Qiangjing tablets ameliorate asthenozoospermia via mitochondrial ubiquitination and mitophagy mediated by LKB1/AMPK/ULK1 signaling.

CONTEXT: Therapeutic effects of Qiangjing tablets (QJT) on sperm vitality and asthenozoospermia (AZS) have been confirmed. However, the mechanism of action remains unclear. OBJECTIVE: This study investigates the effects of QJT on AZS and the underlying mechanism of action. MATERIALS AND METHODS: Sixty Sprague-Dawley rats were randomly divided into six groups: Control, ORN (ornidazole; 200 mg/kg), ORN + QJT-low (0.17 g/mL), ORN + QJT-middle (0.33 g/mL), ORN + QJT-high (0.67 g/mL), and ORN + QJT + Radicicol (0.67 g/mL QJT and 20 mg/kg radicicol) groups. Pathological evaluation and analysis of mitophagy were conducted by H&E staining and transmission electron microscopy, respectively. Reactive oxygen species were detected by flow cytometry. Protein expression was determined by Western blotting. RESULTS: QJT significantly improved ORN-treated sperm motility and kinematic parameters, as well as the pathological symptoms of testicular and epididymal tissues. In particular, QJT mitigated impaired mitochondrial morphology, and increased the PHB, Beclin-1, LC3-II protein, and ROS levels (p < 0.05), and reduced the protein expression levels of LC3-I and p62 (p < 0.05). Mechanistically, QJT antagonized the downregulation of SCF and Parkin protein levels (p < 0.05). Furthermore, QJT significantly increased the protein expressions levels of LKB1, AMPKα, p-AMPKα, ULK1 and p-ULK1 (p < 0.05). The ameliorative effect of QJT on pathological manifestations, mitochondrial morphology, and the expressions of mitophagy and mitochondrial ubiquitination-related proteins was counteracted by radicicol. DISCUSSION AND CONCLUSIONS: QJT improved AZS via mitochondrial ubiquitination and mitophagy mediated by the LKB1/AMPK/ULK1 signaling pathway. Our study provides a theoretical basis for the treatment of AZS and male infertility.

Thymoquinone attenuates hepatic lipid accumulation by inducing autophagy via AMPK/mTOR/ULK1-dependent pathway in nonalcoholic fatty liver disease.

Thymoquinone (TQ) has been proved to exert wide-ranging pharmacological activities, with anti-inflammatory, antioxidant, anticonvulsant, antimicrobial, anti-tumor, and antidiabetic properties. In this study, we investigated the beneficial effects of TQ on a high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) in C57BL/6 N mice in vivo and free fatty acid (FFA)-induced human hepatocellular carcinoma HepG2 cells in vitro. Further, the underlying mechanisms of TQ to promote hepatic autophagy were also discovered. Data showed that TQ caused (p < 0.01) body weight reduction, improved glucose homeostasis, alleviated hepatosteatosis, and decreased hepatic lipid accumulation related to the induction of autophagy in HFD-fed mice. In vitro, TQ obviously increased (p < 0.01) autophagic flux in FFA-induced HepG2 cells and consequently reduced the lipid accumulation in combination with activation of AMPK/mTOR/ULK1 signaling pathways. Moreover, pharmacological inhibition of the AMPK pathway by addition with AMPK inhibitor Compound C (CC) or silence of ULK1 by transfection with siRNA(ULK1) into HepG2 cells reversed these beneficial effects of TQ on triggering hepatic autophagy and reducing lipid accumulation (p < 0.01). Taken together, these results suggested that TQ alleviated hepatic lipid accumulation by triggering autophagy through the AMPK/mTOR/ULK1-dependent signaling pathway. Our study supports a potential role for TQ in ameliorating NAFLD.

Humanin Alleviates Insulin Resistance in Polycystic Ovary Syndrome: A Human and Rat Model-Based Study.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism and insulin resistance (IR); however, the pathogenesis of local ovarian IR in PCOS remains largely unclear. Humanin, a mitochondria-derived peptide, has been reported to be associated with IR. Our previous study confirmed that humanin is expressed in multiple cell types in the ovary and is present in follicular fluid. However, it remains unknown whether humanin participates in the pathogenesis of local ovarian IR or whether humanin supplementation can improve IR in PCOS patients. In this study, we compared humanin concentrations in follicular fluid from PCOS patients with and without IR. We further investigated the effect of humanin analogue (HNG) supplementation on IR in a rat model of dehydroepiandrosterone-induced PCOS. Humanin concentrations in the follicular fluid were found to be significantly lower in PCOS patients with IR than in those without IR. HNG supplementation attenuated both the increases in the levels of fasting plasma glucose and fasting insulin in rats with PCOS and the decreases in phosphorylation of IRS1, PI3K, AKT, and GLUT4 proteins in the granulosa cells of these rats. Combined supplementation with HNG and insulin significantly improved glucose consumption in normal and humanin-siRNA-transfected COV434 cells. In conclusion, downregulated humanin in the ovaries may be involved in the pathogenesis of IR in PCOS, and exogenous supplementation with HNG improved local ovarian IR through modulation of the IRS1/PI3K/Akt signaling pathway in a rat model. This finding supports the potential future use of HNG as a therapeutic drug for PCOS.

Icariin Promotes the Osteogenic Action of BMP2 by Activating the cAMP Signaling Pathway.

Icariin (ICA) is the main active flavonoid glucoside from herbs of the genus Epimedium; in traditional Chinese medicine, these herbs have long been prescribed for the treatment of bone fractures and osteoporosis. Several studies have shown that treatment with ICA can increase osteogenic differentiation and reduce bone loss in vivo and in vitro. However, the definite signaling pathway of this osteogenic effect remains unclear. In this study, we selected bone morphogenetic protein 2 (BMP2)-induced osteoblastic differentiation of multipotent mesenchymal progenitor C2C12 cells as a model of osteoblast differentiation. We investigated the effects of ICA on C2C12 cells osteogenic differentiation and the underlying molecular mechanisms. We found that ICA could enhance BMP2-mediated osteoblastic differentiation of C2C12 cells in a dose-dependent manner. Treatment with ICA activated the cAMP/PKA/CREB signaling axis in a time-dependent manner. Blocking cAMP signaling using the PKA selective inhibitor H89 significantly inhibited the stimulatory effect of ICA on osteogenesis. Therefore, the osteoinductive potential and the low cost of ICA indicate that it is a promising alternative treatment or promoter for enhancing the therapeutic effects of BMP2.

The Inhibition of Melanogenesis Via the PKA and ERK Signaling Pathways by Chlamydomonas reinhardtii Extract in B16F10 Melanoma Cells and Artificial Human Skin Equivalents.

Abnormal melanin synthesis results in several hyperpigmentary disorders such as freckles, melanoderma, age spots, and other related conditions. In this study, we investigated the antimelanogenic effects of an extract from the microalgae Chlamydomonas reinhardtii (CE) and potential mechanisms responsible for its inhibitory effect in B16F10, normal human epidermal melanocyte cells, and human skin-equivalent models. The CE extract showed significant dose-dependent inhibitory effects on α-melanocyte-stimulating, hormone-induced melanin synthesis in cells. Additionally, the CE extract exhibited suppressive effects on the mRNA and protein expression of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. The CE extract also inhibited the phosphorylation of protein kinase A and extracellular signal-related kinase, which function as upstream regulators of melanogenesis. Using a three-dimensional, reconstructed pigmented epidermis model, the CE-mediated, anti-pigmentation effects were confirmed by Fontana-Masson staining and melanin content assays. Taken together, CE extract can be used as an anti-pigmentation agent.

Isolation and lipolytic activity of eurycomanone and its epoxy derivative from Eurycoma longifolia.

Eurycomanone (1) and 13ß,21-epoxyeurycomanone (2) were isolated from Eurycoma longifolia for studies of lipolytic activity. Compound 1 enhanced lipolysis in adipocytes with an EC50 of 14.6µM, while its epoxy derivate, compound 2, had a stronger activity with an EC50 of 8.6µM. Based on molecular mechanistic study using several specific inhibitors to lipolytic signaling pathways, it was found that PKA inhibitor totally diminished the lipolytic activity of 1 and 2. Further immunoblotting analysis confirmed the activation of phosphorylated PKA by both 1 and 2. With the growing need to develop new anti-obesity agents, eurycomanone and its epoxy derivate can be used as promising lead compounds to target lipid catabolism.

[Roles of orexin and effects of orexin receptor antagonists].

Orexin A and orexin B are hypothalamic neuropeptides that were discovered in 1998. Several studies suggested that orexin deficiency causes narcolepsy in humans and other mammalian species, highlighting roles of this hypothalamic neuropeptide in the regulation of sleep and wakefulness. The orexin system regulates sleep and wakefulness through interactions with systems that regulate emotion, reward and energy homeostasis. This system regulates sleep and wakefulness to occur at appropriate times that are in accordance with our internal and external environments. Recent findings have brought about the possibility of novel therapies targeting orexin system for sleep disorder including insomnia and narcolepsy-cataplexy. In this review, I will discuss the current understanding of the integrative physiology and clinical perspectives of the orexin system.

Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators.

Features of the fission yeast Schizosaccharomyces pombe cAMP/PKA pathway make S. pombe particularly amenable for heterologous expression of cAMP pathway proteins such as GαS subunits and their cognate adenylyl cyclases, PKA catalytic and regulatory subunits, and cyclic nucleotide phosphodiesterases. We have constructed two PKA-repressed reporters for use in high-throughput screens to detect compounds that elevate or reduce PKA activity, thus facilitating the discovery of both inhibitors and activators of these target proteins. Here, we describe steps to construct screening strains and to optimize and conduct these screens.

Sleep disorders, obesity, and aging: the role of orexin.

The hypothalamic neuropeptides orexin A and B (hypocretin 1 and 2) are important homeostatic mediators of central control of energy metabolism and maintenance of sleep/wake states. Dysregulation or loss of orexin signaling has been linked to narcolepsy, obesity, and age-related disorders. In this review, we present an overview of our current understanding of orexin function, focusing on sleep disorders, energy balance, and aging, in both rodents and humans. We first discuss animal models used in studies of obesity and sleep, including loss of function using transgenic or viral-mediated approaches, gain of function models using exogenous delivery of orexin receptor agonist, and naturally-occurring models in which orexin responsiveness varies by individual. We next explore rodent models of orexin in aging, presenting evidence that orexin loss contributes to age-related changes in sleep and energy balance. In the next section, we focus on clinical importance of orexin in human obesity, sleep, and aging. We include discussion of orexin loss in narcolepsy and potential importance of orexin in insomnia, correlations between animal and human studies of age-related decline, and evidence for orexin involvement in age-related changes in cognitive performance. Finally, we present a summary of recent studies of orexin in neurodegenerative disease. We conclude that orexin acts as an integrative homeostatic signal influencing numerous brain regions, and that this pivotal role results in potential dysregulation of multiple physiological processes when orexin signaling is disrupted or lost.

Gastroprotective effect of orexin-A and heme oxygenase system.

BACKGROUND: Orexin-A, besides playing an important role in the mechanism of food intake, exhibits a potent gastroprotective action against the formation of acute gastric mucosal injury. The aim of the present study was to determine the effect of administered orexin-A against ischemia-reperfusion (I/R)-induced gastric injury on the expression of heme oxygenase (HO)-1 and HO-2 in gastric tissue. MATERIALS AND METHODS: Wistar rats were subjected to 30 min of ischemia followed by 3 h reperfusion. Orexin-A was infused at a dose of 500 pmol/kg/min during the I/R period. The lesion area was measured by stereomicroscope. The myeloperoxidase activity and 4-hydroxinonenol-malondialdehyde content of gastric mucosa were evaluated spectrophotometrically, and the gastric tumor necrosis factor-α was measured by enzyme linked immune sorbent assay. The expression of HO-1 and HO-2 was determined by Western blotting analysis. RESULTS: Orexin-A significantly decreased the I/R-induced gastric lesions, myeloperoxidase activity, and 4-hydroxinonenol-malondialdehyde concentration in gastric tissue exposed to I/R. The gastroprotective effect of orexin-A in gastric I/R model was accompanied by the increase in HO-2 expression and the decrease in HO-1 expression. CONCLUSIONS: Orexin-A exerts a protective action on gastric mucosa subjected to I/R, and this effect is associated with the reduction of neutrophil infiltration and lipid peroxidation in gastric tissue in addition to the increase in HO-2 expression due to the administration of orexin-A.

Hypothalamic orexin prevents hepatic insulin resistance via daily bidirectional regulation of autonomic nervous system in mice.

Circadian rhythm is crucial for preventing hepatic insulin resistance, although the mechanism remains uncovered. Here we report that the wake-active hypothalamic orexin system plays a key role in this regulation. Wild-type mice showed that a daily rhythm in blood glucose levels peaked at the awake period; however, the glucose rhythm disappeared in orexin knockout mice despite normal feeding rhythm. Central administration of orexin A during nighttime awake period acutely elevated blood glucose levels but subsequently lowered daytime glucose levels in normal and diabetic db/db mice. The glucose-elevating and -lowering effects of orexin A were suppressed by adrenergic antagonists and hepatic parasympathectomy, respectively. Moreover, the expression levels of hepatic gluconeogenic genes, including Pepck, were increased and decreased by orexin A at nanomolar and femtomolar doses, respectively. These results indicate that orexin can bidirectionally regulate hepatic gluconeogenesis via control of autonomic balance, leading to generation of the daily blood glucose oscillation. Furthermore, during aging, orexin deficiency enhanced endoplasmic reticulum (ER) stress in the liver and caused impairment of hepatic insulin signaling and abnormal gluconeogenic activity in pyruvate tolerance test. Collectively, the daily glucose rhythm under control of orexin appears to be important for maintaining ER homeostasis, thereby preventing insulin resistance in the liver.

Hepatic branch vagus nerve plays a critical role in the recovery of post-ischemic glucose intolerance and mediates a neuroprotective effect by hypothalamic orexin-A.

Orexin-A (a neuropeptide in the hypothalamus) plays an important role in many physiological functions, including the regulation of glucose metabolism. We have previously found that the development of post-ischemic glucose intolerance is one of the triggers of ischemic neuronal damage, which is suppressed by hypothalamic orexin-A. Other reports have shown that the communication system between brain and peripheral tissues through the autonomic nervous system (sympathetic, parasympathetic and vagus nerve) is important for maintaining glucose and energy metabolism. The aim of this study was to determine the involvement of the hepatic vagus nerve on hypothalamic orexin-A-mediated suppression of post-ischemic glucose intolerance development and ischemic neuronal damage. Male ddY mice were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Intrahypothalamic orexin-A (5 pmol/mouse) administration significantly suppressed the development of post-ischemic glucose intolerance and neuronal damage on day 1 and 3, respectively after MCAO. MCAO-induced decrease of hepatic insulin receptors and increase of hepatic gluconeogenic enzymes on day 1 after was reversed to control levels by orexin-A. This effect was reversed by intramedullary administration of the orexin-1 receptor antagonist, SB334867, or hepatic vagotomy. In the medulla oblongata, orexin-A induced the co-localization of cholin acetyltransferase (cholinergic neuronal marker used for the vagus nerve) with orexin-1 receptor and c-Fos (activated neural cells marker). These results suggest that the hepatic branch vagus nerve projecting from the medulla oblongata plays an important role in the recovery of post-ischemic glucose intolerance and mediates a neuroprotective effect by hypothalamic orexin-A.

Mechanisms underlying obesity resistance associated with high spontaneous physical activity.

Obesity resistance due to elevated orexin signaling is accompanied by high levels of spontaneous physical activity (SPA). The behavioral and neural mechanisms underlying this observation have not been fully worked out. We determined the contribution of hypothalamic orexin receptors (OXRs) to SPA stimulated by orexin A (OXA), whether OXA-stimulated SPA was secondary to arousal and whether voluntary wheel running led to compensations in 24-h SPA. We further tested whether orexin action on dopamine one receptors (DA1R) in the substantia nigra (SN) plays an important role in the generation of SPA. To test this, SPA response was determined in lean and obese rats with cannulae targeted toward the rostral lateral hypothalamus (rLH) or SN. Sleep/wake states were also measured in rats with rLH cannula and electroencephalogram/electromyogram radiotelemetry transmitters. SPA in lean rats was more sensitive to antagonism of the OX1R and in the early response to the orexin 2 agonist. OXA increased arousal equally in lean and obese rodents, which is discordant from the greater SPA response in lean rats. Obesity-resistant rats ran more and wheel running was directly related to 24-h SPA levels. The OX1R antagonist, SB-334867-A, and the DA1R antagonist, SCH3390, in SN more effectively reduced SPA stimulated by OXA in obesity-resistant rats. These data suggest OXA-stimulated SPA is not secondary to enhanced arousal, propensity for SPA parallels inclination to run and that orexin action on dopaminergic neurons in SN may participate in the mediation of SPA and running wheel activity.

Activation of organum vasculosum of lamina terminalis, median preoptic nucleus, and medial preoptic area in anticipation of nursing in rabbit pups.

Rhythmic feeding in rabbit pups is a natural model to study food entrainment because, similar to rodents under a schedule of food restriction, these animals show food-anticipatory activity (FAA) prior to daily nursing. In rodents, several brain systems, including the orexinergic system, shift their activity to the restricted feeding schedule, and remain active when subjects are hungry. As the lamina terminalis and regions of the preoptic area participate in the control of behavioral arousal, it was hypothesized that these brain regions are also activated during FAA. Thus, the effects of daily milk ingestion on FOS protein expression in the organum vasculosum of lamina terminalis (OVLT), median preoptic nucleus (MnPO), and medial preoptic area (MPOA) were examined using immunohistochemistry before and after scheduled time of nursing in nursed and fasted subjects. Additionally, FOS expression was explored in orexin (ORX) cells in the lateral hypothalamic area and in the supraoptic nucleus (SON) because of their involvement in arousal and fluid ingestion, respectively. Pups were entrained by daily nursing, as indicated by a significant increase in locomotor behavior before scheduled time of nursing in both nursed and fasted subjects. FOS was significantly higher in the OVLT, MnPO, and MPOA at the time of nursing, and decreased 8 h later in nursed pups. In fasted subjects, this effect persisted in the OVLT, whereas in the MnPO and MPOA, values did not drop at 8 h later, but remained at the same level or higher than those at the time of scheduled nursing. In addition, FOS was significantly higher in ORX cells during FAA in nursed pups in comparison with 8 h later, but in fasted subjects it remained high during most fasting time points. Additionally, OVLT, SON, and ORX cells were activated 1.5 h after nursing. We conclude that the OVLT, MnPO, and MPOA, but not SON, may participate in FAA, as they show activation before suckling of periodic milk ingestion, and that sustained activation of the OVLT, MnPO, and MPOA by fasting may contribute to the high arousal state associated with food deprivation. In agreement with this, ORX cells also remain active after expected nursing, which is consistent with reports in other species.

Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure.

Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which L-glutamate (0.25 M; 10 nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207 ± 78), 32.3 ± 2.3% co-expressed L-Enk immunoreactivity and of these, 2.6 ± 1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo-medullary pathway may represent the anatomical substrate by which hcrt-1/L-Enk neurons function in the coordination of autonomic-cardiovascular responses during different behavioral states.

Orexin-A suppresses postischemic glucose intolerance and neuronal damage through hypothalamic brain-derived neurotrophic factor.

Orexin-A (a glucose-sensing neuropeptide in the hypothalamus) and brain-derived neurotrophic factor (BDNF; a member of the neurotrophin family) play roles in many physiologic functions, including regulation of glucose metabolism. We previously showed that the development of postischemic glucose intolerance is one of the triggers of ischemic neuronal damage. The aim of this study was to determine whether there was an interaction between orexin-A and BDNF functions in the hypothalamus after cerebral ischemic stress. Male ddY mice were subjected to 2 hours of middle cerebral artery occlusion (MCAO). Neuronal damage was estimated by histologic and behavioral analyses. Expression of protein levels was analyzed by Western blot. Small interfering RNA directed BDNF, orexin-A, and SB334867 [N-(2-methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea; a specific orexin-1 receptor antagonist] were administered directly into the hypothalamus. The level of hypothalamic orexin-A, detected by immunohistochemistry, was decreased on day 1 after MCAO. Intrahypothalamic administration of orexin-A (1 or 5 pmol/mouse) significantly and dose-dependently suppressed the development of postischemic glucose intolerance on day 1 and development of neuronal damage on day 3. The MCAO-induced decrease in insulin receptor levels in the liver and skeletal muscle on day 1 was recovered to control levels by orexin-A, and this effect of orexin-A was reversed by the administration of SB334867 as well as by hypothalamic BDNF knockdown. These results suggest that suppression of postischemic glucose intolerance by orexin-A assists in the prevention of cerebral ischemic neuronal damage. In addition, hypothalamic BDNF may play an important role in this effect of orexin-A.

EEG effect of orexin A in freely moving rats.

Orexin A and orexin B are neuropeptides produced by a group of neurons located in the lateral hypothalamus which send widespread projections virtually to the whole neuraxis. Several studies indicated that orexins play a crucial role in the sleep-wake regulation and in the pathomechanism of the sleep disorder narcolepsy. As no data are available related to the EEG effects of orexin A in healthy, freely moving rats, the aim of the present experiments was to analyze EEG power changes in the generally used frequency bands after intracerebroventricular orexin A administration.Orexin A administration (0.84 and 2.8 nM/rat) differently affected fronto-occipital EEG waves in the different frequency bands recorded for 24 hours. Delta (1-4 Hz) and alpha (10-16 Hz) power decreased, while theta (4-10 Hz) and beta (16-48 Hz) power increased. Decrease of the delta power was followed by a rebound in case of the higher orexin A dose. This complex picture might be explained by the activation of several systems by the orexin A administration. Among these systems, cortical and thalamic circuits as well as the role of the neurons containing corticotrophin-releasing factor might be of significant importance.

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model.

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.

Amygdalar excitatory/inhibitory circuits interacting with orexinergic neurons influence differentially feeding behaviors in hamsters.

Recently, environmental stimuli on different neurobiological events, via participation of distinct amygdalar (AMY) ORXergic fibers have aroused wide interests in view of their ability to modify neuronal linked stressful and physiological homeostatic conditions. Results of the present study indicate that ORXergic (ORX-A/B) circuits of the facultative hibernating golden hamster (Mesocricetus auratus) central AMY (CeA) and basolateral AMY (BlA) nuclei constitute major sites of feeding behaviors. Indeed, hamsters after treatment of BlA with ORX-A frequently ingested greater quantities of food as compared to controls, while ORX-B in CeA induced a very (p<0.001) great consumption of water. The same nuclei treated separately with either ORX-A or ORX-B ± the selective α(1) GABA(A) benzodiazepine receptor agonist (zolpidem) dedicated less time to eating and drinking sessions. Conversely, hamsters that received the same neuropeptides but this time with the glutamatergic agonist NMDA displayed greater hyperphagic effects above all for ORX-A. When behavioral changes were compared to the expression of the specific ORXergic receptor (ORX-2R), an up-/down-regulating pattern was detected in some limbic areas (AMY, hippocampus and hypothalamus) following treatment with ORX-A or ORX-B plus NMDA. Overall, indications deriving from this study strongly point to hamster BlA-enriched ORX-A fibers in combination with either inhibitory or excitatory signals as main targets of hyperphagic responses while CeA ORX-B activities in presence of these same neuronal signals predominantly induced drinking motivational behaviors. The distinct behavioral activities of these two neuropeptides may have useful clinical bearings toward psychiatric and sleeping disorders such as bulimia and narcolepsy.

Effect of orexin-B-saporin-induced lesions of the lateral hypothalamus on performance on a progressive ratio schedule.

It has been suggested that a sub-population of orexinergic neurones whose somata lie in the lateral hypothalamic area (LHA) play an important role in regulating the reinforcing value of both food and drugs. This experiment examined the effect of disruption of orexinergic mechanisms in the LHA on performance on the progressive ratio schedule of reinforcement, in which the response requirement increases progressively for successive reinforcers. The data were analysed using a mathematical model which yields a quantitative index of reinforcer value and dissociates effects of interventions on motor and motivational processes. Rats were trained under a progressive ratio schedule using food-pellet reinforcement. They received bilateral injections of conjugated orexin-B-saporin (OxSap) into the LHA or sham lesions. Training continued for a further 40 sessions after surgery. Equations were fitted to the response rate data from each rat, and the parameters of the model were derived for successive blocks of 10 sessions. The OxSap lesion reduced the number of orexin-containing neurones in the LHA by approximately 50% compared with the sham-lesioned group. The parameter expressing the incentive value of the reinforcer was not significantly altered by the lesion. However, the parameter related to the maximum response rate was significantly affected, suggesting that motor capacity was diminished in the OxSap-lesioned group. The results indicate that OxSap lesions of the LHA disrupted food-reinforced responding on the progressive ratio schedule. It is suggested that this disruption was brought about by a change in non-motivational (motor) processes.