OXA, OX1R and GLUT 3 in the test is in response to both doses of the antagonist points to their down-regulation causing inefficient uptake of glucose by the testicular cells, thereby affecting gonadal development. In conclusion, our results suggest that the binding of OXA to OX1R is important for the development of the testis.