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Abstract

Background: Hemopressin, VD-hemopressin(α) and RVD-hemopressin(α) are hemoglobin α chain derived-peptides which have been found in mouse brain, and where they modulate cannabinoid (CB) receptor function. The nonapeptide hemopressin has been reported to inhibit feeding after both central and peripheral administration, possibly playing a role of antagonist/inverse agonist of CB1 receptors, and consequently blocking the orexigenic effects of endogenous cannabinoids. VD-hemopressin(α) and RVD- hemopressin(α), are N-terminal extended forms of hemopressin. VD-hemopressin(α) has CB1 agonist activity, and as such it has been shown to stimulate feeding. RVD-hemopressin(α) is reported to play a negative allosteric modulatory function on CB1 receptors, but there are no data on its possible effects on feeding and metabolic control. Methods: We have studied, in rats, the effects of 14 daily intraperitoneal (ip) injections of RVD-hemopressin(α) (10nmol). Results: We found that RVD-hemopressin(α) treatment inhibited food intake while total body weight was not affected. The null effect on body weight despite diminished feeding could be related to decreased uncoupling protein 1 (UCP-1) gene expression in brown adipose tissue (BAT). We also investigated the underlying neuromodulatory effects of RVD-hemopressin(α) and found it to down regulate proopiomelanocortin (POMC) gene expression, together with norepinephrine (NE) levels, in the hypothalamus. Conclusions: In conclusion, RVD-hemopressin(α) administration has an anorectic effect, possibly related to inhibition of POMC and NE levels in the hypothalamus. Despite decreased food intake, body weight is not affected by RVD-hemopressin(α) treatment, possibly due to inhibition of UCP-1 gene expression in BAT.

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... In this regard, Koch and colleagues [29] suggested that cannabinoid-induced feeding could be mediated by increased levels of the POMC-derived peptide β-endorphin. Actually, the inhibition of the POMC gene expression following CBD and CBG treatment is consistent with their putative role as negative allosteric modulators of CB1 receptor [30]. In contrast, the inhibition of the expression of the NPY gene registered after the treatment, although being consistent with the anorexigenic effects ascribed to CBD [6], appears to be discrepant with the orexigenic role of CBG [16,17]. ...
... Having both CBD and CBG demonstrated their effectiveness in reducing NE steady state level in isolated rat hypothalamus, this monoamine can be suggested as a possible mediator of the effects of the two terpenophenols on food intake. In a previous study, the RVD-hemopressin-α, an endogenous anorexigenic peptide, proved to be a negative allosteric modulator of CB1 [43] and to inhibit hypothalamic NE levels following peripheral administration despite being ineffective against DA and 5-HT levels [30]. The two terpenophenols objects of this study were also ineffective against the DA level, whereas the sole CBD stimulated 5-HT levels, and this could explain, albeit partially, the aforementioned anorexigenic effects [14]. ...
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Background: Cannabidiol (CBD) and cannabigerol (CBG) are non-psychotropic terpenophenols isolated from Cannabis sativa, which, besides their anti-inflammatory/antioxidant effects, are able to inhibit, the first, and to stimulate, the second, the appetite although there are no studies elucidating their role in the hypothalamic appetite-regulating network. Consequently, the aim of the present research is to investigate the role of CBD and CBG in regulating hypothalamic neuromodulators. Comparative evaluations between oxidative stress and food intake-modulating mediators were also performed. Methods: Rat hypothalamic Hypo-E22 cells and isolated tissues were exposed to either CBD or CBG, and the gene expressions of neuropeptide (NP)Y, pro-opiomelanocortin (POMC) and fatty acid amide hydrolase were assessed. In parallel, the influence of CBD on the synthesis and release of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) was evaluated. The 3-hydroxykinurenine/kinurenic acid (3-HK/KA) ratio was also determined. Results: Both CBD and CBG inhibited NPY and POMC gene expression and decreased the 3-HK/KA ratio in the hypothalamus. The same compounds also reduced hypothalamic NE synthesis and DA release, whereas the sole CBD inhibited 5-HT synthesis. Conclusion: The CBD modulates hypothalamic neuromodulators consistently with its anorexigenic role, whereas the CBG effect on the same mediators suggests alternative mechanisms, possibly involving peripheral pathways.
... In this regard, Koch and colleagues [29] suggested that cannabinoid-induced feeding could be mediated by increased levels of the POMC-derived peptide β-endorphin. Actually, the inhibition of the POMC gene expression following CBD and CBG treatment is consistent with their putative role as negative allosteric modulators of CB1 receptor [30]. In contrast, the inhibition of the expression of the NPY gene registered after the treatment, although being consistent with the anorexigenic effects ascribed to CBD [6], appears to be discrepant with the orexigenic role of CBG [16,17]. ...
... Having both CBD and CBG demonstrated their effectiveness in reducing NE steady state level in isolated rat hypothalamus, this monoamine can be suggested as a possible mediator of the effects of the two terpenophenols on food intake. In a previous study, the RVD-hemopressin-α, an endogenous anorexigenic peptide, proved to be a negative allosteric modulator of CB1 [43] and to inhibit hypothalamic NE levels following peripheral administration despite being ineffective against DA and 5-HT levels [30]. The two terpenophenols objects of this study were also ineffective against the DA level, whereas the sole CBD stimulated 5-HT levels, and this could explain, albeit partially, the aforementioned anorexigenic effects [14]. ...
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Tanacetum parthenium (feverfew) has traditionally been employed as a phytotherapeutic remedy in the treatment of migraine. In this study, a commercial T. parthenium water extract was investigated to explore its anti-inflammatory and neuromodulatory effects. Isolated mouse cortexes were exposed to a K+ 60 mM Krebs-Ringer buffer and treated with T. parthenium water extract. The prostaglandin E2 (PGE2) level, brain-derived neurotrophic factor (BDNF), interleukin-10 (IL-10), and IL-1β gene expression were evaluated in the cortex. The effects on dopamine (DA) release and dopamine transporter (DAT) gene expression were assayed in hypothalamic HypoE22 cells. A bioinformatics analysis was conducted to further investigate the mechanism of action. The extract was effective in reducing cortex PGE2 release and IL-1β gene expression. In the same experimental system, IL-10 and BDNF gene expressions increased, and in HypoE22 cells, the extract decreased the extracellular dopamine level and increased the DAT gene expression due to the direct interaction of parthenolide with the DAT. Overall, the present findings highlight the efficacy of T. parthenium water extract in controlling the inflammatory pathways that occur during cortical-spreading depression. Additionally, the inhibition of the hypothalamic DA release observed in this study further supports the role of dopaminergic pathways as key targets for novel pharmacological approaches in the management of migraine attacks.
... The discovery of hemopressin (hp), an hemoglobin α chain-derived peptide and RVDhemopressin(α) [RVD-hp(α)], the N-terminally extended peptide of Hp, also known as PEPCAN-12, revealed a promising research field for the pharmacotherapy of obesity [10,11]. Hp and RVD-hp(α) were found to bind CB1 receptors, as antagonist/inverse agonist and negative allosteric modulator, respectively [12][13][14]. ...
... In the present study we have shown that RVD-hp(α), a CB1/CB2 allosteric modulator [13][14][15], is able to modulate emotional and feeding behavior. In confirming the anxiolytic and anorexigenic effects, previously described by our research group [11,16], we also observed a decrease in locomotor activity after central administration. The contrasting results with our recent published studies could be possibly related to the different route of administration, thus further supporting the hypothesis of multiple central and peripheral mechanisms underlying behavioral peptide effects in vivo, including the possible modulation of adipokines and gut-derived hormones, both involved in metabolic and behavioral pathways [28,29,38]. ...
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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) (10 nmol) 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.
... Another strategy to avoid the side effects of CB 1 antagonists is through allosteric modulation of the CB 1 receptor. The negative allosteric modulators ORG27569 [89], RVDhemopressin(α) [90], and PSNCBAM-1 [91] reduce food intake with or without a reduction in body weight in rats. ...
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The cannabinoid 1 (CB1) receptor regulates appetite and body weight; however, unwanted central side effects of both agonists (in wasting disorders) or antagonists (in obesity and diabetes) have limited their therapeutic utility. At the peripheral level, CB1 receptor activation impacts the energy balance of mammals in a number of different ways: inhibiting satiety and emesis, increasing food intake, altering adipokine and satiety hormone levels, altering taste sensation, decreasing lipolysis (fat break down), and increasing lipogenesis (fat generation). The CB1 receptor also plays an important role in the gut–brain axis control of appetite and satiety. The combined effect of peripheral CB1 activation is to promote appetite, energy storage, and energy preservation (and the opposite is true for CB1 antagonists). Therefore, the next generation of CB1 receptor medicines (agonists and antagonists, and indirect modulators of the endocannabinoid system) have been peripherally restricted to mitigate these issues, and some of these are already in clinical stage development. These compounds also have demonstrated potential in other conditions such as alcoholic steatohepatitis and diabetic nephropathy (peripherally restricted CB1 antagonists) and pain conditions (peripherally restricted CB1 agonists and FAAH inhibitors). This review will discuss the mechanisms by which peripheral CB1 receptors regulate body weight, and the therapeutic utility of peripherally restricted drugs in the management of body weight and beyond.
... Given the unique characteristics of pepcan-12 as an allosteric modulator of CB1Rs and CB2Rs, several groups evaluated its therapeutic potential. Pepcan-12 has been shown to reduce food consumption in rats, increase locomotor activity [144,145], and induce anxiolytic and antidepressive effects, as assessed in locomotor activity/open field, light-dark exploration, and forced swim tests [146]. To date, no study has evaluated its therapeutic potential for the treatment of SUDs. ...
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Substance use disorder (SUD) is a major public health crisis worldwide, and effective treatment options are limited. During the past 2 decades, researchers have investigated the impact of a variety of pharmacological approaches to treat SUD, one of which is the use of medical cannabis or cannabinoids. Significant progress was made with the discovery of rimonabant, a selective CB1 receptor (CB1R) antagonist (also an inverse agonist), as a promising therapeutic for SUDs and obesity. However, serious adverse effects such as depression and suicidality led to the withdrawal of rimonabant (and almost all other CB1R antagonists/inverse agonists) from clinical trials worldwide in 2008. Since then, much research interest has shifted to other cannabinoid-based strategies, such as peripheral CB1R antagonists/inverse agonists, neutral CB1R antagonists, allosteric CB1R modulators, CB2R agonists, fatty acid amide hydrolase (FAAH) inhibitors, monoacylglycerol lipase (MAGL) inhibitors, fatty acid binding protein (FABP) inhibitors, or nonaddictive phytocannabinoids with CB1R or CB2R-binding profiles, as new therapeutics for SUDs. In this article, we first review recent progress in research regarding the endocannabinoid systems, cannabis reward versus aversion, and the underlying receptor mechanisms. We then review recent progress in cannabinoid-based medication development for the treatment of SUDs. As evidence continues to accumulate, neutral CB1R antagonists (such as AM4113), CB2R agonists (JWH133, Xie2-64), and nonselective phytocannabinoids (cannabidiol, β-caryophyllene, ∆9-tetrahydrocannabivarin) have shown great therapeutic potential for SUDs, as shown in experimental animals. Several cannabinoid-based medications (e.g., dronabinol, nabilone, PF-04457845) that entered clinical trials have shown promising results in reducing withdrawal symptoms in cannabis and opioid users.
... In fact, the neurosteroid pregnenolone blocked THC-promoted feeding behaviour both in sated Wistar rats as well as in 24-h fasting mice, although pregnenolone did not have any effect when it was administrated alone [212]. In addition, hemopressin is a small eCB-like peptide, called pepcans, and it has been demonstrated to reduce food intake in rats [51, 57,106,175]. Further experiments are required to determine the effects of such negative CB1 allosteric modulators in the context of obesity and their use as potential anti-obesity treatment. ...
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The endocannabinoid (eCB) system is widely expressed in many central and peripheral tissues, and is involved in a plethora of physiological processes. Among these, activity of the eCB system promotes energy intake and storage, which, however, under pathophysiological conditions, can favour the development of obesity and obesity-related disorders. It is proposed that eCB signalling is evolutionary beneficial for survival under periods of scarce food resources. Remarkably, eCB signalling is increased both in hunger and in overnutrition conditions, such as obesity and type-2 diabetes. This apparent paradox suggests a role of the eCB system both at initiation and at clinical endpoint of obesity. This review will focus on recent findings about the role of the eCB system controlling whole-body metabolism in mice that are genetically modified selectively in different cell types. The current data in fact support the notion that eCB signalling is not only engaged in the development but also in the maintenance of obesity, whereby specific cell types in central and peripheral tissues are key sites in regulating the entire body’s energy homeostasis.
... The allosteric binding site for Pepcan-12 is still unknown, whereas, since the introduction of chemical modifications at the N-terminus greatly reduced its affinity toward CB1, this latter portion is likely responsible for receptor binding (Bauer et al., 2012). Exogenous administration of Pepcan-12 induces anorexigenic and antinociceptive effects in rat models, with little or no side effects (Han et al., 2014;Ferrante et al., 2017), and has been able to restore impaired memory functions in Aβ 1−42 treated mice, highlighting its pharmacological versatility also in the treatment of Alzheimer Disease-induced memory deficits (Zhang et al., 2016). Given the proven involvement of the ECS in several neurodegenerative diseases, the full pharmacological potential of pepcans may yet to be fully revealed. ...
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Endocannabinoid peptides, or “pepcans,” are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1.
... The field is now entering a new stage in which this new knowledge is being translated into novel drugs -many entering clinical development phase-, and drug discovery programs are being designed with the resolution approach in mind . The melanocortin system (MC) constitutes one of such endogenous pro-resolving mechanisms, in addition to its multiple roles including energy homeostasis, skin pigmentation or steroidogenesis, thus presenting very diverse potential therapeutic applications (Leone et al., 2013;Montero-Melendez, 2015;Ferrante et al., 2016Ferrante et al., , 2017. However, the development of the "ideal" MC molecule is a proven challenge. ...
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... In fact, this peptide is able to displace [ 3 H]SR141716 binding at CB1 receptor with a subnanomolar affinity value and shows a biological profile similar to that of rimonabant [67]. Hp 19 has proved to inhibit food intake in normal and obese animal models [68,69] as well as to exhibit antinociceptive effects in inflammatory pain models [70] without causing the neurological drawbacks normally associated with the CB1 activation [66]. This interesting aspect together with the fact that Hp antinociceptive effects are ...
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Endocannabinoid System (ES) has gained over the years a leading position in scientific research thanks to its involvement in numerous patho/physiological conditions. Accordingly, its main components, such as receptors, enzymes and mediators, have become important drug targets for the management of diseases where it is dysregulated. Within the manuscript, several classes of cannabinergic ligands are examined, emphasizing molecules coming from the natural world, unique source of active compounds. Firstly, the endogenous lipid ES modulators are described, starting from the major endocannabinoids to the plethora of endocannabinoid congeners. Afterwards, Cannabis-derived cannabinoids, namely well-known phytocannabinoids and new constituents from different varieties of Cannabis, are reviewed also mentioning the huge effort of pharmaceutical research in obtaining synthetic analogues. Finally, an overview of peptides and miscellaneous natural products points out new opportunities to modulate ES, offering an enormous chemical heterogeneity. Accordingly, hemopressin and related peptides, plant-derived alkylamides, terpenoid derivatives, neolignans and examples from the marine world can provide interesting hints and original ideas to develop new cannabinergic compounds.
... Hp and RVD-hp(a) have been obtained in our laboratory by Fmoc-solid phase peptide synthesis (Fmoc-SPPS) strategy on 2-CTC (2-chlorotrityl chloride) resin, as previously reported [16][17][18][19]. Chromatographic purification was performed by RP-HPLC semipreparative C18 column (eluent: ACN/H 2 O gradient, 5-95% over 32 min) at a flow gradient of 4 ml/min. ...
Article
Background: The endocannabinoid (eCB) system plays an important role in regulating emotional disorders, and is involved, directly or indirectly, in psychiatric diseases, such as anxiety and depression. Hemopressin, a hemoglobin α chain-derived peptide, and RVD-hemopressin(α), a N-terminally extended form of hemopressin, act as antagonist/inverse agonist and negative allosteric modulator of the cannabinoid 1 (CB1) receptor, respectively. Methods: Considering the possible involvement of these peptides on emotional behaviour, the aim of our study was to investigate the behavioural effects of a single intraperitoneal (ip) injection of hemopressin (0.05mg/kg) and RVD-hemopressin(α) (0.05mg/kg), using a series of validated behavioural tests (locomotor activity/open field test, light-dark exploration test, forced swim test) in rats. Prefrontal cortex levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and the gene expression of monoamine oxidase (MAO-B) and catechol-O-methyltransferase (COMT) were measured by high performance liquid chromatography (HPLC) analysis and real-time reverse transcription polymerase chain reaction (RT-PCR), respectively. Results: Hemopressin administration induced anxiogenic and depressive behaviour, decreased monoamine steady state levels in prefrontal cortex, and increased the gene expression of the enzymes involved in their catabolism. By contrast, RVD- hemopressin(α) induced anxiolytic and antidepressive effects, increased monoamines and decreased the enzymes in prefrontal cortex. Conclusion: In conclusion, in the present study we demonstrated behavioral effects induced by peripheral hemopressin and RVD-hemopressin(α) injections, that could involve modulatory effects on monoaminergic signaling, in the prefrontal cortex.
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New information regarding neuronal circuits that control food intake and their hormonal regulation has extended our understanding of energy homeostasis, the process whereby energy intake is matched to energy expenditure over time. The profound obesity that results in rodents (and in the rare human case as well) from mutation of key signalling molecules involved in this regulatory system highlights its importance to human health. Although each new signalling pathway discovered in the hypothalamus is a potential target for drug development in the treatment of obesity, the growing number of such signalling molecules indicates that food intake is controlled by a highly complex process. To better understand how energy homeostasis can be achieved, we describe a model that delineates the roles of individual hormonal and neuropeptide signalling pathways in the control of food intake and the means by which obesity can arise from inherited or acquired defects in their function.
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Cannabidiol has been reported to act as an antagonist of cannabinoid agonists at type 1 cannabinoid receptors (CB1 ). We hypothesized that cannabidiol can inhibit cannabinoid agonist activity through negative allosteric modulation of CB1 . CB1 internalization, arrestin2 recruitment, and PLCβ3 and ERK1/2 phosphorylation, were quantified in HEK 293A cells heterologously expressing CB1 and in the STHdh(Q7/Q7) cell model of striatal neurons endogenously expressing CB1 . Cells were treated with 2-arachidonylglycerol or Δ(9) -tetrahydrocannabinol alone and in combination with different concentrations of cannabidiol. Cannabidiol reduced the efficacy and potency of 2-arachidonylglycerol and Δ(9) -tetrahydrocannabinol on PLCβ3- and ERK1/2-dependent signaling in cells heterologously (HEK 293A) or endogenously (STHdh(Q7/Q7) ) expressing CB1 . By reducing arrestin2 recruitment to CB1 , cannabidiol treatment prevented CB1 internalization. The allosteric activity of cannabidiol depended upon polar residues being present at positions 98 and 107 in the extracellular amino-terminus. Cannabidiol behaved as a non-competitive negative allosteric modulator of CB1 . Allosteric modulation, in conjunction with non-CB1 effects, may explain the in vivo effects of cannabidiol. Allosteric modulators of CB1 have the potential to treat central nervous system and peripheral disorders while avoiding the adverse effects associated with orthosteric agonism or antagonism of CB1 . This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Central regulatory mechanisms for neurotransmit-ters of food intake vary among animals. Endocannabinoids have crucial role on central food intake regulation in mammals but its role has not been studied in layer-type chicken. Thus, in this study 6 experiments designed to evaluate effects of intra-cerebroventricular (ICV) administration of 2-AG (2-Arachidonoylglycerol, selective CB 1 receptors agonist), SR141716A (selective CB 1 receptors antagonist), JWH015 (selective CB 2 receptors agonist), AM630 (selective CB 2 receptors antagonist) on feeding behavior in 3 h food deprived neonatal layer-type chickens. In experiment 1, birds ICV injected with control solution and 2-AG (0.25, 0.5 and 1 μg). In experiment 2: control solution, SR141716A (6.25, 12.5 and 25 μg) were ICV injected to birds. In experiment 3 animals received: control solution, SR141716A (6.25 μg), 2-AG (1 μg) and co-injection of SR141716A+2-AG. In experiment 4, chickens received control solution and JWH015 (6.25, 12.5 and 25 μg). In experiment 5, control solution and AM630 (1.25, 2.5 and 5 μg) were injected. In experiment 6, the birds received control solution, AM630 (1.25 μg), JWH015 (25 μg) and co-administration of AM630+ JWH015. Then, cumulative food intake was recorded until 120 min after injection. According to the results, 2-AG dose dependently increased cumulative food intake while SR141716A reduced appetite compared to control group (P<0.05). Injection of 2-AG (1 μg) amplified food intake and its effect minimized by SR141716A (6.25 μg) (P<0.05). Also, ICV injection of JWH015 (25 μg) dose dependently increased food intake and co-injection of JWH015+ AM630 decreased JWH015-induced food intake (P<0.05). These results suggest CB 1 and CB 2 receptors have an important role on ingestive behavior in FD 3 neonatal layer-type chicken.
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Hemopressin (PVNFKFLSH; HP) is an orally active peptide derived from rat hemoglobin α-chain that could act as an inverse agonist at cannabinoid type 1 receptors (CB1). Here, we aim to investigate possible behavioral effects of HP in male Wistar rats tested in the elevated plus maze (EPM), following HP intraperitoneal (i.p., 0.05 mg/kg), oral (P.O., 0.05 and 0.5 mg/kg) or intracerebroventricular (I.C.V., 3 and 10 nmol) administration. HP induced a decrease in EPM open arm exploration, indicating an anxiogenic-like effect. However, i.p. administration of HP (1 mg/kg) followed by mass spectrometry analysis of brain-peptide extracts suggested that the intact HP does not cross the blood brain barrier. I.C.V. administrated HP produced anxiogenic-like effects that were prevented by Transient Receptor Potential Vanilloid Type 1 (TRPV1) antagonists, 6-iodonordihydrocapsaicin (1 nmol) or SB366791 (1 nmol), but not by the CB1 receptor antagonist AM251 (0.1 and 1 nmol). Altogether, these data suggest that I.C.V. administrated HP induces anxiogenic-like effects by activating TRPV1 receptors. The similar anxiogenic effects observed after i.p. or P.O. administration could be due to HP fragment(s) crossing the blood brain barrier. The present results advance our knowledge about HP pharmacology and suggest concerns in future clinical studies.
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N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multi-target analgesic compounds, we designed the first ω-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of ω-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity. This article is protected by copyright. All rights reserved.
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Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are opioid peptides which are selective partial agonists of μ-opioid receptor. We studied the effects of EM-2 injected into the arcuate nucleus (ARC) of the hypothalamus on feeding behavior and gene expression of orexigenic [agouti-related peptide (AgRP), neuropeptide Y (NPY) and orexin-A] and anorexigenic [cocaine and amphetamine-regulated transcript (CART), corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC)] peptides in male Wistar rats fed a standard laboratory diet. Furthermore, we evaluated the effects of EM-2 on dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) steady state concentrations, in the hypothalamus. 64 rats (16 for each group of treatment) were injected into the ARC, at 9.00 am, with either vehicle or EM-2 (0.50-0.75μmol/kg) or EM-2 (0.50μmol/kg) plus β-funaltrexamine (0.20μmol/kg). Food intake was recorded through 24h following injection, and hypothalamic DA, NE, 5-HT levels and neuropeptide gene expression were evaluated 24h after EM-2 administration. Compared to vehicle, EM-2 significantly increased food intake, throughout 24h post-injection. Furthermore, EM-2 treatment led to a significant increase of DA and NE concentrations and a decrease of CRH mRNA levels. On the other hand, β-funaltrexamine administration reverted both feeding stimulatory and neuromodulatory effects induced by EM-2. We can conclude that the orexigenic effect of μ-opioid receptor activation by EM-2 could be related to both inhibition of CRH and stimulation of dopamine and norepinephrine levels, in the hypothalamus.
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Allosteric ligands bind to G protein-coupled receptors (GPCRs; also known as seven-transmembrane receptors) at sites that are distinct from the sites to which endogenous ligands bind. The existence of allosteric ligands has enriched the ways in which the functions of GPCRs can be manipulated for potential therapeutic benefit, yet the complexity of their actions provides both challenges and opportunities for drug screening and development. Converging avenues of research in areas such as biased signalling by allosteric ligands and the mechanisms by which allosteric ligands modulate the effects of diverse endogenous ligands have provided new insights into how interactions between allosteric ligands and GPCRs could be exploited for drug discovery. These new findings have the potential to alter how screening for allosteric drugs is performed and may increase the chances of success in the development of allosteric modulators as clinical lead compounds.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
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Obesity represents nowadays one of the most devastating health threats. Published reports even project a decline in life expectancy of US citizens due to the rapidly increasing prevalence of obesity. This alarming increase is intimately linked with recent changes of environment and lifestyle in western countries. In this context, the rewarding or even addictive properties of popular food may represent one of the most serious obstacles to overcome for an effective anti-obesity therapy. Therefore, in addition to molecular networks controlling energy homeostasis, now researchers are starting to define central nervous mechanisms governing hedonic and addictive components of food intake. A recently emerging body of data suggests that the endogenous cannabinoid and opioid systems both represent key circuits responding to the rewarding value of food. This review focuses on the role of these two systems for the homeostatic and hedonic aspects of eating behavior and includes their anatomical and functional interactions. Independent from the degree to which eating can be considered an addiction, cannabinoid and opioid receptor antagonists are promising anti-obesity drugs, since they are targeting both hedonic and homeostatic components of energy balance control.
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An interactive blood-brain barrier (BBB) helps regulate the passage of peptides from the periphery to the CNS and from the CNS to the periphery. Many peptides cross the BBB by simple diffusion, mainly explained by their lipophilicity and other physicochemical properties. Other peptides cross by saturable transport systems. The systems that transport peptides into or out of the CNS can be highly specific, transporting MIF-1 but not Tyr-MIF-1, PACAP38 but not PACAP27, IL-1 but not IL-2, and leptin but not the smaller ingestive peptides NPY, orexin A, orexin B, CART (55-102[Met(O)(67)]), MCH, or AgRP(83-132). Although the peptides EGF and TGF-alpha bind to the same receptor, only EGF enters by a rapid saturable transport system, suggesting that receptors and transporters can represent different proteins. Even the polypeptide NGF enters faster than its much smaller subunit beta-NGF. The saturable transport of some compounds can be upregulated, like TNF-alpha in EAE (an animal model of multiple sclerosis) and after spinal cord injury, emphasizing the regulatory role of the BBB. As has been shown for CRH, saturable transport from brain to blood can exert effects in the periphery. Thus, the BBB plays a dynamic role in the communication of peptides between the periphery and the CNS.
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Although the neuronal pathways within the hypothalamus critical in controlling feeding and energy expenditure and projecting to brown adipose tissue (BAT) have been identified and their peptidergic content characterized, endogenous action of such peptides in the control of BAT activity has not been elucidated. Here male Sprague Dawley rats received infusions of either melanin-concentrating hormone antagonist (SNAP-7941) (1 microg/microl x h), orexin A receptor antagonist (SB-334867-A; 1 microg/microl x h), combined SB-334867-A (1 microg/microl x h), and SNAP-7941 (1 microg/microl x h), or melanocortin-3/4 receptor antagonist (SHU9119) (1 microg/microl x h) via an indwelling cannula in the lateral ventricle attached to s.c. implanted osmotic minipump. BAT temperature, physical activity, body weight, food intake, and changes in uncoupling protein (UCP)-1 were measured. SB-334867-A and SNAP-7941 significantly increased BAT temperature and UCP1 expression and reduced food intake and body weight. Combined infusion of SB-334867-A and SNAP-7941 produced a pronounced response that was greater than the addition of the individual effects in all parameters measured. SHU9119 significantly decreased BAT temperature and UCP1 expression and increased feeding and body weight. In a second series of experiments, the effect of SB-334867-A and SNAP-7941 alone or combination on the expression of the Fos protein was determined. SB-334867-A and SNAP-7941 increased Fos expression in key hypothalamic and brainstem feeding-related regions. In combination, these antagonists produced a greater than additive elevation of Fos expression in most of the regions evaluated. These findings support a role for endogenous orexigenic and anorexigenic hypothalamic peptides acting in concert to create a thermogenic tone via BAT activity.
Article
We have investigated the effects of the gastric peptide obestatin injected into the arcuate nucleus of the rat hypothalamus on the hypothalamic mRNA expression of peptides which play master roles as feeding behavior modulators. We have also evaluated the effects of obestatin on dopamine, norepinephrine and serotonin release from rat hypothalamic synaptosomes in vitro. After 4 daily intrahypothalamic injections of obestatin (1 nmol/kg), we recorded a significant reduction of daily caloric intake and body weight gain. Gene expressions of either anorexigenic (cocaine- and amphetamine-regulated transcript, corticotropin releasing hormone, proopiomelanocortin) or orexigenic (agouti-related peptide, neuropeptide Y, orexin-A) peptide mRNAs in the hypothalamus, as evaluated by real-time quantitative PCR, were not different in respect to vehicle treated rats. Moreover, ghrelin/obestatin prepropeptide gene expression in the hypothalamus was not affected by obestatin treatment. In hypothalamic synaptosomes perfused with obestatin (1-100 nM), we found a dose-dependent inhibition of depolarization-induced dopamine release, while norepinephrine and serotonin releases were not modified by obestatin treatment. When ghrelin (1 nM) and obestatin (1 nM) were co-perfused, we observed that ghrelin reversed obestatin-induced inhibition of dopamine release, and obestatin was able to block ghrelin-induced inhibition of serotonin release. We can conclude that obestatin plays an anorectic role in the hypothalamus which could be partially mediated by the acute inhibition of dopamine release, with the possible involvement of antagonism of the hypothalamic serotonin inhibitory effects of ghrelin.
Article
The locus coeruleus (LC)-norepinephrine system is a target of both cannabinoid and opioid actions. The present study investigated the anatomical distribution of cannabinoid-1 receptor (CB1r) in the LC and its association with mu-opioid receptor (MOR). Immunoreactivity for CB1r was localized to pre- and postsynaptic cellular profiles in the LC, 82% of which were dual-labeled for tyrosine hydroxylase (TH). Of the CB1r-immunoreactive structures, 66% were somatodendritic profiles, 22% were axon terminals, and the remaining 12% were associated with glial and small unmyelinated axon-like structures. CB1r immunoreactivity (-ir) in somatodendritic profiles was more often localized to the cytoplasm, whereas CB1r-ir located in axon terminals was more commonly localized on the plasma membrane. Somatodendritic profiles with CB1r-ir typically received input from axon terminals forming asymmetric-type synapses. In contrast, presynaptic profiles with CB1r-ir typically formed symmetric synaptic specializations. Anatomical studies confirmed the co-existence of MOR and CB1r-ir in common somatodendritic compartments of catecholaminergic neurons in the LC, and also revealed CB1r-positive axon terminals forming synaptic contact with MOR-containing dendrites. Our results provide evidence for a heterogeneous distribution of CB1r in the LC and demonstrate that CB1r and MOR co-exist in cellular profiles in this region. These data suggest important potential interactions between cannabinoid and opioid systems in LC neuronal profiles that may impact noradrenergic tone.
Article
Numerous studies have demonstrated that administration of rimonabant (SR 141716), a CB(1) receptor antagonist, causes a decrease in energy intake. However, the mechanisms by which rimonabant exerts its anorectic actions are unclear. The main focus of the study reported here was to establish the chemical identity of neurons that may subserve the anorectic effects of rimonabant. As such three approaches were utilised: (i) the identification of rimonabant-activated neurons using Fos as a marker of neuronal activity; (ii) the identification of the chemical phenotype of rimonabant-activated neurons by combining immunocytochemical identification of Fos and feeding-related peptides; and (iii) the evaluation of the effect of rimonabant on messenger RNA (mRNA) and protein for a number of feeding-related peptides. Rimonabant-induced Fos-positive nuclei were localized within a range of discrete hypothalamic regions with a predominance in the parvocellular part of the paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus, arcuate nucleus and lateral hypothalamic area. Furthermore, Fos labelling within these hypothalamic regions was colocalized with anorexigenic and orexigenic peptides including melanin-concentrating hormone (MCH), orexin, cocaine- and amphetamine-regulated transcript (CART) and alpha-melanocyte-stimulating hormone (alpha-MSH). Rimonabant specifically induced a decrease in NPY and an increase in CART and alpha-MSH mRNA and protein, consistent with its effect in reducing food intake and increasing energy expenditure. As such these data provide insights into the mechanisms of action that may underpin rimonabant's effects on energy balance and body weight.
Article
Leptin, or OB protein, is produced by fat cells and may regulate body weight by acting on the brain. To reach the brain, circulating leptin must cross the blood-brain barrier (BBB). Intravenously injected radioiodinated leptin (125I-leptin) had an influx constant (Ki) into brain of (5.87)10(-4) ml/g-min, a rate 20 times greater than that of labeled albumin. Unlabeled leptin inhibited the influx of 125I-leptin in a dose-dependent manner whereas unlabeled tyrosine and insulin, which have saturable transport systems, were without effect. HPLC and acid precipitation showed that the radioactivity in brain and serum represented intact 125I-leptin. About 75% of the extravascular 125I-leptin in brain completely crossed the BBB to reach brain parenchyma. Autoradiography detected uptake at the choroid plexus, arcuate nuclei of the hypothalamus, and the median eminence. Saturable transport did not occur out of the brain. The results show that leptin is transported intact from blood to brain by a saturable system.
Article
Noradrenergic and dopaminergic afferents arising from the brain stem to the hypothalamus still play a poorly characterised role in food intake control. We have studied the effect of leptin, an adipocyte-derived hormone which has been implicated in the regulation of feeding behaviour, on [3H]norepinephrine and [3H]dopamine release from perfused hypothalamic neuronal endings (synaptosomes) in vitro. We have found that leptin (0.01-10 nM) does not modify basal, while it inhibits depolarization-induced norepinephrine and dopamine release. We can hypothesize that at least part of the anorectic activity of leptin in the hypothalamus is effected through an inhibition of noradrenergic and dopaminergic firing.
Article
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Article
A large body of evidence supports the notion that Delta9-tetrahydrocannabinol (THC) stimulates food intake by its actions on CB1 cannabinoid receptors. Indirect evidence also suggests a role for dopamine (DA) receptors in mediating THC-induced feeding. In the present study, a series of experiments involving intraperitoneal drug administration in rats were conducted to further investigate the interaction between cannabinoid and dopamine receptors in feeding behaviour. Male Wistar rats were habituated to the test environment and injection procedure, and then were injected with vehicle alone, the dopamine D1-like receptor antagonist SCH 23390 (0.005, 0.01, 0.5 or 0.1 mg/kg), THC (0.1, 0.5 or 1.0 mg/kg) or SCH 23390 and THC combined. Food intake and locomotor activity were then measured for 120 min. Results revealed that administration of SCH 23390 dose-dependently decreased food intake while THC dose-dependently increased feeding. Furthermore, SCH 23390 attenuated feeding induced by THC at a dose that did not affect feeding on its own. These findings provide direct evidence for the existence of cannabinoid-dopamine interactions in feeding behaviour and suggest that dopamine D1 signalling is necessary for cannabinoids to stimulate food intake.
Article
Feeding and energy expenditures are modulated by the interplay of hormones and neurotransmitters in the central nervous system (CNS), where the hypothalamus plays a pivotal role in the transduction of peripheral afferents into satiety and feeding signals. Aminergic neurotransmitters such as dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) are historically considered to play a key role, but a number of peptides are involved in finely tuning feeding regulation. This review summarizes the current understanding of the CNS mechanisms of orexigenic peptides, such as neuropeptide Y, orexins, and ghrelin, as well as anorectic peptides, such as leptin, neurotensin (NT), cocaine- and amphetamine regulated transcript (CART) peptide, thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), urocortin, amylin.
Article
Peptide YY (1-36) and peptide YY (3-36) are gut-derived hormones which are involved in feeding control in the hypothalamus. The hypothalamic mechanisms of feeding have been shown to be modulated by aminergic neurotransmitters, which could mediate the anorectic or orexigenic effects of neuropeptides and hormones. We have investigated the role of peptide YY (1-36) and peptide YY (3-36) on dopamine, norepinephrine, and serotonin release from hypothalamic synaptosomes in vitro. We found that peptide YY (3-36) inhibited depolarization-induced dopamine and norepinephrine release, leaving unaffected serotonin release, while peptide YY (1-36) did not modify either basal or stimulated amine release. We can hypothesize that the effects of peptide YY (3-36) could be mediated by inhibited hypothalamic dopamine and norepinephrine release, which could partially account for the anorectic activity of the peptide. On the other hand, peptide YY (1-36), which has a feeding stimulatory role, does not affect aminergic neurotransmission in the hypothalamus.
Article
The present study examined the impact of repeated administration of a synthetic cannabinoid agonist, WIN 55,212-2 on the coeruleo-cortical pathway, a circuit implicated in anxiety. Male Sprague-Dawley rats received repeated systemic injections of WIN 55,212-2 (3.0 mg/kg). A separate group of rats received repeated WIN 55,212-2 injections followed by a period of abstinence. Control animals received vehicle injections. Ninety minutes following the last injection on day 8, anxiety-related behavior was assessed using the elevated plus maze. The abstinent group was tested after another 8 days. Following behavioral testing, brain tissue was extracted from the locus coeruleus (LC) and probed for tyrosine hydroxylase (TH) expression. In a separate group of animals, in vivo microdialysis was used to monitor extracellular norepinephrine efflux in the frontal cortex following repeated WIN 55,212-2 administration and following a period of abstinence. Repeated administration of WIN 55,212-2 evoked an anxiogenic-like response that was accompanied by an increase in TH protein expression in the LC. A similar neurochemical profile was observed using in vivo microdialysis where an augmented increase in cortical norepinephrine efflux was identified in response to a systemic injection of WIN 55,212-2 on day 8. Anxiety-like behavior, catecholamine synthesizing enzyme levels and NE efflux returned to control values after 8 days of abstinence. The present findings indicate that repeated administration of a synthetic cannabinoid receptor agonist induces transient anxiety-like behaviors that correlate with increases in catecholamine synthesizing enzyme expression in the LC and augmented norepinephrine efflux in response to a challenge injection of WIN 55,212-2.
Article
In 2005, the first evidence was obtained that the cannabinoid CB(1) receptor contains an allosteric binding site. The site can be recognized by synthetic small molecules, which display a markedly divergent effect on orthosteric ligand affinity versus efficacy; these small molecules are allosteric enhancers of agonist binding affinity and allosteric inhibitors of agonist signalling efficacy. Allosteric modulation heralds a new approach to the manipulation of the endocannabinoid system for therapeutic benefit; it promises to augment the existing portfolio of selective direct agonists, competitive antagonists and enzyme inhibitors.
Article
Appetite is regulated by a complex system of central and peripheral signals which interact in order to modulate the individual response to nutrient ingestion. Peripheral regulation includes satiety signals and adiposity signals, while central control is accomplished by several effectors, including the neuropeptidergic, monoaminergic and endocannabinoid systems. Satiety signals, including cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), originate from the gastrointestinal (GI) tract during a meal and, through the vagus nerve, reach the nucleus tractus solitarius (NTS) in the caudal brainstem. From NTS afferents fibers project to the arcuate nucleus (ARC), where satiety signals are integrated with adiposity signals, namely leptin and insulin, and with several hypothalamic and supra-hypothalamic inputs, thus creating a complex network of neural circuits which finally elaborate the individual response to a meal. As for the neuropeptidergic system, ARC neurons secrete orexigenic substances, such as neuropeptide Y (NPY) and agouti-related peptide (AGRP), and anorexigenic peptides such as pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Other brain areas involved in the control of food intake are located downstream the ARC: among these, the paraventricular nucleus (PVN), which produces anorexigenic peptides such as thyrotropin releasing hormone (TRH), corticotrophin releasing hormone (CRH) and oxytocin, the lateral hypothalamus (LHA) and perifornical area (PFA), secreting the orexigenic substances orexin-A (OXA) and melanin concentrating hormone (MCH). A great interest in endocannabinoids, important players in the regulation of food intake, has recently developed. In conclusion, the present work reviews the most recent insights into the complex and redundant molecular mechanisms regulating food intake, focusing on the most encouraging perspectives for the treatment of obesity.
Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla
  • M Koch
  • L Varela
  • J G Kim
  • J D Kim
  • F Hernández-Nuño
  • S E Simonds
Koch M, Varela L, Kim JG, Kim JD, Hernández-Nuño F, Simonds SE, et al. Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla. Neuropharmacology 2015;98:78-89.
Leptin enters the brain by a saturable system independent of insulin
  • Banks
Allosterism and cannabinoind CB(1) receptors: the shape of things to come
  • Ross