Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.     

Ventricular, paraventricular and circumventricular structures involved in peptide-induced satiety

Cholecystokinin, bombesin or gastrin (2 microliter of 50 ng/microliter) was injected stereotaxically into the paraventricular nucleus of the hypothalamus, the arcuate/ventromedial area, the subfornical organ, the area postrema and the cerebral aqueduct of Sprague-Dawley rats and the effects of these injections on food and water intake were studied. While the injection of cholecystokinin reduced food intake when it was injected into both hypothalamic loci, food and water intake were most severely affected by the injection of this peptide into the cerebral aqueduct. Bombesin reduced food intake after its injection into all areas except the subfornical organ and reliable reductions in water intake were seen after injection of this peptide into all areas except the paraventricular nucleus. Minor reductions in food intake were seen following gastrin injection into the paraventricular nucleus while increased water consumption was observed after this peptide was injected into the paraventricular nucleus and cerebral aqueduct. In a second study 6-hydroxydopamine injections (2 microliter of 8 micrograms/microliter were made into the five areas studied 10 days before animals were injected with 100 micrograms/kg of cholecystokinin (i.p.). All 6-hydroxydopamine-injected animals reduced their food and water intake in response to the cholecystokinin challenge as did intact controls. These results indicate that while the changes in food and water intake produced by the central injection of cholecystokinin, bombesin or gastrin may involve central catecholamine systems, those occurring after its systemic administration do not. Therefore, if the release of gastrointestinal peptides during natural feeding is part of a homeostatic mechanism regulating hunger and satiety, this mechanism may operate without directly involving central catecholamine systems.     

Anti-peptide antibodies specific for the gastrin/cholecystokinin-B receptor

Summary Seven synthetic peptides, between 7–22 residues long, corresponding to six different parts of the gastrin/CCK_B receptor molecule which are conserved among the species, were used for raising antibodies. The peptides were coupled to keyhole limpet hemocyanine and injected into rabbits. ELISA analysis demonstrated that all peptides produced an immune response after three to six injections given at biweekly intervals. The titer ranged from 1:10⁴ to 1:10⁵. All antibodies recognized a 78 kDa protein on immunoblots of NIH 3T3 cells stably transfected with human gastrin/CCK_B receptor cDNA, as well as human and guinea pig stomach mucosal extracts. Preincubation of the sera with the corresponding peptides abolished the staining. Indirect immunofluorescence staining revealed that four antibodies out of the seven tested recognized the receptor in fixed COS-7 cells transiently transfected with human gastrin/CCK_B receptor cDNA. The reactive antibodies were raised against the peptides corresponding to receptor residues 40–58, 153–160, 288–294 and 356–372. Immunohistochemical staining of guinea pig stomach using these antisera resulted in intense staining of parietal cells in the fundus and cardia regions.Abbreviations CCK cholecystokinin - GR gastrin/CCK_B receptor - TFA trifluoroacetic acid - BSA bovine serum albumin - PBS phosphate-buffered saline - PAGE polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay - EDTA ethylenediamino tetraacetic acid - CLSM confocal laser scanning microscopy - KLH keyhole limpet hemocyanine     

Long-term comparative effect of cholecystokinin and gastrin on mouse stomach, antrum, intestine, and exocrine pancreas

Mice were injected three times a day for 12 days with 300 micrograms/kg body weight of gastrin G17 or 37.5 Ivy dog U/kg body weight of CCK or saline. Other mice were also injected four times an hr for 1 hr with 7.5 micrograms/kg of gastrin, nine Ivy dog U/kg of CCK or saline; 1 hr before killing, they were injected with tritiated thymidine to evaluate the labelling indices in peptic, antral, duodenal, jejunal, and ileal mucosae. Four hours after the first injection of the two peptides, the peptic labelling indices increased while those of intestinal mucosa increased 8 hr after these injections. Long-term injections of CCK had a trophic effect on secretory cells of the digestive tract: the number of gastric zymogenic cells, Paneth cells, and the mucous cells of Brünner glands were hypertrophied. The pepsin, amylase, chymotrypsin, and lysozyme activities increased in stomach, exocrine pancreas, and intestine, respectively. Neither parietal cells nor intestinal enterocytes and hydrolase activities were affected. The trophic effect of long-term injections of gastrin is confirmed on parietal cells and exocrine pancreatic parenchyma and is demonstrated in Paneth cells. Confirming cytological results, pancreatic lipase and amylase activities and intestinal lysozyme activity were increased after gastrin. Although CCK and gastrin have a structural analogy, these two peptides did not affect the same cellular types. A specific action of CCK on the main secretory cells of the digestive mucosa is demonstrated.     

针刺对术后胃肠功能紊乱大鼠胃肠传输功能及脑肠肽的影响

摘要 目的：探讨针刺三里穴、中脘对大鼠胃大部切除术后胃肠传输功能恢复的影响及可能的作用机制。方法：将60只 SD 大鼠随机分为空白组、模型组和针刺组,每组 20 只。造模成功后第3天开始,针刺组进行针刺足三里、中脘,连续治疗14天。于末次针刺结束后,各组记录进食量、体重等;后各组禁食24 h后进行胃残留率和小肠推进率测定,腹主动脉取血测定胃泌素、胃动素、食欲素A及食欲素1型受体。结果：造模前,三组大鼠体重和进食量差异无统计学意义,P>0.05。造模后3天,模型组及针刺组体重和进食量低于空白组,差异有统计学意义,P<0.05。针刺干预后,模型组体重和进食量低于空白组和针刺组,差异有统计学意义,P<0.05。针刺干预后,针刺组大鼠胃残留率、小肠推进率、胃泌素、胃动素、食欲素A及食欲素1型受体高于模型组,差异有统计学意义,P<0.05;模型组胃残留率、小肠推进率、胃泌素、胃动素、食欲素A及食欲素1型受体低于空白组,差异有统计学意义,P<0.05;针刺组与空白组胃残留率、小肠推进率、胃泌素、胃动素、食欲素A及食欲素1型受体差异无统计学意义,P>0.05。结论：针刺胃大部切除术后大鼠足三里穴、中脘穴,改善胃排空和小肠推进功能,促进术后胃肠功能的恢复,其作用机制可能为改变脑肠肽代谢,增加食欲素A水平,激活食欲素1型受体,促进胃泌素、胃动素分泌。     

下丘脑室旁核胃动素对消化功能影响的研究

目的：研究下丘脑室旁核注入胃动素及其拮抗剂对大鼠消化功能和体重增长的研究。方法：将剂量为0.005-5nmol的motilin和GM109注入大鼠下丘脑室旁核,1小时后可观察到大鼠摄食量显著增加并持续到两小时后。进食量的计算是通过预先称量好的鼠粮和应用药物20分钟、1小时、两小时后剩余数量比较而得出。实验持续一周。将实验组和对照组的进食量和体重进行比较。结果：室旁核注入胃动素5nmol的实验组和合并应用GM1090.005nmol的实验组在应用药物后1小时和2小时,可观察到摄食量显著增加（p〈0.01）,一周后体重也增加（p〉0.05）,然而摄食量的增加有显著性差异,体重的增加并无显著性差异。其他实验组也没有观察到显著性差异。结论：胃动素有调节消化运动,促进胃肠排空,促进食欲的作用。可能由于胃肠排空是频繁的,没有充裕的时间消化吸收,从而体重增加无显著性差异。     

下丘脑室旁核胃动素对消化功能影响的研究

目的:研究下丘脑室旁核注入胃动素及其拮抗剂对大鼠消化功能和体重增长的研究。方法:将剂量为0.005-5nmol的motilin和GM109注入大鼠下丘脑室旁核,1小时后可观察到大鼠摄食量显著增加并持续到两小时后。进食量的计算是通过预先称量好的鼠粮和应用药物20分钟、1小时、两小时后剩余数量比较而得出。实验持续一周。将实验组和对照组的进食量和体重进行比较。结果:室旁核注入胃动素5nmol的实验组和合并应用GM1090.005nmol的实验组在应用药物后1小时和2小时,可观察到摄食量显著增加(p<0.01),一周后体重也增加(p>0.05),然而摄食量的增加有显著性差异,体重的增加并无显著性差异。其他实验组也没有观察到显著性差异。结论:胃动素有调节消化运动,促进胃肠排空,促进食欲的作用。可能由于胃肠排空是频繁的,没有充裕的时间消化吸收,从而体重增加无显著性差异。     

Ovarian hormones and food intake in female guinea pigs and rhesus monkeys

Food intake was measured during ovarian cycles of female guinea pigs and rhesus monkeys. In both species, the period of lowest food intake occurred around the expected time of ovulation. Feeding levels were lowest in the guinea pig during the time of estrus, estrus being estimated on the basis of vaginal membrane changes. Minimum food intake in the rhesus occurred prior to the midpoint of the menstrual cycle, on Day 13, approximately 15–16 days before the next menstruation. In ovariectomized females of both species, either multiple or single estradiol injections significantly lowered food intake. Single progesterone injections to such females were found to have no significant effect on levels of feeding.     

Rhesus monkey gastroenteropancreatic hormones: relationship to human sequences

The amino acid sequences of the gastroenteropancreatic peptides of Old World mammals are generally well-conserved. However, only the glucagons and vasoactive intestinal polypeptides (VIP) have been shown to be identical among the species studied to date. Rhesus monkey (Macaca mulatta) insulin has been shown to be identical with human insulin. The question addressed in this study is whether other gastroenteropancreatic peptides are identical to the human peptides. Purification and sequencing of glucagon, pancreatic polypeptide, VIP and insulin confirmed their identity with the corresponding human peptides. However, the 17 amino acid monkey gastrin is identical to dog gastrin and differs from human gastrin by substitution of methionine for leucine at position 5 from the N-terminus and alanine for glutamic acid in position 10. If additional rhesus monkey tissues become available, it would be of interest to determine whether other gastrointestinal peptides also differ from the corresponding human peptides.     

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/ VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22–79, PHM, prepro-VIP 111–122, VIP and prepro-VIP 156–170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single-or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.     

A large proportion of afferent neurons innervating the uterine cervix of the cat contain VIP and other neuropeptides

Summary Axonal tracing techniques were used in combination with immunohistochemistry to examine the distribution of neuropeptides in afferent pathways from the uterine cervix of the cat. Primary afferent neurons innervating the uterine cervix were identified by axonal transport of the dye, fast blue, injected into the cervix. Fifteen to twenty-five days after the injection, dorsal root ganglia (L1–S3) were removed and incubated for 48–72 h in culture medium containing colchicine to increase the levels of peptides. Calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), leucine-enkephalin (LENK), somatostatin, substance P and vasoactive intenstinal polypeptide (VIP) were identified by use of indirect immunohistochemical techniques. Eighty-four percent of uterine cervix afferent neurons were identified in the sacral dorsal root ganglia (S1–S3), and 16% in the middle lumbar dorsal root ganglia (L3–L4). In sacral dorsal root ganglia, VIP was present in the highest percentage of dye-labeled cells (71%), CGRP in 42%, and substance P in 18% of the cells. CCK and LENK were present in 13% of the cells. In lumbar dorsal root ganglia, CGRP (51%) was most prominent peptide followed by VIP (34%), substance P (28%), LENK (17%) and CCK (13%). Somatostatin was present in the ganglia but did not occur in dye-labeled neurons. In conclusion, the uterine cervix of the cat receives a prominent VIP-and CGRP-containing afferent innervation. The percentage of neurons containing VIP is three to five times higher than the percentage of these neurons in afferent pathways to other pelvic organs. These observations coupled with the results of physiological studies suggest that VIP is an important transmitter in afferent pathways from the cervix.     

Regulatory peptides in the gastrointestinal tract of Alligator mississipiensis

Summary The gastrointestinal tract of the alligator Alligator mississipiensis has been investigated for the presence of immunoreactivity to fourteen regulatory peptides all known to occur in the mammalian gut system.Mucosal endocrine cells reacting specifically with the antisera to neurotensin, C-terminal gastrin, somatostatin, bombesin, secretin, pancreatic glucagon and enteroglucagon were detectable, the distribution of these cells being, in general, similar to the mammalian pattern. Peripheral nerve cell bodies and nerve fibres were detected with the antisera to vasoactive intestinal polypeptide, substance P, bombesin and somatostatin again with a distribution similar to that seen in mammals.No immunoreactivity was observed with the available antisera to glicentin, motilin, gastric inhibitory polypeptide, gastrin 34, cholecystokinin 9–20 and met-enkephalin.     

The role of some central catecholamine systems in cholecystokinin-induced satiety

Cholecystokinin (CCK), bombesin and gastrin were stereotaxically injected into catecholamine (CA) innervated areas of the lateral hypothalamus (LH), the nucleus caudatus putamen (NP) and the olfactory tubercle (OT) in male Sprague Dawley rats. Bilateral injections of 100 ng of CCK in 2 μl of vehicle into the LH produced a slight but significant decrease in food intake during the first hour of a 4 hour eating test. The other peptides when injected into any of the brain areas did not significantly alter food intake. Water intake was affected by the injection of all three hormones although differentially in all 3 sites. The observed changes in drinking were not related to the prandial characteristics of drinking typically seen in rodents. Denervation of the CA innervation of the OT, LH or NP with 6-hydroxydopamine did not change the satiety response to peripherally administered CCK displayed by intact animals. These results suggest that the satiety which occurs after the central and peripheral administration of CCK may be mediated by different mechanisms and that central CA systems may not be necessary for CCK-induced satiety to occur during natural feeding.     

The effects of prior weight reduction on the running wheel-induced feeding suppression in rats

Adult male rats given ad lib access to food and a running wheel show an initial feeding and weight suppression. Over 6-10 days feeding recovers, but body weight remains low. It is not clear which effect is primary, the wheel-induced feeding or weight change. To test this, rats were first restricted to 15 g of food a day for 8 or 16 days to reduce their weight relative to control non-restricted rats. They were then returned to ad lib feeding and half the restricted and non-restricted control rats were introduced to the wheel either immediately (Experiment 1) or 4 days later (Experiment 2). Food intake, body weight, and wheel running were monitored throughout the experiments. At the return to ad lib feeding, prior food restriction elevated feeding. Both immediate and delayed wheel access suppressed feeding in both groups of wheel access rats compared to the appropriate control rats. Feeding history did not have a significant effect on wheel running. The wheel-induced reductions in feeding from baseline were similar in the weight reduced and normal weight animals suggesting that prior weight restriction did not prevent the onset of the wheel-induced feeding suppression. It is therefore suggested that the feeding suppression is not driven by a reduced weight set point.     

Xenopsin immunoreactivity in antral G-cells may reside in the N-terminus of gastrin 17

Summary The nature of xenopsin immunoreactivity in mammalian antral G-cells has been reassessed. Xenopsin immunostaining was most intense in human antral G-cells, present in those of the dog and pig and not detected in guinea pig or rat tissues. Rigorous specificity controls for ionic binding of immunoglobulins to antral G-cell granules indicated that this mechanism was not responsible for xenopsin immunostaining. Preincubation of the xenopsin antiserum with xenopsin, human gastrin 1–13 and gastrin 2–17 completely abolished immunostaining at similar molar concentrations. Gastrin 34 was ineffective at much higher concentrations. These results infer that xenopsin-immunoreactivity in antral G-cells resides in the N-terminal region of gastrin 17. Examination of the primary structures of xenopsin and the N-terminal regions of some mammalian gastrins reveals a hitherto unrecognized homology.     

Ability of calcitonins to alter food and water consumption in the rat

Synthetic salmon calcitonin (sCT) given SC (30 MRC U/kg) or ICV (1.4 U, 300 ng) inhibited 24-hr food consumption in the rat by 50–100%. Furthermore, ICV doses of sCT ranging from 75 ng (0.35 U) to 300 ng (1.4 U) lowered both food and water intake in a dose-dependent manner. Use of various forms of CT give an apparent order of potency of salmon>porcine≥human with sCT being at least 40 fold more potent than the mammalian forms. Measurement of food and water intake over a brief (30–90 min) period showed that ICV sCT was effective in reducing food and water consumption regardless of whether it had been given 1, 12, or 23 hr previously. Daily administration of sCT for 5 days caused marked suppression of food and water intake for 2 days; thereafter, consumption returned toward normal, becoming equivalent to vehicle injected controls by day 3 or 4 despite continued daily injections of sCT. The results show that CT can act centrally to modify certain types of behavior and are of special interest since CT-like peptides have been described in the pituitary and hypothalamus and since CT receptors have been reported in hypothalamic and other brain regions.     

Dorsomedial hypothalamic corticotropin-releasing factor mediation of exercise-induced anorexia

Running wheel access and resulting voluntary exercise alter food intake and reduce body weight. The neural mechanisms underlying these effects are unclear. In this study, we first assessed the effects of 7 days of running wheel access on food intake, body weight, and hypothalamic gene expression. We demonstrate that running wheel access significantly decreases food intake and body weight and results in a significant elevation of CRF mRNA expression in the dorsomedial hypothalamus (DMH) but not the paraventricular nucleus. Seven-day running wheel access also results in elevated arcuate nucleus and DMH neuropeptide Y gene expression. To assess a potential role for elevated DMH CRF activity in the activity-induced changes in food intake and body weight, we compared changes in food intake, body weight, and hypothalamic gene expression in rats receiving intracerebroventricular (ICV) CRF antagonist alpha-helical CRF or vehicle with or without access to running wheels. During a 4-day period of running wheel access, we found that exercise-induced reductions of food intake and body weight were significantly attenuated by ICV injection of the CRF antagonist. The effect on food intake was specific to a blockade of activity-induced changes in meal size. Central CRF antagonist injection further increased DMH CRF mRNA expression in exercised rats. Together, these data suggest that DMH CRF play a critical role in the anorexia resulting from increased voluntary exercise.     

Effects of beta-chlornaltrexamine on food intake, body weight and opioid-induced feeding

beta-Chlornaltrexamine (beta-CNA) is a non-equilibrium opioid receptor antagonist which alkylates and inactivates opioid receptors. Because opioid peptides are thought to contribute to the regulation of food intake, we examined the effects of intracerebroventricular (icv) injections of beta-CNA on the food intake and body weight of male rats. We also tested the ability of beta-CNA to block food intake stimulated by selective agonists of kappa, mu and delta opioid receptors: dynorphin A2 (DYN), Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAGO), and [(D-Ser2,Leu5]-enkephalin-Thr6 (DSLET). Treatment with beta-CNA caused a long-term (2-4 days) reduction in daily food intake and a concomitant reduction in body weight. An additional experiment indicated that the weight loss after beta-CNA treatment could be completely accounted for by the reduction in intake. beta-CNA treatment also abolished or greatly attenuated the feeding effects of DAGO, DSLET and DYN, even when these peptides were tested 26 hours after beta-CNA administration. The long duration of the effects of beta-CNA suggests that this compound will be a useful pharmacological tool in further study of the opioid feeding system.     

Effects of vasopressin replacement during food-restriction stress

The effects of subcutaneous injections of vasopressin in vasopressin-deficient (Brattleboro or DI) rats were observed during nonstress (habituation) and stress (food-restriction) conditions as compared to other rats. Four groups of animals were employed: 1) Long-Evans (LE) rats that were food restricted with no injections (normal control animals), 2) DI rats that were food restricted with no injections, 3) DI rats injected with vasopressin, and 4) DI rats injected with peanut oil (vehicle). The parameters studied were: body weight, food intake, water intake, and gastric ulcer formation. With respect to body weight, water intake, and ulcer formation, two sets of animals emerged. The vasopressin-injected DI rats resembled the LE control rats, whereas the peanut oil-injected DI rats were similar to the DI rats with no injections. The former set of animals showed a higher body weight, reduced water intake, and fewer gastric ulcers than the latter set of animals. Thus the vasopressin-injected DI rats and the LE control rats could cope with the stress of food restriction, but the peanut oil-injected DI rats and the DI rats with no injections could not.     

Identifying Predictors of Activity Based Anorexia Susceptibility in Diverse Genetic Rodent Populations

Animal studies are very useful in detection of early disease indicators and in unravelling the pathophysiological processes underlying core psychiatric disorder phenotypes. Early indicators are critical for preventive and efficient treatment of progressive psychiatric disorders like anorexia nervosa. Comparable to physical hyperactivity observed in anorexia nervosa patients, in the activity-based anorexia rodent model, mice and rats express paradoxical high voluntary wheel running activity levels when food restricted. Eleven inbred mouse strains and outbred Wistar WU rats were exposed to the activity-based anorexia model in search of identifying susceptibility predictors. Body weight, food intake and wheel running activity levels of each individual mouse and rat were measured. Mouse strains and rats with high wheel running activity levels during food restriction exhibited accelerated body weight loss. Linear mixed models for repeated measures analysis showed that baseline wheel running activity levels preceding the scheduled food restriction phase strongly predicted activity-based anorexia susceptibility (mice: Beta  =  −0.0158 (±0.003 SE), P<0.0001; rats: Beta  =  −0.0242 (±0.004 SE), P<0.0001) compared to other baseline parameters. These results suggest that physical activity levels play an important role in activity-based anorexia susceptibility in different rodent species with genetically diverse background. These findings support previous retrospective studies on physical activity levels in anorexia nervosa patients and indicate that pre-morbid physical activity levels could reflect an early indicator for disease severity.