| Apelin, a novel identified bioactive peptide, is the endogenous ligand for the APJ. Apelin/APJ system is widely distributed within the central nervous system (CNS) and peripheral tissues in human and rodents. The precursor of apelin (preproapelin) is processed to several molecular forms in different tissues, including apelin-36, apelin-17, apelin-13and apelin-12. However, the apelin-13has a greater biological activity, and the amino acids sequence of apelin-13is more conserved across all species. In this study, apelin-13was synthesized by the solid-phase peptide synthesis method and purified by high performance liquid chromatography (HPLC). We have investigated the mechanisms as well as regulatory effects of apelin-13on feeding behavior, gastrointestinal motility, pain (visceral pain, acute pain and inflammatory pain) and depression.The apelin/APJ mRNA has been detected in hypothalamus, indicating that apelin may play a potential role in regulating feeding behavior and gastrointestinal motility. However, the effect of acute central injection of apelin-13on food intake and gastrointestinal motility in mice remain unknown till now. Our results show that intracerebroventricular (i.c.v.) administration of apelin-13decreased the cumulative food intake and cumulative water intake at4hours after injection in the freely feeding and fasted mice during the dark period, while i.c.v. apelin-13had no effect on food intake in freely feeding mice during the light period. The inhibitory effect on cumulative food intake/0-4h induced by apelin-13in freely feeding mice during the dark period could be blocked by the APJ receptor antagonist apelin-13(F13A) and corticotropin-releasing factor (CRF) receptor antagonist a-helical CRF9-41, but not arginine vasopressin (AVP) receptor antagonist deamino(CH2)5Tyr(Me)AVP. I.c.v. injection with apelin-13could inhibited the gastric emptying, gastrointestinal transit, bead expulsion and fecal pellet output, and these inhibitory effects could be reversed by apelin-13(F13A) and the non-selective opioid receptor blocker naloxone. These results indicate that i.c.v. apelin-13inhibited food intake in mice, the APJ and CRF receptor, but not AVP receptor were involved in this process, and i.c.v. apelin-13inhibited gastrointestinal motility via APJ and opioid receptor in mice.Apelin has a distribution in pain-related areas in the CNS, such as dorsal raphe nucleus, amygdala, hypothalamus and spinal cord, suggesting that apelin may play a role in regulation of pain. The present study investigated the mechanisms and effects of apelin-13in the model of visceral pain, acute pain and inflammatory pain, using the acetic acid-induced writhing response, warm-water tail-flick test and formalin test. The results show that,(1) in the acetic acid-induced writhing assay, i.c.v. apelin-13inhibited visceral pain in mice, which could be antagonized by apelin-13(F13A), naloxone and β-FNA, a μ opioid receptor antagonist. In addition, low dose of i.c.v. apelin-13could significantly potentiate the antinociceptive potencies of modest and even relatively ineffective doses of morphine administrated at supraspinal level, which could be blocked by naloxone.(2) In the warm-water tail-flick test, intrathecally (i.t.) administered apelin-13significantly induced antinociception, which could be antagonized by apelin-13(F13A) and naloxone.(3) In the formalin test, i.t. apelin-13obviously induced hyperalgesic effect in the phase Ⅱ in mice, which could be blocked by apelin-13(F13A) and bicuculline methiodide, a GABAA receptor antagonist, but not naloxone. These results indicate that i.c.v. apelin-13inhibited visceral pain, and i.t. apelin-13inhibited acute pain. Both of the analgestic effects were mediated by APJ and opioid receptor. The synergistic analgesic relationship of morphine and apelin-13was mediated by opioid-responsive neurons in the acetic acid-induced visceral pain model. In the inflammatory pain model (formalin test), interestingly, i.t. apelin-13produced hyperalgesic effect through APJ and GABAA receptor, but not opioid receptor in mice.In the CNS, APJ mRNA was found in amygdala, dentate gyrus, Ammon’s horn and hypothalamus et al., which was closely related with emotion, indicating a possible role of apelin in emotional behavior. We investigated the mechanism and effect of i.c.v. apelin-13on depression in mice, using the classical animal model of depression-forced swimming test and tail-suspension test. At the same time, in order to exclude the possibility of a false-positive result, we evaluated the spontaneous activity counts and motor function by the spontaneous activity test, rotarod test and wire hanging test. The results show that i.c.v. apelin-13increased the immobility time, which could be abolished by apelin-13(F13A), naloxone and nor-BNI, a κ opioid receptor antagonist, but not a-helical CRF9-41. I.c.v. injection of apelin-13(F13A) did influence the immobility time in mice. Apelin-13had no influence on spontaneous activity and motor function in mice. These results demonstrate that i.c.v. apelin-13produced depression-like behavior, APJ and κ opioid receptor, but not CRF receptor, were involved in this process. The depression-like behavior induced by apelin-13was not due to spontaneous activity inhibition or motor function impairment, so we can eliminate the false-positive result.GBR12909is a potent and selective dopamine re-uptake inhibitor. Dopamine is closely related to reward and hyperactivity. The anorexia nervosa is often characterized by hyperactivity and body weight loss. Therefore, the present study was designed to investigate the mechanism and effect of GBR12909in activity-based anorexia (ABA) model. Mice were provided free access to a running wheel and then calorie is restricted. We measured the fat mass and lean mass using the EchoMRI Animal Tissue Composition Analyzer. After intraperitoneal (i.p.) injection with GBR12909, the gene expression of factors was determined using the quantitative real-time PCR. After one week calorie restriction, the body weight, fat mass and lean mass were decreased, and the running wheel activity were increased, which were appeared as the feature of anorexia nervosa. GBR12909(2mg/kg) significantly increased the1h running wheel activity in mice exposed ABA, but not under ad libitum feeding or overnight fast condition. The GBR12909induced a significant increase of hypothalamic NPY and AgRP mRNA expression, but not POMC, CART, MCH or LeptinR, in mice exposed ABA. GBR12909did not influence D1R, D2R and DAT mRNA expression in caudate-putamen (CPu) in mice, and GBR12909had no effect on D1R, D2R, VMAT2, BDNF, CREB, FosB, GluR2or MCHR1mRNA level in accumbens (Acb). GBR12909induced an increase of the DAT mRNA expression in Acb, but it is not reach a statistically significant level. All the results demonstrate that acute treatment with GBR12909led to an exaggerated increase in running wheel activity in mice exposed to ABA, which was associated with orexigenic neuropeptides NPY and AgRP gene expression in hypothalamus, but not related to dopamine.In conclusion, apelin-13regulated food intake, gastrointestinal motility, pain and depression via different receptors in mice. This study could provide preclinical experiment data for apelin-13which is a potential drug developing for obesity, gastrointestinal disorder and pain treatment. In addition, this study shows that GBR12909exaggerated the ABA due to the change of neuropeptide NPY and AgRP gene expression in hypothalamus, which could provide a theoretical reference for the clinical application of GBR12909. |
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