Irisin inhibits CCK-8-induced TNF-α production via integrin αVß5-NF-κB signaling pathways in Nile tilapia (Oreochromis niloticus).

Irisin, a secreted myokine generated by fibronectin type III domain-containing protein 5, has recently shown the potential to alleviate inflammation. Cholecystokinin-octapeptide (CCK-8) is closely associated with the inflammatory factor TNF-α, a central cytokine in inflammatory reactions. However, the interactions between irisin and CCK-8 in regulating TNF-α production and the underlying mechanism have not yet been elucidated. In the present study, irisin treatment inhibited the basal and the CCK-8-induced TNF-α production in vivo. Additionally, neutralizing circulating irisin using an irisin antiserum significantly augmented the CCK-8-induced stimulation of TNF-α levels. Moreover, the incubation of head kidney cells with irisin or CCK-8 has opposite effects on TNF-α secretion. Notably, irisin treatment inhibited basal and CCK-8-stimulated TNF-α release and gene transcription in head kidney cells. Mechanistically, the inhibitory actions of irisin on basal and CCK-8-induced TNF-α production could be negated by co-administered with the selective integrin αVß5 inhibitor cilengitide. In addition, the inhibitory effect of irisin on basal and CCK-8-triggered TNF-α production could be abolished by the inhibition of the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, irisin impeded CCK-8-induced phosphorylation and degradation of IκBα, simultaneously inhibiting NF-κB phosphorylation, preventing its translocation into the nucleus, and suppressing its DNA-binding activity induced by CCK-8. Collectively, these results suggest that the inhibitory effect of irisin on TNF-α production caused by CCK-8 is mediated via the integrin αVß5-NF-κB signaling pathways in tilapia.

CCK-8 enhances acid-sensing ion channel currents in rat primary sensory neurons.

Cholecystokinin (CCK) is a peptide that has been implicated in pain modulation. Acid sensitive ion channels (ASICs) also play an important role in pain associated with tissue acidification. However, it is still unclear whether there is an interaction between CCK signaling and ASICs during pain process. Herein, we report that a functional link between them in rat dorsal root ganglion (DRG) neurons. Pretreatment with CCK-8 concentration-dependently increased acid-evoked ASIC currents. CCK-8 increased the maximum response of ASICs to acid, but did not changed their acid sensitivity. Enhancement of ASIC currents by CCK-8 was mediated by the stimulation of CCK2 receptor (CCK2R), rather than CCK1R. The enhancement of ASIC currents by CCK-8 was prevented by application of either G-protein inhibitor GDP-ß-S or protein kinase C (PKC) inhibitor GF109203×, but not by protein kinase A (PKA) inhibitor H-89 or JNK inhibitor SP600125. Moreover, CCK-8 increased the number of action potentials triggered by acid stimuli by activating CCK2R. Finally, CCK-8 dose-dependently exacerbated acid-induced nociceptive behavior in rats through local CCK2R. Together, these results indicated that CCK-8/CCK2R activation enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats. The enhancement effect depended on G-proteins and intracellular PKC signaling rather than PKA and JNK signaling pathway. These findings provided that CCK-8/CCK2R is an important therapeutic target for ASIC-mediated pain.

An investigation on the changes of serum CCK-8, substance P, and 5-HT in patients with post-stroke insomnia.

BACKGROUND: At present, the pathogenesis of post-stroke insomnia (PSI) is still inconclusive. OBJECTIVE: To explore the changes and significance of serum cholecystokinin-8 (CCK-8), substance P (SP), and 5-hydroxytryptamine (5-HT) in patients with PSI. METHODS: Ninety-one patients with stroke were selected as the research subjects, and according to the score of the Athens Insomnia Scale (AIS), they were divided into the insomnia group and the non-insomnia group. The serum levels of CCK-8, SP, and 5-HT in the two groups were compared to explore their relationships with PSI. RESULTS: Among the 91 patients, 56 were in the insomnia group and 35 were in the non-insomnia group, and the incidence of insomnia was 61.5%. There was no significant difference in the serum levels of CCK-8, SP, and 5-HT between the two groups (P= 0.696, 0.980, and 0.809, respectively). One-way analysis of variance showed that there was no significant correlation between the serum levels of CCK-8, SP, 5-HT, and the AIS score (P= 0.7393, 0.9581, and 0.5952, respectively). CONCLUSION: The incidence of PSI was relatively high, but it could not be proved that CCK-8, SP, and 5-HT were involved in the pathogenesis of PSI. There might exist other neurotransmitters involved in the pathophysiological process of PSI, which should be further explored.

Expression of oxytocin in hypothalamus and reduction of nociceptive stress following administration of Kamikihi-to in female rats.

Hypothalamo-neurohypophysial oxytocin (OXT) plays an essential role in reproduction and in several socio-physiological functions, including stress reduction, anxiety relief, feeding suppression, social recognition, and trust building. Recent studies suggest that the central OXT system is also involved in antinociceptive and anti-inflammatory functions. Kamikihi-to (KKT), a Japanese traditional herbal (Kampo) medicine composed of 14 herbal ingredients, is clinically prescribed for patients with psychological symptoms, including anxiety, depression, and insomnia, and it has been associated with OXT expression. We investigated the antinociceptive response and OXT expression according to sex and the effects of KKT pre administration in a rat model. We found that nociceptive responses measured via the hot plate and formalin tests were attenuated following the administration of KKT-enriched feed for 4 weeks. The observation of mRFP1 fluorescence in OXT-mRFP1 transgenic rats revealed that KKT-administered rats showed increased expression of OXT in the magnocellular and parvocellular paraventricular nucleus of the hypothalamus. Food intake in the KKT-pre-administered group significantly decreased after cholecystokinin (CCK)-8 administration. Our results suggest that KKT is involved in the attenuation of nociceptive stress in female rats by enhancing the expression of OXT in the hypothalamus.

Contribution of Histamine to Nociceptive Behaviors Induced by Intrathecally Administered Cholecystokinin-8.

The involvement of spinal release of histamine in the nociceptive behaviors induced by cholecystokinin-8 (CCK-8) was investigated in mice. Intrathecal (i.t.) injection of CCK-8 elicited the nociceptive behaviors consisting of biting and licking. The nociceptive behaviors induced by i.t. treatment with CCK-8 showed two bell-shaped patterns. The histamine H3 receptor antagonist significantly promoted the nociceptive behaviors induced by CCK-8 at doses of 1-100 fmol and 100 pmol. The nociceptive behaviors elicited by CCK-8 was inhibited by i.t. administration of the CCK-B receptor antagonist in a dose-dependent manner, but not by the CCK-A receptor antagonist. The nociceptive behaviors induced by CCK-8 were markedly suppressed by i.t. pretreatment with antiserum against histamine and were abolished in histidine decarboxylase-deleted gene mice. In histamine H1 receptor-deleted gene mice, the nociceptive behaviors induced at both 10 amol and 10 pmol of CCK-8 were not affected. The tachykinin neurokinin-1 (NK1) receptor antagonists inhibited CCK-8 (10 pmol)-induced nociceptive behaviors in a dose-dependent manner. CCK-8 (10 amol)-induced nociceptive behaviors was not antagonized by co-administration with the tachykinin NK1 receptor antagonists. The nociceptive behaviors elicited by CCK-8 were inhibited by i.t. administration of the antagonist for the N-methyl-D-aspartate (NMDA) receptor in a dose-dependent manner. Our results suggest that the nociceptive behaviors induced by i.t. administration of CCK-8 (10 pmol) are mediated through the spinal release of histamine and are elicited via activation of the tachykinin NK1 and NMDA receptors, whereas the nociceptive behaviors induced by i.t. administration of CCK-8 (10 amol) are mediated through the spinal release of histamine and elicited via NMDA receptor activation.

Cholecystokinin-8 attenuates methamphetamine-induced inflammatory activation of microglial cells through CCK2 receptor.

Methamphetamine (METH) exposure reportedly promotes microglial activation and pro-inflammatory cytokines secretion. Sustained inflammation in abusers of psychostimulant drugs further induces neural damage. Cholecystokinin-8 (CCK-8) is a gut-brain peptide which exerts a wide range of biological activities in the gastrointestinal tract and central nervous system. We previously found that pre-treatment with CCK-8 inhibited behavioural and histologic changes typically induced by repeated exposure to METH. Here, we aimed to estimate the effects of CCK-8 on METH-induced neuro-inflammation, which is markedly characterized by microglia activation and increased pro-inflammatory cytokines production in vivo and in vitro. Moreover, we assessed the subtypes of the CCK receptor mediating the regulatory effects of CCK-8, and the changes in the NF-κB signalling pathway. We found that CCK-8 inhibited METH-induced microglial activation and IL-6 and TNF-α generation in vivo and in vitro in a dose-dependent manner. Furthermore, co-treatment of CCK-8 with METH significantly attenuated the activation of the NF-κB signalling pathway by activating the CCK2 receptor subtype in N9 cells. In conclusion, our findings indicated the inhibitory effect of CCK-8 on METH-induced neuro-inflammation in vivo and in vitro, and suggested the underlying mechanism may involve the activation of the CCK2 receptor, which downregulated the NF-κB signalling pathway induced by METH stimulation.

Ghrelin signaling contributes to fasting-induced attenuation of hindbrain neural activation and hypophagic responses to systemic cholecystokinin in rats.

In rats, overnight fasting reduces the ability of systemic cholecystokinin-8 (CCK) to suppress food intake and to activate cFos in the caudal nucleus of the solitary tract (cNTS), specifically within glucagon-like peptide-1 (GLP-1) and noradrenergic (NA) neurons of the A2 cell group. Systemic CCK increases vagal sensory signaling to the cNTS, an effect that is amplified by leptin and reduced by ghrelin. Since fasting reduces plasma leptin and increases plasma ghrelin levels, we hypothesized that peripheral leptin administration and/or antagonism of ghrelin receptors in fasted rats would rescue the ability of CCK to activate GLP-1 neurons and a caudal subset of A2 neurons that coexpress prolactin-releasing peptide (PrRP). To test this, cFos expression was examined in ad libitum-fed and overnight food-deprived (DEP) rats after intraperitoneal CCK, after coadministration of leptin and CCK, or after intraperitoneal injection of a ghrelin receptor antagonist (GRA) before CCK. In fed rats, CCK activated cFos in ~60% of GLP-1 and PrRP neurons. Few or no GLP-1 or PrRP neurons expressed cFos in DEP rats treated with CCK alone, CCK combined with leptin, or GRA alone. However, GRA pretreatment increased the ability of CCK to activate GLP-1 and PrRP neurons and also enhanced the hypophagic effect of CCK in DEP rats. Considered together, these new findings suggest that reduced behavioral sensitivity to CCK in fasted rats is at least partially due to ghrelin-mediated suppression of hindbrain GLP-1 and PrRP neural responsiveness to CCK.

Cholecystokinin-8 treatment reduces acinar necrosis and edema of pigs with induced pancreatitis.

BACKGROUND: Acute pancreatitis is an inflammatory process of the pancreas and a leading cause of hospitalization amongst gastrointestinal disorders. Previously, cholecystokinin (CCK) has been described to play a role in regeneration of pancreas. The aim of this study was to analyse the function of cholecystokinin octapeptide (CCK-8) during induced pancreatitis in an animal model. METHODS: Overall acute pancreatitis was induced in 38 pigs. After the induction of acute pancreatitis, half of the animals were treated with CCK-8. Intraoperative clinical data, postoperative blood parameters, 'Porcine Well-being' (PWB) and fitness score and post-mortal histopathological data were analysed. RESULTS: At baseline, physiologically parameters of the pigs of both groups were comparable. No differences were observed regarding the overall survival of animals (p = 0.97). Postoperative PWB score were significantly enhanced in animals treated with CCK-8 as compared to the control group (p = 0.029). Moreover, histopathological analysis of the pancreatic tissue revealed that acinar necrosis and edema were significant reduced in the CCK-8 group in comparison to the control group (p = 0.016 and p = 0.019). CONCLUSIONS: In conclusion, we found that CCK-8 treatment reduces acinar necrosis and edema of pancreatic tissue after induction of an acute pancreatitis in pigs.

[Nutrient Sensing and Anorexia via Neuropeptides].

Various neuropeptides play an essential role in the nutrient sensing mechanism and related homeostasis. Nesfatin-1 is a newly identified neuropeptide having anorectic activity, and nesfatin-1-containing neurons are widely distributed in the brain, including the hypothalamus and brain stem. Our previous study showed that dehydration-induced anorectic effects are mediated via the central nesfatin-1 pathway in rats. Our recent studies have also shown that peripheral anorectic peptides (cholecystokinin-8, glucagon-like peptide-1, and leptin) and an antineoplastic agent (cisplatin) caused inhibition of feeding via the central nesfatin-1 pathway in rats. Nesfatin-1-containing neurons in the central nervous system, in particular the hypothalamus and the brain stem, may mediate peripheral nutrient signals and regulate feeding behavior.

Common Hepatic Branch of Vagus Nerve-Dependent Expression of Immediate Early Genes in the Mouse Brain by Intraportal L-Arginine: Comparison with Cholecystokinin-8.

Information from the peripheral organs is thought to be transmitted to the brain by humoral factors and neurons such as afferent vagal or spinal nerves. The common hepatic branch of the vagus (CHBV) is one of the main vagus nerve branches, and consists of heterogeneous neuronal fibers that innervate multiple peripheral organs such as the bile duct, portal vein, paraganglia, and gastroduodenal tract. Although, previous studies suggested that the CHBV has a pivotal role in transmitting information on the status of the liver to the brain, the details of its central projections remain unknown. The purpose of the present study was to investigate the brain regions activated by the CHBV. For this purpose, we injected L-arginine or anorexia-associated peptide cholecystokinin-8 (CCK), which are known to increase CHBV electrical activity, into the portal vein of transgenic Arc-dVenus mice expressing the fluorescent protein Venus under control of the activity-regulated cytoskeleton-associated protein (Arc) promotor. The brain slices were prepared from these mice and the number of Venus positive cells in the slices was counted. After that, c-Fos expression in these slices was analyzed by immunohistochemistry using the avidin-biotin-peroxidase complex method. Intraportal administration of L-arginine increased the number of Venus positive or c-Fos positive cells in the insular cortex. This action of L-arginine was not observed in CHBV-vagotomized Arc-dVenus mice. In contrast, intraportal administration of CCK did not increase the number of c-Fos positive or Venus positive cells in the insular cortex. Intraportal CCK induced c-Fos expression in the dorsomedial hypothalamus, while intraportal L-arginine did not. This action of CCK was abolished by CHBV vagotomy. Intraportal L-arginine reduced, while intraportal CCK increased, the number of c-Fos positive cells in the nucleus tractus solitarii in a CHBV-dependent manner. The present results suggest that the CHBV can activate different brain regions depending on the nature of the peripheral stimulus.

Serotonergic modulation of the activity of GLP-1 producing neurons in the nucleus of the solitary tract in mouse.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) and 5-HT are potent regulators of food intake within the brain. GLP-1 is expressed by preproglucagon (PPG) neurons in the nucleus tractus solitarius (NTS). We have previously shown that PPG neurons innervate 5-HT neurons in the ventral brainstem. Here, we investigate whether PPG neurons receive serotonergic input and respond to 5-HT. METHODS: We employed immunohistochemistry to reveal serotonergic innervation of PPG neurons. We investigated the responsiveness of PPG neurons to 5-HT using in vitro Ca2+ imaging in brainstem slices from transgenic mice expressing the Ca2+ indicator, GCaMP3, in PPG neurons, and cell-attached patch-clamp recordings. RESULTS: Close appositions from 5-HT-immunoreactive axons occurred on many PPG neurons. Application of 20 µM 5-HT produced robust Ca2+ responses in NTS PPG dendrites but little change in somata. Dendritic Ca2+ spikes were concentration-dependent (2, 20, and 200 µM) and unaffected by blockade of glutamatergic transmission, suggesting 5-HT receptors on PPG neurons. Neither activation nor blockade of 5-HT3 receptors affected [Ca2+]i. In contrast, inhibition of 5-HT2 receptors attenuated increases in intracellular Ca2+ and 5-HT2C receptor activation produced Ca2+ spikes. Patch-clamp recordings revealed that 44% of cells decreased their firing rate under 5-HT, an effect blocked by 5-HT1A receptor antagonism. CONCLUSIONS: PPG neurons respond directly to 5-HT with a 5-HT2C receptor-dependent increase in dendritic [Ca2+]i. Electrical responses to 5-HT revealed additional inhibitory effects due to somatic 5-HT1A receptors. Reciprocal innervation between 5-HT and PPG neurons suggests that the coordinated activity of these brainstem neurons may play a role in the regulation of food intake.

Exogenous glucagon-like peptide-1 reduces body weight and cholecystokinin-8 enhances this reduction in diet-induced obese male rats.

The sites of action regulating meal size (MS) and intermeal interval (IMI) length by glucagon like peptide-1 (7-36) (GLP-1 (7-36)) and cholecystokinin-8 (CCK-8) reside in the areas supplied by the two major branches of the abdominal aorta, celiac and cranial mesenteric arteries. We hypothesized that infusing GLP-1 near those sites reduces body weight (BW) and adding CCK-8 to this infusion enhances the reduction. Here, we measured BW in diet-induced obese (DIO) male rats maintained and tested on normal rat chow and infused with saline, GLP-1 (0.5nmol/kg) and GLP-1+CCK-8 (0.5nmol/kg each) in the aorta once daily for 21days. We found that GLP-1 and GLP-1+CCK-8 decrease BW relative to saline vehicle and GLP-1+CCK-8 reduced it more than GLP-1 alone. Reduction of BW by GLP-1 alone was accompanied by decreased 24-h food intake, first MS, duration of first meal and number of meals, and an increase in latency to first meal. Reduction of BW by the combination of the peptides was accompanied by decrease 24-h food intake, first MS, duration of first meal and number of meals, and increase in the IMI length, satiety ratio and latency to first meal. In conclusion, GLP-1 reduces BW and CCK-8 enhances this reduction if the peptides are given near their sites of action.

Infusion of exogenous cholecystokinin-8, gastrin releasing peptide-29 and their combination reduce body weight in diet-induced obese male rats.

We hypothesized that exogenous gastrin releasing peptide-29 (GRP-29), cholecystokinin-8 (CCK-8) and their combination reduce body weight (BW). To test this hypothesis, BW was measured in four groups of diet-induced obese (DIO) male rats infused in the aorta (close to the junctions of the celiac and cranial mesenteric arteries) with saline, CCK-8 (0.5 nmol/kg), GRP-29 (0.5 nmol/kg) and CCK-8+GRP-29 (0.5 nmol/kg each) once daily for a total of 23 days. We found that CCK-8, GRP-29 and CCK-8+GRP-29 reduce BW relative to saline control. In conclusion, CCK-8, GRP-29 and their combination reduce BW in the DIO rat model. If infused near their gastrointestinal sites of action CCK-8, GRP-29 and their combination may have a role in regulating BW.

A prospective study on changes in blood levels of cholecystokinin-8 and leptin in patients with refractory epilepsy treated with the ketogenic diet.

OBJECTIVE: To evaluate the changes in serum CCK-8 and leptin levels in patients with refractory epilepsy treated with the ketogenic diet (KD). METHODS: In this prospective study, patients aged between one and 40 years with refractory epilepsy were included. CCK-8 and leptin were measured in serum at baseline and after three and 12 months of treatment with the KD using an enzyme-linked Immunoabsorbant Assay. Seizure frequency and responsiveness were calculated. RESULTS: Fifty-four patients were included; 26 patients (48%) were still on the KD at 12 months. After three and 12 months, respectively, 39% and 26% were responders. CCK-8 values were statistically significantly increased at three months (p=0.005) and 12 months (p=0.012). In responders, at three months follow-up, this increase of CCK-8 was significant (p=0.020), whereas in the non-responders it was not (p=0.34). Leptin values were decreased at three and 12 months, the decrease at three months being statistically significant in post-pubertal men (p=0.028) and post-pubertal women (p=0.007). SIGNIFICANCE: In responders to the KD, serum CCK-8 increased statistically significantly during treatment at three months. Serum leptin decreased statistically significantly at three months in men and in post-pubertal women. It is plausible that the increase of CCK-8 and the decrease of leptin contribute to the anti-convulsive effect of the KD.

Influences of CCK-8 on expressions of apoptosis-related genes in prefrontal cortex neurons of morphine-relapse rats.

In order to elucidate the influences of CCK-8 on expressions of apoptosis-related genes, Bax, Bcl-2 and Caspase-3, of prefrontal cortex neurons in morphine-relapse rats, an effective, successful morphine-relapse-rat model using the conditioned place preference (CPP) under CCK-8 (0.01, 0.1 and 1.0µg, i.c.v) intervention was established. The prefrontal cortexes were made into slices with the cellular plasmas immunohistochemically stained. The expressions of Bax, Bcl-2, Caspase-3 of neurons were evaluated through their scores, and each corresponding ratio of Bax and Bcl-2 (Bax/Bcl-2) was also computed. The results showed that the expression of Bcl-2 was very weak and those of Bax and Caspase-3 were hardly seen in group normal saline; the expressions of Bax and Caspase-3 were strong and that of Bcl-2 was weak in group morphine and compared to group normal saline, there were significant differences (P<0.05); the expressions of Bax, Caspase-3 and the ratios of Bax/Bcl-2 have a gradually-decreased trend in the sequence of group 0.01µg, group 0.1µg and group 1.0µg, but the expression of Bcl-2 has an opposite trend in the same sequence, and compared to group morphine, there were significant differences (P<0.05) excluding group 0.01µg. So we draw a conclusion that CCK-8 (0.1 and 1.0µg, i.c.v) could protect neurons of prefrontal cortex through up-regulating the expression of Bcl-2, down-regulating those of Bax and Caspase-3 and reducing Bax/Bcl-2 ratio in the model of morphine-relapse rats.

Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50µg/kg) and secretin (100µg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.

A1-adenosine acute withdrawal response and cholecystokinin-8 induced contractures are regulated by Ca(2+)- and ATP-activated K(+) channels.

In isolated guinea-pig ileum (GPI), the A1-adenosine acute withdrawal response is under the control of several neuronal signalling systems, including the µ/κ-opioid and the cannabinoid CB1 systems. It is now well established that after the stimulation of the A1-adenosine system, the indirect activation of both µ/κ-opioid and CB1 systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated the involvement of the Ca(2+)/ATP-activated K(+) channels in the regulation of both acute A1-withdrawal and CCk-8-induced contractures in the GPI preparation. Interestingly, we found that: (a) the A1-withdrawal contracture is inhibited by voltage dependent Ca(2+)-activated K(+) channels, Kv, while it is enhanced by the voltage independent Ca(2+)-activated K(+) channels, SKCa; (b) in the presence of CCk-8, the inhibitory effect of the A1 agonist, CPA, on the peptide induced contracture is significantly enhanced by the voltage independent Ca(2+)-activated K(+) channel, SKCa; and (c) the A1-withdrawal contracture precipitated in the presence of CCk-8 is controlled by the ATP-sensitive potassium channels, KATP. Our data suggest, for the first time, that both Ca(2+)- and ATP-activated K(+) channels are involved in the regulation of both A1-withdrawal precipitated and CCk-8 induced contractures.