In vitro gastrointestinal digestion of buckwheat (Fagopyrum esculentum Moench) protein: release and structural characteristics of novel bioactive peptides stimulating gut cholecystokinin secretion.

Satiety hormone cholecystokinin (CCK) plays a vital role in appetite inhibition. Its secretion is regulated by dietary components. The search for bioactive compounds that stimulate CCK secretion is currently an active area of research. The objective of this study was to evaluate the ability of buckwheat (Fagopyrum esculentum Moench) protein digest (BPD) to stimulate CCK secretion in vitro and in vivo and clarify the structural characteristics of peptides stimulating CCK secretion. BPD was prepared by an in vitro gastrointestinal digestion model. The relative molecular weight of BPD was <10 000 Da, and peptides with <3000 Da accounted for 70%. BPD was rich in essential amino acids Lys, Leu, and Val but lacked sulfur amino acids Met and Cys. It had a stimulatory effect on CCK secretion in vitro and in vivo. Chromatographic separation was performed to isolate peptide fractions involved in CCK secretion, and five novel CCK-releasing peptides including QFDLDD, PAFKEEHL, SFHFPI, IPPLFP, and RVTVQPDS were successfully identified. A sequence length range of 6-8 and marked hydrophobicity (18-28) were observed among the most CCK-releasing peptides. The present study demonstrated for the first time that BPD could stimulate CCK secretion and clarify the structural characteristics of bioactive peptides having CCK secretagogue activity in BPD.

Cultured Vagal Afferent Neurons as Sensors for Intestinal Effector Molecules.

The gut-brain axis embodies the bi-directional communication between the gastrointestinal tract and the central nervous system (CNS), where vagal afferent neurons (VANs) serve as sensors for a variety of gut-derived signals. The gut is colonized by a large and diverse population of microorganisms that communicate via small (effector) molecules, which also act on the VAN terminals situated in the gut viscera and consequently influence many CNS processes. However, the convoluted in vivo environment makes it difficult to study the causative impact of the effector molecules on VAN activation or desensitization. Here, we report on a VAN culture and its proof-of-principle demonstration as a cell-based sensor to monitor the influence of gastrointestinal effector molecules on neuronal behavior. We initially compared the effect of surface coatings (poly-L-lysine vs. Matrigel) and culture media composition (serum vs. growth factor supplement) on neurite growth as a surrogate of VAN regeneration following tissue harvesting, where the Matrigel coating, but not the media composition, played a significant role in the increased neurite growth. We then used both live-cell calcium imaging and extracellular electrophysiological recordings to show that the VANs responded to classical effector molecules of endogenous and exogenous origin (cholecystokinin serotonin and capsaicin) in a complex fashion. We expect this study to enable platforms for screening various effector molecules and their influence on VAN activity, assessed by their information-rich electrophysiological fingerprints.

A parabrachial to hypothalamic pathway mediates defensive behavior.

Defensive behaviors are critical for animal's survival. Both the paraventricular nucleus of the hypothalamus (PVN) and the parabrachial nucleus (PBN) have been shown to be involved in defensive behaviors. However, whether there are direct connections between them to mediate defensive behaviors remains unclear. Here, by retrograde and anterograde tracing, we uncover that cholecystokinin (CCK)-expressing neurons in the lateral PBN (LPBCCK) directly project to the PVN. By in vivo fiber photometry recording, we find that LPBCCK neurons actively respond to various threat stimuli. Selective photoactivation of LPBCCK neurons promotes aversion and defensive behaviors. Conversely, photoinhibition of LPBCCK neurons attenuates rat or looming stimuli-induced flight responses. Optogenetic activation of LPBCCK axon terminals within the PVN or PVN glutamatergic neurons promotes defensive behaviors. Whereas chemogenetic and pharmacological inhibition of local PVN neurons prevent LPBCCK-PVN pathway activation-driven flight responses. These data suggest that LPBCCK neurons recruit downstream PVN neurons to actively engage in flight responses. Our study identifies a previously unrecognized role for the LPBCCK-PVN pathway in controlling defensive behaviors.

Neonatal overnutrition, but not neonatal undernutrition, disrupts CCK-induced hypophagia and neuron activation of the nucleus of the solitary tract and paraventricular nucleus of hypothalamus of male Wistar rats.

Metabolic programming may be induced by reduction or enhancement of litter size, which lead to neonatal over or undernutrition, respectively. Changes in neonatal nutrition can challenge some regulatory processes in adulthood, such as the hypophagic effect of cholecystokinin (CCK). In order to investigate the effects of nutritional programming on the anorexigenic function of CCK in adulthood, pups were raised in small (SL, 3 pups per dam), normal (NL, 10 pups per dam), or large litters (LL, 16 pups per dam), and on postnatal day 60, male rats were treated with vehicle or CCK (10 µg/Kg) for the evaluation of food intake and c-Fos expression in the area postrema (AP), nucleus of solitary tract (NTS), and paraventricular (PVN), arcuate (ARC), ventromedial (VMH), and dorsomedial (DMH) nuclei of the hypothalamus. Overnourished rats showed increased body weight gain that was inversely correlated with neuronal activation of PaPo, VMH, and DMH neurons, whereas undernourished rats had lower body weight gain, inversely correlated with increased neuronal activation of PaPo only. SL rats showed no anorexigenic response and lower neuron activation in the NTS and PVN induced by CCK. LL exhibited preserved hypophagia and neuron activation in the AP, NTS, and PVN in response to CCK. CCK showed no effect in c-Fos immunoreactivity in the ARC, VMH, and DMH in any litter. These results indicate that anorexigenic actions, associated with neuron activation in the NTS and PVN, induced by CCK were impaired by neonatal overnutrition. However, these responses were not disrupted by neonatal undernutrition. Thus, data suggest that an excess or poor supply of nutrients during lactation display divergent effects on programming CCK satiation signaling in male adult rats.

Modulation of hypothalamic AMPK and hypothalamic neuropeptides in the control of eating behavior: A systematic review.

Eating behavior is regulated by central and peripheral signals, which interact to modulate the response to nutrient intake. Central control is mediated by the hypothalamus through neuropeptides that activate the orexigenic and anorexigenic pathways. Energy homeostasis depends on the efficiency of these regulatory mechanisms. This neuroendocrine regulation of hunger and appetite can be modulated by nutritional sensors such as adenosine monophosphate-activated protein kinase (AMPK). Thus, this systematic review discusses the literature on correlations between AMPK and hypothalamic neuropeptides regarding control of eating behavior. Lilacs, PubMed/Medline, ScienceDirect, and Web of Science were searched for articles published from 2009 to 2021 containing combinations of the following descriptors: "eating behavior," "hypothalamus," "neuropeptide," and "AMPK." Of the 1330 articles found initially, 27 were selected after application of the inclusion and exclusion criteria. Of the selected articles, 15 reported decreased AMPK activity, due to interventions using angiotensin II infusion, fructose, glucose, cholecystokinin, leptin, or lipopolysaccharide (LPS) injection; dietary control through a low-protein diet or a high-fat diet (60 % fat); induction of hyperthyroidism; or injection of AMPK inhibitors. Seven studies showed a decrease in neuropeptide Y (NPY) through CV4 AICAR administration; fructose, glucose, leptin, or angiotensin II injections; or infusion of LPS from Escherichia coli and liver kinase B1 (LKB1) overexpression. Eleven studies reported a decrease in food consumption due to a decrease in AMPK activity and/or hypothalamic neuropeptides such as NPY. The results indicate that there is a relationship between AMPK and the control of eating behavior: a decrease in AMPK activity due to a dietary or non-dietary stimulus is associated with a consequent decrease in food intake. Furthermore, AMPK activity can be modulated by glucose, thyroid hormones, estradiol, leptin, and ghrelin.

Chemosensing of fat digestion by the expression pattern of GPR40, GPR120, CD36 and enteroendocrine profile in sheep.

Gastrointestinal tract (GIT) epithelial cells detect nutrients in the lumen via G-protein coupled receptors (GPRs) located in the gut epithelial cells especially in enteroendocrine cells. Dietary free fatty acids (FFA) are the major energy source and also acts as signalling molecules for FFA receptors. Long chain fatty acids (LCFA) activate LCFA receptors, GPR40/FFAR1 and GPR120/FFAR4 which trigger intracellular signalling and release gut hormones or modifies gene expression that facilitate fat digestion and absorption. However, there is a paucity of information on chemosensing of nutrients and digestion in ruminants. Hence, present study was aimed to evaluate chemosensing of fat digestion and absorption by the expression pattern of GPR40, GPR120, chylomicron forming genes, fatty acid translocase (CD36/FAT), microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (APOB) in the various segments of GIT in sheep supplemented with calcium salts of long chain fatty acids (CSLCFAs) along with the secretory patterns of gut peptides cholecystokinin (CCK) and peptide tyrosine tyrosine (PYY). The study was carried out for a period 60 days with eighteen adult ewes of 8-12 months of age and they were divided into three groups with six animals each as group-I, group-II and group-III. All the experimental animals were stall fed with a basal diet and maintained as per animal husbandry standards. Group-II and group-III were supplemented additionally with 3% and 5% CSLCFAs, respectively on dry matter intake. The results from the study indicated that the supplementation of CSLCFAs upregulated (P < 0.05) the relative mRNA expression of GPR40 and GPR120 in the various segments of GIT of sheep in correspondence to level of dietary fat. Abundance of mRNA expression of CD36, MTTP and APOB increased (P < 0.05) in the GIT of sheep in accordance to quantity of LCFAs in the diet where these genes facilitate fatty acid uptake. Feeding of CSLCFAs enhanced (P < 0.05) pre-feeding level of CCK from day 15 onwards, whereas, post-feeding CCK and PYY increased in all the experimental sheep. However, the increase was higher (P < 0.05) in sheep supplemented with CSLCFAs by 10.80 ± 1.45% and 14.25 ± 1.17%, respectively in comparison to group-I. The comprehensive results of the study concluded that feeding of additional CSLCFAs upregulated the expression of GPR40, GPR120, CD36, and chemosensing of LCFAs by these genes triggered the signalling transduction that enhanced CCK and PYY levels to facilitate fat digestion and absorption in accordance with quantity of dietary fat. This was further evident from the significant upregulation of MTTP and APOB in the various segments of GIT supported the high content of dietary fat at cellular fat metabolism in the gut that regulates the fatty acid uptake.

Digestion characteristics of quinoa, barley and mungbean proteins and the effects of their simulated gastrointestinal digests on CCK secretion in enteroendocrine STC-1 cells.

The demand for plant-based proteins has been rapidly increasing due to sustainability, ethical and health reasons. The present study aimed to investigate the digestion characteristics of three plant proteins (quinoa, barley and mungbean) based on an in vitro digestion model and the effect of their simulated gastrointestinal digests on satiety hormone cholecystokinin (CCK) secretion in enteroendocrine STC-1 cells. The nitrogen distribution in the digestion process, the relative molecular weight (MW) of peptides and the amino acid composition in simulated gastrointestinal digests were characterized. Quinoa protein had the highest proportion of soluble nitrogen after gastrointestinal digestion (85.79%), followed by barley protein (74.98%) and mungbean protein (64.14%), suggesting that quinoa protein was more easily digested than barley and mungbean proteins. The peptides but not free amino acids were the main components in the gastrointestinal digests of quinoa, barley, and mungbean proteins. The gastrointestinal digest of quinoa protein had a well balanced amino acid pattern, whereas that of barley protein was lacking Lys, and that of the mungbean protein was short of sulfur amino acids (Phe + Tyr) but rich in Lys. In terms of the ability to stimulate CCK secretion, the gastrointestinal digest of barley protein had a strong stimulatory effect on CCK secretion, while that of quinoa and mungbean proteins had only a weak stimulatory effect. After pretreatment with a specific calcium-sensing receptor (CaSR) antagonist NPS 2143, CCK secretion induced by the barley protein digest was greatly suppressed, indicating that CaSR was involved in barley protein digest-induced CCK secretion. These results show that quinoa protein has good nutritional quality, while barley protein is an excellent plant protein source to stimulate CCK secretion and has a potential application as a dietary supplement for obesity management.

Initiation and severity of experimental pancreatitis are modified by phosphate.

Proper mitochondrial function and adequate cellular ATP are necessary for normal pancreatic protein synthesis and sorting, maintenance of intracellular organelles and enzyme secretion. Inorganic phosphate is required for generating ATP and its limited availability may lead to reduced ATP production causing impaired Ca2+ handling, defective autophagy, zymogen activation, and necrosis, which are all features of acute pancreatitis. We hypothesized that reduced dietary phosphate leads to hypophosphatemia and exacerbates pancreatitis severity of multiple causes. We observed that mice fed a low-phosphate diet before the induction of pancreatitis by either repeated caerulein administration or pancreatic duct injection as a model of pressure-induced pancreatitis developed hypophosphatemia and exhibited more severe pancreatitis than normophosphatemic mice. Pancreatitis severity was significantly reduced in mice treated with phosphate. In vitro modeling of secretagogue- and pressure-induced pancreatic injury was evaluated in isolated pancreatic acini using cholecystokinin and the mechanoreceptor Piezo1 agonist, Yoda1, under low and normal phosphate conditions. Isolated pancreatic acini were more sensitive to cholecystokinin- and Yoda1-induced acinar cell damage and mitochondrial dysfunction under low-phosphate conditions and improved following phosphate supplementation. Importantly, even mice on a normal phosphate diet exhibited less severe pancreatitis when treated with supplemental phosphate. Thus, hypophosphatemia sensitizes animals to pancreatitis and phosphate supplementation reduces pancreatitis severity. These appear to be direct effects of phosphate on acinar cells through restoration of mitochondrial function. We propose that phosphate administration may be useful in the treatment of acute pancreatitis.NEW & NOTEWORTHY Impaired ATP synthesis disrupts acinar cell homeostasis and is an early step in pancreatitis. We report that reduced phosphate availability impairs mitochondrial function and worsens pancreatic injury. Phosphate supplementation improves mitochondrial function and protects against experimental pancreatitis, raising the possibility that phosphate supplementation may be useful in treating pancreatitis.

Roles of Cholecystokinin in the Nutritional Continuum. Physiology and Potential Therapeutics.

Cholecystokinin is a gastrointestinal peptide hormone with important roles in metabolic physiology and the maintenance of normal nutritional status, as well as potential roles in the prevention and management of obesity, currently one of the dominant causes of direct or indirect morbidity and mortality. In this review, we discuss the roles of this hormone and its receptors in maintaining nutritional homeostasis, with a particular focus on appetite control. Targeting this action led to the development of full agonists of the type 1 cholecystokinin receptor that have so far failed in clinical trials for obesity. The possible reasons for clinical failure are discussed, along with alternative pharmacologic strategies to target this receptor for prevention and management of obesity, including development of biased agonists and allosteric modulators. Cellular cholesterol is a natural modulator of the type 1 cholecystokinin receptor, with elevated levels disrupting normal stimulus-activity coupling. The molecular basis for this is discussed, along with strategies to overcome this challenge with a corrective positive allosteric modulator. There remains substantial scope for development of drugs to target the type 1 cholecystokinin receptor with these new pharmacologic strategies and such drugs may provide new approaches for treatment of obesity.

Excitatory cholecystokinin neurons of the midbrain integrate diverse temporal responses and drive auditory thalamic subdomains.

The central nucleus of the inferior colliculus (ICC) integrates information about different features of sound and then distributes this information to thalamocortical circuits. However, the lack of clear definitions of circuit elements in the ICC has limited our understanding of the nature of these circuit transformations. Here, we combine virus-based genetic access with electrophysiological and optogenetic approaches to identify a large family of excitatory, cholecystokinin-expressing thalamic projection neurons in the ICC of the Mongolian gerbil. We show that these neurons form a distinct cell type, displaying uniform morphology and intrinsic firing features, and provide powerful, spatially restricted excitation exclusively to the ventral auditory thalamus. In vivo, these neurons consistently exhibit V-shaped receptive field properties but strikingly diverse temporal responses to sound. Our results indicate that temporal response diversity is maintained within this population of otherwise uniform cells in the ICC and then relayed to cortex through spatially restricted thalamic subdomains.

Effects of fasting and re-feeding on the expression of CCK, PYY, hypothalamic neuropeptides, and IGF-related genes in layer and broiler chicks.

Cholecystokinin (CCK) and peptide YY (PYY) have been investigated as gut hormones that send satiation signals to the brain in mammals. There is evidence that chicken PYY mRNA expression was the highest in the pancreas compared to other tissues. We recently suggested that insulin-like growth factor (IGF)-1 and its binding proteins (IGFBPs) may be involved in the appetite regulation system in chicks. In the present study, in order to evaluate the possible roles of CCK, PYY, and IGF-related proteins in the appetite regulation system in chicks, we analyzed changes in the mRNA levels of these genes in response to fasting and re-feeding in layer and hyperphagic broiler chicks. In layer chicks, 12 h of fasting reduced the mRNA levels of intestinal CCK, PYY, Y2 receptor, and pancreatic PYY, and these changes were reversed by 12 h of re-feeding. On the other hand, in broiler chicks 12 h of fasting reduced the mRNA levels of intestinal PYY and Y2 receptor, but not intestinal CCK and pancreatic PYY, and these changes were reversed by 12 h of re-feeding. Hypothalamic NPY mRNA significantly increased by 12 h of fasting in both chicks, and these changes were reversed by re-feeding. Also, 12 h of fasting significantly increased the mRNA levels of hypothalamic agouti-related protein and reduced the mRNA levels of hepatic IGF-1 only in broiler chicks, and 12 h of re-feeding did not change these. IGFBP-1 and -2 mRNA levels were markedly increased by 12 h of fasting in both chicks, and these changes were reversed by re-feeding. IGFBP-3 mRNA levels were increased by 12 h of fasting only in layer chicks, while re-feeding reduced the mRNA levels of IGFBP-3 in both types of chicks. These results suggest that several peripheral hormones, such as pancreatic PYY and intestinal CCK, may not play important roles in the regulation of food intake in broiler chicks.

Neonatal overnutrition programming impairs cholecystokinin effects in adultmale rats.

Overfeeding and rapid weight gain during early life are risk factors for the development of obesity in adulthood. This metabolic malprogramming may be mediated by endocrine disturbances during critical periods of development. Cholecystokinin (CCK) acts on the central nervous system by elevating thermogenesis and the activity of anorectic neurons, modulating overall energy balance. Therefore, we tested the hypothesis that postnatal overfeeding impaired CCK effects. Pups were raised in either a litter of three (neonatal overnutrition/small litter group) or 12 (controls/normal litter group) pups per dam to study the effects of postnatal overfeeding on the central and peripheral CCK systems in adulthood. Rats raised in small litters became overweight during lactation and remained overweight as adults, with increased adiposity and plasma levels of lipids, glucose, insulin, and leptin. Neonatally over-nourished rats showed attenuation of gastric emptying and anorexigenic response to CCK, suggesting that offspring from the SL group may present CCK resistance as adult male rats. Consistent with this idea, overweight rats displayed impaired central response in c-Fos immunoreactivity on the nucleus tractus solitarius, area postrema, paraventricular nucleus, central amygdala, arcuate nucleus, and dorsomedial hypothalamus in response to peripheral CCK at adulthood. The small litter group of adult male rats also exhibited reduced norepinephrine- and CCK-stimulated thermogenesis. Unresponsiveness to the effects of CCK may contribute to overweight and metabolic dysfunctions observed in postnatally over-nourished adult rats. Thus, the involvement of an impaired CCK system, among other neurohormonal failures, may contribute to the development of obesity.

[Effect of electroacupuncture of "biaoben acupoint combination" on central sensitivity of cholecystokinin in obese rats with insulin resistance].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "biaoben acupoint combination" on appetite, body fat, insulin sensitivity and central sensitivity of cholecystokinin (CCK) in obese rats with insulin resistance (IR), and to explore the mechanism of EA on improving obesity with insulin resistance. METHODS: Among the fifty 8-week-old healthy SPF male Wistar rats, 10 rats were randomly selected and fed with normal diet; after 8 weeks, 8 rats were randomly selected as a normal group. The remaining 40 rats were fed with high-fat diet to establish the model of obsesity IR; after 8 weeks, 24 rats with successful model of obsesity IR were randomly divided into a model group, an EA group and a sham EA group, 8 rats in each group. Eight weeks after model establishment, the rats in the EA group were intervened with EA at "Fenglong" (ST 40), "Zhongwan" (CV 12), "Guanyuan" (CV 4) and "Zusanli" (ST 36), with continuous wave, in frequency of 2 Hz, and current intensity of 1 mA, for 10 min each time. The rats in the sham EA group were intervened with EA at the points 5 mm next to the acupoints used in the EA group and no electricity was given; the sham EA was given for 10 min each time. Both the treatments were given once every other day for 8 weeks. The Lee's index and food intake were observed before the intervention as well as 2 weeks, 4 weeks, 6 weeks and 8 weeks into intervention; after the intervention, serum insulin (INS) and glucose infusion rate (GIR) were detected; serum cholecystokinin (CCK) level was detected by ELISA; c-fos expression in the area postrema (AP) and nucleus tractus solitarius (NTS) of medulla oblongata was detected by immunohistochemistry. RESULTS: Before the intervention as well as 2 weeks, 4 weeks, 6 weeks and 8 weeks into intervention, the Lee's index and food intake in the model group were higher than those in normal group (P<0.01). The Lee's index (6 weeks and 8 weeks into intervention) and food intake (4 weeks, 6 weeks and 8 weeks into intervention) in the EA group were lower than those in the model group and the sham EA group (P<0.05, P<0.01). After intervention, compared with the normal group, serum level of INS was increased (P<0.01), while GIR, serum CCK level, c-fos expression in AP and NTS in the model group were decreased (P<0.01, P<0.05). Compared with the model group and the sham EA group, serum level of INS in the EA group was decreased (P<0.01), and GIR, serum CCK level, c-fos expression in AP and NTS were increased (P<0.01, P<0.05). CONCLUSION: EA of "biaoben acupoint combination" could effectively reduce appetite, body fat and enhance insulin sensitivity in obese rats with IR. The mechanism may be related to the regulation of central sensitivity of CCK.

Oral D/L-3-Hydroxybutyrate Stimulates Cholecystokinin and Insulin Secretion and Slows Gastric Emptying in Healthy Males.

BACKGROUND: D-3-hydroxybutyrate (D-3-OHB) is a ketone body that serves as an alternative nutritional fuel but also as an important signaling metabolite. Oral ketone supplements containing D/L-3-OHB are becoming a popular approach to achieve ketosis. AIM: To explore the gut-derived effects of ketone supplements. METHODS: Eight healthy lean male volunteers were investigated on 2 separate occasions:An acetaminophen test was performed to evaluate gastric emptying and blood samples were obtained consecutively throughout the study period. RESULTS: We show that oral consumption of D/L-3-OHB stimulates cholecystokinin release (P = 0.02), elevates insulin (P = 0.03) and C-peptide (P < 0.001) concentrations, and slows gastric emptying (P = 0.01) compared with matched intravenous D/L-3-OHB administration. Measures of appetite and plasma concentrations of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) were unaffected by interventions. CONCLUSION: Our findings show that D/L-3-OHB exert incretin effects and indicate luminal sensing in the gut endothelium. This adds to our understanding of ketones as signaling metabolites and displays the important difference between physiological ketosis and oral ketone supplements.

Effects of Flavanols on Enteroendocrine Secretion.

Some beneficial effects of grape seed proanthocyanidin extract (GSPE) can be explained by the modulation of enterohormone secretion. As GSPE comprises a combination of different molecules, the pure compounds that cause these effects need to be elucidated. The enterohormones and chemoreceptors present in the gastrointestinal tract differ between species, so if humans are to gain beneficial effects, species closer to humans-and humans themselves-must be used. We demonstrate that 100 mg/L of GSPE stimulates peptide YY (PYY) release, but not glucagon-like peptide 1 (GLP-1) release in the human colon. We used a pig ex vivo system that differentiates between apical and basolateral intestinal sides to analyse how apical stimulation with GSPE and its pure compounds affects the gastrointestinal tract. In pigs, apical GSPE treatment stimulates the basolateral release of PYY in the duodenum and colon and that of GLP-1 in the ascending, but not the descending colon. In the duodenum, luminal stimulation with procyanidin dimer B2 increased PYY secretion, but not CCK secretion, while catechin monomers (catechin/epicatechin) significantly increased CCK release, but not PYY release. The differential effects of GSPE and its pure compounds on enterohormone release at the same intestinal segment suggest that they act through chemosensors located apically and unevenly distributed along the gastrointestinal tract.

Long-acting CCK analogue NN9056 lowers food intake and body weight in obese Göttingen Minipigs.

BACKGROUND/OBJECTIVES: Cholecystokinin (CCK) is a regulator of appetite and energy intake in man. The aim of this study was to determine the effect of NN9056, a long-acting CCK-1 receptor-selective CCK analogue, on food intake and body weight (BW) in obese Göttingen Minipigs. SUBJECTS/METHODS: Tolerability of NN9056 and acute effects on food intake, pancreas histology, amylase and lipase levels were assessed in lean domestic pigs in doses up to 100 nmol/kg (n = 3-4). Subsequently, obese Göttingen Minipigs were treated subcutaneously (s.c.) once daily for 13 weeks with vehicle, NN9056 low dose (regulated from 5 to 2 nmol/kg) or NN9056 high dose (10 nmol/kg) (n = 7-8). Food intake was measured daily and BW twice weekly. At the end of the treatment period, an intravenous glucose tolerance test (IVGTT) and a 24-h exposure profile was obtained. Data are mean ± SD. RESULTS: The acute studies in domestic pigs showed significant and dose-dependent effect of NN9056 on food intake, acceptable tolerability and no histopathological signs of pancreatitis. Sub-chronic treatment in obese Göttingen Minipigs was also well tolerated and accumulated food intake was significantly lower in both treated groups compared to vehicle, with no significant difference between the dose levels of NN9056 (41.8 ± 12.6, 51.5 ± 13.8 and 86.5 ± 19.5 kg in high-dose, low-dose and vehicle groups, respectively, p = 0.012 and p < 0.0001 for low and high dose vs. vehicle, respectively). Accordingly, there was a weight loss in both treated groups vs. a weight gain in the vehicle group (-7.2 ± 4.6%, -2.3 ± 3.2% and 12.3 ± 3.9% in the high-dose, low-dose and vehicle groups, respectively, p < 0.0001 for both vs. vehicle). IVGTT data were not significantly different between groups. CONCLUSION: NN9056, a long-acting CCK-1 receptor-selective CCK analogue, significantly reduced food intake and BW in obese Göttingen Minipigs after once daily s.c. dosing for 13 weeks.

Tibetan medicine Ru-yi-Zhen-bao Pills exhibits anti-migraine effect through mediating PAG anti-nociceptive channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Migraine is a disabling neurovascular disorder, which increases risk of cardiovascular events and is a social burden worldwide. The present first-line anti-migraine medications can cause overwhelming side-effects, of which one includes the onset of cardiovascular disease. As one of the marketed Tibetan drugs, Ru-yi-Zhen-bao Pills (RYZBP) have been clinically used to treat cardiovascular disorders and as anti-migraine medication. However, there is currently no research exploring the anti-migraine actions of RYZBP. AIM OF THE STUDY: The current research was designed to assess the anti-migraine roles of RYZBP and explore the underlying mechanisms in a nitroglycerin (NTG)-induced migraine rat model trial. MATERIALS AND METHODS: 120 rats were randomly divided into the following six groups of 20 rats each: normal control group, model control group, positive control group, and RYZBP high/medium/low-dose groups (Ru-yi-Zhen-bao Pills; TH 1.00 g/kg, TM 0.50 g/kg and TL 0.25 g/kg). All rats were administered intragastrically for 7 consecutive days, which were subcutaneously injected with the NTG (10 mg/kg) after the last gavage (except in the normal control group). 3min after NTG treatment, 30 rats (5 rats from each group) were anesthetized and devoted to electroencephalogram(EEG) testing, which was used to evaluate the analgesic effect of RYZBP. One hour after NTG treatment, the rest of the 90 rats (15 rats from each group) were anesthetized and midbrain tissue sample was dissected. The dissection was then washed with physiological saline and collected. The histopathological changes in the periaqueductal gray(PAG) of 5 tissue samples were determined by aematoxylin-eosin (H&E) staining, as well as an estimation of substance P (SP) and neurokinin 1 receptor (NK1R) expression through immunohistochemically staining(IHC). Another 5 midbrain preparations were carried out to evaluate calcitonin gene-related peptide (CGRP), proenkephalin (PENK), SP, and cholecystokinin (CCK) expressions by real-time quantitative polymerase chain reaction (RT-qPCR). The rest of the 5 brainstem tissues were then used to measure CCK, CGRP, and opioid peptide receptor (DORR) levels by western blotting(WB). RESULTS: In the EEG test, RYZBP (TM 0.50 g / kg) treatment transformed the EEG pain-wave of the NTG-induced migraine model rats in different time period. In the mechanism assay, compared with the model control group, RYZBP pretreatment reduced inflammatory cell infiltration, fibrosis and vacuolation of neuronal cells of PAG tissue seen by HE staining. IHC experiments further showed that RYZBPTM up-regulated SP expression levels and enhanced NK1R levels in the NTG-induced migraine rats (P < 0.05). Therapeutic administration of RYZBP also increased PENK mRNA expression and DORR protein level. Both RT-qPCR and western blotting trials indicated that RYZBP treatment significantly decreased CCK and CGRP expression levels (P < 0.01 or P < 0.05) in the NTG-induced migraine rats. CONCLUSIONS: RYZBP has the potential to be an effective anti-migraine treatment through suppressing the EEG pain-wave, increasing the levels of SP, PENK, DORR and reducing expression of CCK and CGRP. Mediating the PAG anti-nociceptive channel and inhibiting central sensitization were the two potential mechanisms, which offers further evidence for clinical therapy.

Cholecystokinin in the central nervous system of the sea lamprey Petromyzon marinus: precursor identification and neuroanatomical relationships with other neuronal signalling systems.

Cholecystokinin (CCK) is a neuropeptide that modulates processes such as digestion, satiety, and anxiety. CCK-type peptides have been characterized in jawed vertebrates and invertebrates, but little is known about CCK-type signalling in the most ancient group of vertebrates, the agnathans. Here, we have cloned and sequenced a cDNA encoding a sea lamprey (Petromyzon marinus L.) CCK-type precursor (PmCCK), which contains a CCK-type octapeptide sequence (PmCCK-8) that is highly similar to gnathostome CCKs. Using mRNA in situ hybridization, the distribution of PmCCK-expressing neurons was mapped in the CNS of P. marinus. This revealed PmCCK-expressing neurons in the hypothalamus, posterior tubercle, prethalamus, nucleus of the medial longitudinal fasciculus, midbrain tegmentum, isthmus, rhombencephalic reticular formation, and the putative nucleus of the solitary tract. Some PmCCK-expressing neuronal populations were only observed in adults, revealing important differences with larvae. We generated an antiserum to PmCCK-8 to enable immunohistochemical analysis of CCK expression, which revealed that GABA or glutamate, but not serotonin, tyrosine hydroxylase or neuropeptide Y, is co-expressed in some PmCCK-8-immunoreactive (ir) neurons. Importantly, this is the first demonstration of co-localization of GABA and CCK in neurons of a non-mammalian vertebrate. We also characterized extensive cholecystokinergic fibre systems of the CNS, including innervation of habenular subnuclei. A conspicuous PmCCK-8-ir tract ascending in the lateral rhombencephalon selectively innervates a glutamatergic population in the dorsal isthmic grey. Interestingly, this tract is reminiscent of the secondary gustatory/visceral tract of teleosts. In conclusion, this study provides important new information on the evolution of the cholecystokinergic system in vertebrates.

An effect of dietary phloretin supplementation on feed intake in mice.

Phloretin, abundantly present in apples, pears and other fruits, has been found to have antioxidant, immunosuppressive and anti-inflammatory activities. It has been reported that oral administration of phloretin dose-dependently increased feed intake in mice, but the mechanism is unclear yet. The aim of this study was to investigate the effect of dietary phloretin supplementation on the feed intake in C57BL/6J mice and to identify its mechanism. Here, sixty C57BL/6J mice (28-day age) were randomly chosen for four dietary treatments and fed a basal diet or a basal diet supplemented with 0.1%, 0.2%, and 0.3% phloretin, respectively, in a 6-week trial. We showed that mice in the 0.1%, 0.2%, and 0.3% phloretin-supplemented groups had increased accumulative feed intake compared with the control group. Furthermore, dietary phloretin supplementation significantly increased the ghrelin mRNA level in the stomach and hypothalamus, and decreased the cholecystokinin (CCK) mRNA level in the duodenum in a dose-dependent manner. The mRNA levels of neuropeptide Y (NPY), agouti-related protein (AgRP), pro-opiomelanocortin and melanocortin receptors 4 (MC4R), and pro-opiomelanocortin (POMC) in the hypothalamus were altered in response to dietary phloretin supplementation. Moreover, we confirmed that dietary phloretin supplementation reduced the expressions of miR-488 and miR-103, two feed intake-related miRNAs. Our present study provides evidence that dietary phloretin supplementation could increase feed intake in mice, which might be attributed to the stimulation of the hypothalamic feeding center via ghrelin, miRNAs (miR-103 and miR-488) and feeding signal factor-related genes (NPY, AgRP, MC4R and POMC), and to the inhibition of CCK to increase gastric emptying.

Branched chain amino acids stimulate gut satiety hormone cholecystokinin secretion through activation of the umami taste receptor T1R1/T1R3 using an in vitro porcine jejunum model.

Branched chain amino acids (BCAAs) are essential amino acids involved in regulation of feed intake. The function of BCAAs on the central nervous system has been extensively studied, but effects of BCAAs on secretion of gut satiety hormones and their underlying mechanisms are largely unknown. In this study, we evaluated the distribution of gut hormones and amino acid receptors in the porcine GI tract and found cholecystokinin (CCK) and taste dimeric receptor type 1 member 1/3 (T1R1/T1R3) were predominantly expressed in the jejunum and functionally interrelated. We further evaluated the effects of l-leucine, l-isoleucine, l-valine, and BCAAs on CCK and T1R1/T1R3 expression in porcine jejunum tissue. Our data demonstrated that stimulation of porcine jejunum tissue with 10 mM l-leucine, l-isoleucine or BCAAs mix (l-leucine : l-isoleucine : l-valine = 1 : 0.51 : 0.63) for 2 hours significantly increased mRNA expression and protein abundance of T1R1/T1R3 and secretion of CCK (P < 0.05). However, the l-valine treatment only increased the mRNA and protein abundance of T1R1 and T1R3 (P < 0.05), but not CCK secretion (P > 0.10). l-Leucine-, l-isoleucine- or BCAAs mix-induced CCK secretion was significantly decreased after tissues were pretreated with lactisole, a T1R1/T1R3 inhibitor (P < 0.05). Furthermore, the increased mRNA and protein abundance of T1R1/T1R3 were also largely attenuated by blocking T1R1/T1R3 with lactisole (P < 0.05). l-Leucine, l-isoleucine and BCAAs mix appeared to induce the gut satiety hormone CCK secretion through jejunal T1R1/T1R3. These results indicate over-supplementation with BCAAs in the diet might decrease food intake in swine and humans through gastrointestinal feedback.