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STUDY OBJECTIVES: The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability. METHODS: Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV <25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs. RESULTS: The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%-17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%-11.6%, p = .013). The CV around the 110 pg/mL cutoff was <7%. CONCLUSIONS: Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as "undetectable" when measured with the Phoenix Pharmaceuticals RIA. CLINICAL TRIAL INFORMATION: This study is not registered in a clinical trial register, as it has a retrospective database design.

STUDY OBJECTIVES: The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability. METHODS: Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV <25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs. RESULTS: The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%-17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%-11.6%, p = .013). The CV around the 110 pg/mL cutoff was <7%. CONCLUSIONS: Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as "undetectable" when measured with the Phoenix Pharmaceuticals RIA. CLINICAL TRIAL INFORMATION: This study is not registered in a clinical trial register, as it has a retrospective database design.

The lateral hypothalamus products various neuropeptides, among of these the Orexin-A (hypocretin-1) is a protein that regulates numerous physiological and behavioural processes involving in energy balance and metabolic status, physical activity, blood glucose levels, and food intake. Many data literature reported that the production of Orexin-A increases following regular and structured physical activity. Indeed, it improves energy metabolism and insulin sensitivity, and also modulate energy expenditure and thermogenesis. The higher Orexin-A tone is an endogenous factor that predicts physical activity, improving BMI and the energy expenditure aspect. It is well known that physical activity has positive effects on health and body weight, it improves cognitive performance increasing Orexin-A levels. Given these evidences, the aim of this study is evaluated Orexin-A serum levels in a cohort of karate athletes. Fort these reasons, were recruited 10 young karate athletes and 10 matched non-athletes as control. We evaluated biochemical and anthropometric parameters, and Orexin-A serum levels through ELISA-test. Our results showed that there is a statistical difference between karate athletes and non-athletes in BMI and in biochemical parameters such as glycaemic and lipid profile. Interestingly, Orexin-A serum levels are statistical higher in karate athletes compared to non- athletes. The differences between athlete and non-athletes in this study could be attributed to a better training-related hypothalamic activity in karate athletes. In addition, these evidences indicate that the physical activity has beneficial effects not only on anthropometric and biochemical parameters but also on orexin-A levels, and then on central nervous system.

The Orexin-A (hypocretin-1) is a neuropeptide secreted by neurons in the lateral hypothalamus. This protein regulates physiological and behavioural processes that have an essential impact on energy balance and metabolic status, physical activity, blood glucose levels, and food intake. Furthermore, that orexin-A regulates insulin sensitivity, energy expenditure and metabolic rate and is involved in immune processes and then regulate inflammatory response, with an anti-inflammatory action. Diabetes mellitus (T2DM) is a worldwide health problem associated with obesity and sedentary lifestyle. High glycaemic levels and lipid serum profile, low col-HDL, or hypertension and increased body mass index (BMI) are significantly associated with increased T2DM risk and with increased cardiovascular mortality and morbidity in T2DM patients. For these reasons the aim of this study is to evaluate the biochemical and anthropometric parameters, orexin-A levels by ELISA test and western blotting analysis, and inflammatory cytokines levels such as TNF-a, IL-8 and IL-10 by ELISA test in subjects affected by diabetes mellitus following an accurate physical activity program at baseline, after 3 months and after 6 months. We found that there is a ameliorate of many anthropometric and biochemical parameters; furthermore, there is a statistical increase of orexin-A serum levels already after 3 months compared to baseline in T2DM subjects and also there is a strongly modulation in inflammatory cytokines expression. These found indicates that the physical activity has beneficial effects not only on anthropometric and biochemical parameters but also on orexin-A levels, and then on CNS.

The lateral hypothalamus products various neuropeptides, among of these the Orexin-A (hypocretin-1) is a protein that regulates numerous physiological and behavioural processes involving in energy balance and metabolic status, physical activity, blood glucose levels, and food intake. Many data literature reported that the production of Orexin-A increases following regular and structured physical activity. Indeed, it improves energy metabolism and insulin sensitivity, and also modulate energy expenditure and thermogenesis. The higher Orexin-A tone is an endogenous factor that predicts physical activity, improving BMI and the energy expenditure aspect. It is well known that physical activity has positive effects on health and body weight, it improves cognitive performance increasing Orexin-A levels. Given these evidences, the aim of this study is evaluated Orexin-A serum levels in a cohort of karate athletes. Fort these reasons, were recruited 10 young karate athletes and 10 matched non-athletes as control. We evaluated biochemical and anthropometric parameters, and Orexin-A serum levels through ELISA-test. Our results showed that there is a statistical difference between karate athletes and non-athletes in BMI and in biochemical parameters such as glycaemic and lipid profile. Interestingly, Orexin-A serum levels are statistical higher in karate athletes compared to non- athletes. The differences between athlete and non-athletes in this study could be attributed to a better training-related hypothalamic activity in karate athletes. In addition, these evidences indicate that the physical activity has beneficial effects not only on anthropometric and biochemical parameters but also on orexin-A levels, and then on central nervous system.

The Orexin-A (hypocretin-1) is a neuropeptide secreted by neurons in the lateral hypothalamus. This protein regulates physiological and behavioural processes that have an essential impact on energy balance and metabolic status, physical activity, blood glucose levels, and food intake. Furthermore, that orexin-A regulates insulin sensitivity, energy expenditure and metabolic rate and is involved in immune processes and then regulate inflammatory response, with an anti-inflammatory action. Diabetes mellitus (T2DM) is a worldwide health problem associated with obesity and sedentary lifestyle. High glycaemic levels and lipid serum profile, low col-HDL, or hypertension and increased body mass index (BMI) are significantly associated with increased T2DM risk and with increased cardiovascular mortality and morbidity in T2DM patients. For these reasons the aim of this study is to evaluate the biochemical and anthropometric parameters, orexin-A levels by ELISA test and western blotting analysis, and inflammatory cytokines levels such as TNF-a, IL-8 and IL-10 by ELISA test in subjects affected by diabetes mellitus following an accurate physical activity program at baseline, after 3 months and after 6 months. We found that there is a ameliorate of many anthropometric and biochemical parameters; furthermore, there is a statistical increase of orexin-A serum levels already after 3 months compared to baseline in T2DM subjects and also there is a strongly modulation in inflammatory cytokines expression. These found indicates that the physical activity has beneficial effects not only on anthropometric and biochemical parameters but also on orexin-A levels, and then on CNS.

Background: Interactions between the orexin peptides and their cognate OX1 and OX2 receptors remain poorly characterized. Site-directed mutagenesis studies on orexin peptides and receptors have indicated amino acids important for ligand binding and receptor activation. However, a better understanding of specific pairwise interactions would benefit small molecule discovery. Results: We constructed a set of three-dimensional models of the orexin 1 receptor based on the 3D-structures of the orexin 2 receptor (released while this manuscript was under review), neurotensin receptor 1 and chemokine receptor CXCR4, conducted an exhaustive docking of orexin-A(16-33) peptide fragment with ZDOCK and RDOCK, and analyzed a total of 4301 complexes through multidimensional scaling and clustering. The best docking poses reveal two alternative binding modes, where the C-terminus of the peptide lies deep in the binding pocket, on average about 5-6 angstrom above Tyr(6.48) and close to Gln(3.32). The binding modes differ in the about 100 degrees rotation of the peptide; the peptide His26 faces either the receptor's fifth transmembrane helix or the seventh helix. Both binding modes are well in line with previous mutation studies and partake in hydrogen bonding similar to suvorexant. Conclusions: We present two binding modes for orexin-A into orexin 1 receptor, which help rationalize previous results from site-directed mutagenesis studies. The binding modes should serve small molecule discovery, and offer insights into the mechanism of receptor activation.

Despite the recent successes in producing orexin receptor subtype-selective antagonists, these are not commonly available, and therefore, agonist ligands are regularly used to ascribe cell and tissue responses to OX(1) or OX(2) receptors. In the current study, we have compared the native "subtype-selective" agonist, orexin-B, and its reputedly enhanced synthetic variant, Ala(11), d-Leu(15)-orexin-B, in two different recombinant cell lines. Ca(2+) elevation was used as readout, and the two "selective" ligands were compared to the subtype-non-selective orexin-A, as is customary with these ligands. In transiently transfected HEK-293 cells, orexin-B showed 9-fold selectivity for the OX(2) receptor and Ala(11), d-Leu(15)-orexin-B 23-fold selectivity, when the potency ratios of ligands were compared between OX(1) and OX(2). In stable CHO-K1 cells, the corresponding values were only 2.6- and 14-fold, respectively. In addition to being low, the selectivity of the ligands was also variable, as indicated by the comparison of the two cell lines. For instance, the relative potency of Ala(11), d-Leu(15)-orexin-B at OX(2) in CHO cells was only 2.3-fold higher than its relative potency at OX(1) in HEK-293 cells; this indicates that Ala(11), d-Leu(15)-orexin-B does not show high enough selectivity for OX(2) to be useful for determination of receptor subtype expression. Comparison of the potencies of orexin-A and -B with respect to a number of published responses in OX(1)-expressing CHO cells, demonstrates that these show great variation: i.e., orexin-A is 1.6-18-fold more potent than orexin-B, depending on the response assessed. These data together suggest that orexin receptor ligands show signal trafficking, which makes agonist-based pharmacology unreliable.

We wanted to analyze the basis for the distinction between OX(1) and OX(2) orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), d-Leu(15)-orexin-B, of which the latter two show some selectivity for OX(2). For this, chimaeric OX(1)/OX(2) and OX(2)/OX(1) orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca(2+) elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.

We wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), D-Leu(15)-orexin-B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Obesity and metabolic diseases are known to be tightly linked to arousal-sleep cycles. Further, both metabolic disease and arousal are known to have significant impacts on cognitive function in humans and animals. Importantly, the armed forces represent a population at significant risk for increased stress and disrupted arousal-sleep cycles. Because the incidence of metabolic disease and obesity is increasing, even in these physically fit individuals, understanding the interactions between these systems is highly significant. Further, some anti-fatigue pharmacologies (e.g.,modafinil) are already used in military settings, though their long-term effects on metabolism or central nervous system function are not well-understood. We have completed Year 1 of the proposed funding period to assess the physiological and behavioral effects of this pharmacology on rat subjects. Our first year data demonstrate that chronic administration of intraperitoneal modafinil decreases food intake and body weight in rats. Additionally, we observed that acute central modafinil has deleterious effects on some hippocamapaldependent forms of learning. These findings support out overall hypothesis that pharmacological activation of the central orexin system may modulate energy balance. Ongoing studies are assessing the effects of treatment on insulin sensitivity and also the effects of drug withdrawal on body weight regulation and cognitive function., The original document contains color images.

Obesity and metabolic diseases are known to be tightly linked to arousal-sleep cycles. Further both metabolic disease and arousal are known to have significant impacts on cognitive function in humans and animals. Importantly the armed forces represent a population at significant risk for increased stress and disrupted arousal-sleep cycles. Because the incidence of metabolic disease and obesity is increasing even in these physically fit individuals understanding the interactions between these systems is highly significant. Further some anti-fatigue pharmacologies (e.g. modafinil) are already used in military settings though their long-term effects on metabolism or central nervous system function are not well-understood. We have completed Year 2 of the proposed funding period to assess the physiological and behavioral effects of this pharmacology on rat subjects and identify potential molecular mechanisms activated by nutrients. Our data demonstrate that chronic administration of intraperitoneal modafinil does not alter dietary induced obesity or impair glucose tolerance. Additionally we observed that chronic central modafinil does not increase stress in rats but does attenuate object recognition memory. Ongoing studies are assessing the effects of pharmacological inhibition or activation of a potential molecular mechanism mTOR., The original document contains color images.

Obesity and metabolic diseases are known to be tightly linked to arousal-sleep cycles. Further, both metabolic disease and arousal are known to have significant impacts on cognitive function in humans and animals. Importantly, the armed forces represent a population at significant risk for increased stress and disrupted arousal-sleep cycles. Because the incidence of metabolic disease and obesity is increasing, even in these physically fit individuals, understanding the interactions between these systems is highly significant. Further, some anti-fatigue pharmacologies (e.g. modafinil) are already used in military settings, though their long-term effects on metabolism or central nervous system function are not well-understood. We have completed Year 1 of the proposed funding period to assess the physiological and behavioral effects of this pharmacology on rat subjects. Our first year data demonstrate that chronic administration of intraperitoneal modafinil decreases food intake and body weight in rats. Additionally, we observed that acute central modafinil has deleterious effects on some hippocamapal-dependent forms of learning. These findings support our overall hypothesis that pharmacological activation of the central orexin system may modulate energy balance. Ongoing studies are assessing the effects of treatment on insulin sensitivity and also the effects of drug withdrawal on body weight regulation and cognitive function., The original document contains color images.