- Email: [email protected]
A novel simultaneous measurement method to assess the influence of intracerebroventricular obestatin on colonic motility and secretion in conscious rats
Peptides 31 (2010) 1113–1117
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
A novel simultaneous measurement method to assess the influence of intracerebroventricular obestatin on colonic motility and secretion in conscious rats Chih-Yen Chen a,b,∗ , Ming-Luen Doong c,1 , Chung-Pin Li a,b,1 , Wen-Jinn Liaw d,1 , Hsing-Feng Lee a,b , Full-Young Chang a,b , Han-Chieh Lin a,b , Shou-Dong Lee a,b a
Faculty of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taiwan c Department and Institute of Physiology, National Yang-Ming University School of Medicine, Taipei, Taiwan d Department of Anesthesiology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 22 February 2010 Received in revised form 17 March 2010 Accepted 17 March 2010 Available online 23 March 2010 Keywords: Obestatin Colonic transit time Fecal pellet output Colonic secretion Intracerebroventricular
a b s t r a c t Obestatin, a novel putative 23-amino acid peptide, is derived from mammalian preproghrelin gene via a bioinformatics approach. Although obestatin regulates thirst, sleep, memory, anxiety, activates cortical neurons in the brain and stimulate proliferation of retinal pigment epithelial cells, there is no study to explore its central impacts on the lower gut motility and secretion. We investigated the influence of intracerebroventricular (ICV) injection of obestatin on rat colonic motor and secretory functions. Colonic transit time, fecal pellet output and fecal content were assessed in freely fed, conscious rats, which were implanted with ICV and colonic catheters chronically. Human/rat corticotropin-releasing factor (h/rCRF) was applied as a stimulatory inducer of colonic motility and secretion. ICV injection of obestatin (0.1, 0.3, 1.0 nmol/rat) did not modify the colonic transit time, whereas ICV injection of h/rCRF (0.3 nmol/rat) significantly shortened colonic transit time. ICV obestatin in any dose we tested did not affect the fecal pellet output, frequency of watery diarrhea, total fecal weight, fecal dried solid weight, or fecal fluid weight in the first hour post-injection, either. In contrast, ICV injection of h/rCRF effectively stimulated fecal pellet output, as well as increased total fecal weight, fecal dried solid weight and fecal fluid weight during the first hour post-injection, compared to ICV saline controls. In conclusion, using our novel simultaneous measurement method, acutely central administration of obestatin exhibits no influence on colonic motility and secretion in conscious rats. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Obestatin, a novel 23-amino acid peptide recently identified from rat stomach, is derived from C-terminal part of mammalian preproghrelin gene that also encodes ghrelin, by comparative genomic analyses [35]. It was originally projected that acyl ghrelin and obestatin exhibit opposite influence on ingestive behaviors, e.g. acyl ghrelin elicits food intake whereas obestatin inhibits it [35], although the effects of obestatin on food intake are debatable [12,13]. Acyl ghrelin binds to growth hormone secretagogue receptor 1a, and, then, signals via a Gq/11 ␣-subunit that results in the release of inositol trisphosphate and Ca2+ [9]. Obestatin
∗ Corresponding author at: Division of Gastroenterology, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Tel.: +886 2 28712121x3341; fax: +886 2 28711058. E-mail address: [email protected] (C.-Y. Chen). 1 These authors contributed equally to this work.
was suggested binding to an orphan G protein-coupled receptor (GPR), termed GPR39 [7,35], and induced the increase of intracellular cAMP [35]. GPR39 expression has been detected in peripheral organs such as the duodenum and kidney but not in the pituitary or hypothalamus [22]. But recent studies reveal that obestatin is not the endogenous cognate ligand for GPR39 [8,22,23,34]. Obestatin manifested various biological functions at the central nervous system, such as inhibition of thirst [30], increase of nonrapid eye movement sleep episodes and decrease of sleep latency [29], as well as improvement of memory retention and reduction of anxiety [6] in rats. Besides, obestatin has also been reported to activate cortical neurons [15], and stimulate proliferation of retinal pigment epithelial cells [5]. In addition, a recent study indicates that mice lacking the preproghrelin gene have impaired abilities to manifest and integrate normal sleep and thermoregulatory responses to metabolic challenges [28]. When considered together, these results imply that obestatin may have some unknown but important novel roles on the central nervous system in the uninvestigated field, which deserve further exploration.
0196-9781/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.03.024
1114
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117
Regarding gastrointestinal motility, many studies of obestatin focus on its effects on the upper gut [1,4,14,20]. Peripheral obestatin inhibited gastroduodenal motility in the fed state but not in the fasted state of conscious rats through activating corticotropinreleasing factor (CRF) receptor 1 and 2 in the brain, while vagal afferent pathways might be partially involved [1,16]. On the other hand, intracisternal injection of obestatin did not affect gastric phasic contraction of anesthetized fasted rats [19]. Our previous paper showed that peripheral administration of obestatin has no impact on colonic motility and secretion in rats [10]. From our PubMed search, there is no study to address the influence of centrally administered obestatin on rat colonic motor and secretory functions. As the above-mentioned multifaceted functions of obestatin in the brain, it is very interesting to investigate the potential, central effects of obestatin on colon motility and secretion. In the present study, we used our newly established animal model to simultaneously measure colonic transit time, fecal pellet output and fecal content in conscious fed rats [10]. Corticotropin-releasing factor (CRF), a well-known colonoprokinetic peptide, was applied to serve as a stimulatory agent to test the validity of this in vivo model. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (National Laboratory Animal Center, Taipei, Taiwan) weighing 250–320 g at the initial period of experiment were used and housed in group cages under controlled illumination (light cycle: 08:00–20:00), humidity and temperature (22.5 ± 1.5 ◦ C) with free access to water and laboratory chow pellets (LabDiet® , Brentwood, MO, USA). All experiments were performed since 9 a.m. in freely moving conscious fed rats, in accordance to guidelines, which have been approved by the Institutional Animal Care and Use Committee (IACUC), Taipei Veterans General Hospital. 2.2. Surgery 2.2.1. Implantation of intracerebroventricular (ICV) catheter Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg, Nembutal; Abbott Laboratories, Abbott Park, IL, USA), placed in a stereotaxic apparatus, and implanted with a guide cannula (25-gauge; Eicom, Kyoto, Japan), which reached the right lateral ventricle. Stereotaxic coordinates were 0.8 mm posterior to bregma, 1.4 mm right lateral to the midline, and 4.5 mm below the outer surface of the skull using a stereotaxic frame (BenchmarkTM , myNeuroLab, St. Louis, MO, USA) with the incisor bar set at the horizontal plane passing through bregma and lambda. The guide cannule was secured with dental cement anchored by two stainless steel screws fixed on the dorsal surface of the skull. After surgery, a dummy cannula (Eicom) was inserted into each guide cannula, and a screw cap (Eicom) was put on the guide cannula to prevent blockade [11]. The correctness of ICV cannula placement was verified by injection of 100 l dye (0.05% cresyl violet, Sigma) into the right lateral ventricle by the brain sections at the end of experiments after euthanasia [11]. Before beginning all colonic motor and secretory function tests, those animals receiving implantation of colonic tubes with ICV catheters were allowed 7 days for recovery. All ICV injections were performed over 60 s in 5 l using the AMI-5 (Eicom). 2.2.2. Implantation of colonic catheter Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg, Nembutal; Abbott). After the lower abdomen laparatomy, a catheter (3 Fr, 1-mm diameter; ATOM) was implanted into the proximal colon 2 cm distal from the ceco-colonic junction chronically. The catheter was fixed at the colonic wall by a
purse-string suture and routed subcutaneously to the interscapular region, where it was exteriorized through the skin using for intracolonic administration of dye marker [10]. Before simultaneous measurement of colonic motor and secretory functions, rats were allowed 7 days to recovery. 2.3. Preparation of drugs Rat obestatin (Peptides International, Inc., Louisville, KY, USA) and human/rat CRF (h/r CRF) (American Peptide Company, Sunnyvale, CA, USA) were kept in powder form at −20 ◦ C, and dissolved in sterile, pyrogen-free 0.9% saline (Otsuka, Tokyo, Japan) immediately before use. The doses of obestatin, 0.1, 0.3, and 1.0 nmol/rat, were selected similar to those effective in elevated plus maze test [6] and thirst study [30], while the dose of human/rat CRF (0.3 nmol/rat) was chosen mimicking the effective one in manometric recording [2]. 2.4. Colonic motor and secretory function tests 2.4.1. Measurement of colonic transit time Colonic transit time was calculated using an enteral dye marker, trypan blue (Sigma Chemical Co, St. Louis, MO, USA), a nonabsorbable dye. The dye was injected in 0.2 ml volume through the catheter positioned in the proximal colon, and followed by a 0.2 ml saline flush 10 min after the ICV injection of either saline, obestatin, or h/rCRF [10]. Colonic transit time was defined as the time interval between the dye injection and the discharge of the first blue pellet. 2.4.2. Measurement of fecal pellet output Rats were accustomed to single housing cage individually for 7 days before the experiment. The total number of pellets was recorded every hour after an intracolonic injection of trypan blue for 2 h. 2.4.3. Measurements of characteristics of fecal content The frequency of watery diarrhea was assessed by the number of rats expelling loose watery stool. The total fecal material was collected every hour after the intracolonic injection of trypan blue for 2 h, and its content was weighed then desiccated overnight at 50 ◦ C, and the fecal fluid and solid output were calculated from the total and dry weights [10,26]. 2.5. Statistical analysis All results are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post hoc test was used to analyze difference among groups. Differences were considered statistically significant with p < 0.05. 3. Results 3.1. Colonic transit time ICV injection of obestatin at 0.1, 0.3, and 1.0 nmol/rat did not change the colonic transit time (290.7 ± 29.9, 265.6 ± 29.4, and 292.9 ± 19.8 min vs. 300.3 ± 21.0 min, p > 0.05), compared to ICV saline controls (Fig. 1). Contrary, ICV injection of h/rCRF at 0.3 nmol/rat significantly shortened colonic transit time (190.3 ± 23.5 vs. 300.3 ± 21.0 min, p < 0.05) in conscious fed rats. 3.2. Fecal pellet output ICV obestatin (0.1, 0.3, and 1.0 nmol/rat) did not affect the number of fecal pellet output (1.17 ± 1.08, 0.92 ± 0.47, and 0.58 ± 0.29 vs. 1.00 ± 0.48 per hour), whereas ICV injection of h/rCRF at 0.3
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117
1115
Fig. 1. The influence of intracerebroventricular (ICV) injection of obestatin and human/rat corticotropin-releasing factor (h/rCRF) on colonic transit time in conscious rats. ICV obestatin did not modify colonic transit time, whereas h/rCRF significantly speeded colonic transit time. *p < 0.05, compared to all the other groups, n = 12 rats in each group.
nmol/rat effectively increased the number of fecal pellet output (2.75 ± 0.58 vs. 1.00 ± 0.48 per hour, p < 0.05) during the first hour post-injection in conscious fed rats (Fig. 2). However, neither any dose of ICV obestatin nor h/rCRF injection modified the number of fecal pellet output (0.33 ± 0.26, 0.58 ± 0.50, 0.67 ± 0.58, and 1.33 ± 0.62 vs. 0.33 ± 0.33 per hour), though h/rCRF treatment showed a tendency to enhance the number of fecal pellet output during the second hour post-injection (Fig. 2). 3.3. Fecal content ICV obestatin (0.1, 0.3, and 1.0 nmol/rat) did not have any impact on the total fecal weight (0.10 ± 0.07, 0.23 ± 0.12, and 0.18 ± 0.09 g/h), dried solid weight (0.05 ± 0.05, 0.09 ± 0.05, and 0.07 ± 0.03 g/h) and fecal fluid weight (0.05 ± 0.03, 0.14 ± 0.08, and 0.11 ± 0.05 g/h) during the first hour in conscious rats (Fig. 3A). In contrary, ICV injection of h/rCRF at 0.3 nmol/rat significantly increased total fecal weight (0.95 ± 0.23 and 0.18 ± 0.08 g/h, p < 0.001), dried solid weight (0.30 ± 0.06 g/h vs. 0.06 ± 0.03 g/h, p < 0.01) and fecal fluid weight (0.65 ± 0.17 g/h vs. 0.12 ± 0.05 g/h, p < 0.001) during
Fig. 3. The influence of intracerebroventricular (ICV) injection of obestatin and human/rat corticotropin-releasing factor (h/rCRF) on colonic secretion 1 h (A) and 2 h (B) after the injection. (A) ICV obestatin did not modify any colonic secretory parameters, whereas h/rCRF significantly enhanced total fecal weight, fecal dried weight, and fecal fluid weight during the first hour after injection. **p < 0.01, ***p < 0.001, compared to all the other groups, n = 12 rats in each group. (B) Neither ICV obestatin nor h/rCRF treatment affected any parameters of colonic secretion during the second hour after injection; n = 12 rats in each group.
the first hour post-injection, compared to ICV saline controls (Fig. 3A). Neither any dose of ICV obestatin nor h/rCRF injection influenced total fecal weight (0.11 ± 0.09 g/h, 0.14 ± 0.12 g/h, 0.18 ± 0.15 g/h, and 0.35 ± 0.14 g/h vs. 0.08 ± 0.08 g/h), dried solid weight (0.04 ± 0.03 g/h, 0.04 ± 0.03 g/h, 0.08 ± 0.06 g/h, and 0.12 ± 0.05 g/h vs. 0.03 ± 0.03 g/h) and fecal fluid weight (0.07 ± 0.06 g/h, 0.10 ± 0.08 g/h, 0.10 ± 0.08 g/h, and 0.23 ± 0.09 g/h vs. 0.04 ± 0.04 g/h), though h/rCRF treatment showed a tendency to increase these fecal parameters during the second hour postinjection (Fig. 3B). Besides, ICV obestatin (0.1, 0.3, and 1.0 nmol/rat) did not change the frequency of watery diarrhea during the first hour post-injection, while ICV injection of h/rCRF at 0.3 nmol/rat exhibited a mild tendency to enhance the frequency of watery diarrhea during the first hour post-injection (Table 1). Similarly, neither any dose of ICV obestatin nor h/rCRF treatment modified the frequency of watery diarrhea during the second hour post-injection (Table 1). 4. Discussion
Fig. 2. The influence of intracerebroventricular (ICV) injection of obestatin and human/rat corticotropin-releasing factor (h/rCRF) on fecal pellet output in conscious rats. ICV obestatin did not affect the fecal pellet output during the first and second hour after injection. However, ICV h/rCRF significantly increased fecal pellet output during the first but not the second hour after injection. *p < 0.05, compared to all the other groups, n = 12 rats in each group.
Previous studies could only just explore either colonic motor or secretory functions in rodents. Our newly established animal model makes a breakthrough over this limitation and is capable of simultaneously measuring both functions [10]. In the present study, we tried to investigate the central, potential effects of obestatin on colonic transit and secretion in rats. The mean obtained colonic
1116
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117
Table 1 Effects of ICV injection of saline, obestatin and h/rCRF on the frequency of watery diarrhea in conscious fed rats; n = 12 rats in each group.
The first hour post-injection The second hour post-injection
Saline (5 l/rat)
Obestatin (0.1 nmol/rat)
Obestatin (0.3 nmol/rat)
Obestatin (1.0 nmol/rat)
h/rCRF (0.3 nmol/rat)
0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00
0.25 ± 0.25 0.25 ± 0.25
transit time of ICV saline-injected control rats as 300 min in our study was comparable to the previous study showing 320 min using the similar trypan blue dye method [31]. The data of fecal pellet output in our ICV saline-injected controls were also comparable with those obtained by the other study group (1.33 vs. 1.28 n/2 h) [21]. In addition, our study could be the first to investigate ICV injections of peptides on fecal content in rats, because no previous literature is found. Moreover, none of our controls demonstrated watery diarrhea, implying that these animals had acclimated and been well handled before and during experiments without any stressful factors. Thus, our measurement for ICV injections of peptides on colonic motor and secretory functions in conscious fed rats should be valid. The first preproghrelin gene product, acyl ghrelin, poses prokinetic activity along the entire gastrointestinal tract [9]. Acyl ghrelin, when ICV administered as well as microinjected inside, but not outside the paraventricular nucleus, does accelerate colonic transit time in rats [31,32]. Acyl ghrelin acts in the central nervous system to modulate colonic motility via hypothalamic neuropeptide Y1 and CRF1 receptors pathways [31]. In addition to stimulating colonic motor functions, oral administration of a centrally acting ghrelin receptor agonist, GSK894281, significantly increases fecal weight in rats [27]. Moreover, ghrelin receptor agonists have been shown to improve colonic functions in rats with post-operative ileus. Intravenous administration of TZP-101 and ipamorelin, another two ghrelin receptor agonists, were demonstrated to effectively reverse laparotomy-induced delay of colonic transit time [14] and inhibition of fecal pellet output [17,33] in rats. In the current study, the influence of the third preproghrelin gene product, obestatin, on colonic motility and secretion in rats were examined. On the contrary, our study indicated that ICV injection of obestatin at low doses (0.1–1.0 nmol), different from ghrelin and/or ghrelin receptor agonists, did not modify colonic transit time. ICV obestatin did not change the fecal pellet output, either. In addition, ICV obestatin did not impact any fecal content parameters during the first hour post-injection, such as the frequency of watery diarrhea, fecal total weight, dried weight and fluid weight. Though both colonic transit time and fecal pellet output belong to colonic motor functions, some differences of physiological significance between the measurement of colonic transit time and fecal pellet output exist. The fecal pellet output is considered as the distal colonic motor function, whereas the colonic transit time corresponds to the motor function of the entire colon [24]. Although central obestatin has important roles in influencing thirst [30], sleep [29], memory and anxiety [6], our study suggested that obestatin does not act in the forebrain to affect colonic motor and secretory functions in conscious rats. However, higher sensitivity of manometric method than solid gastric emptying in detecting the inhibitory effects of anorexigenic peptides, such as neuromedin S, on the upper gut motility has been clearly demonstrated by a recent report [3]. Further studies using the potentially more sensitive manometric method to investigate central effects of obestatin on gastric and colonic motility, including applying the higher effective dose (3 nmol/rat) shown in the elevated plus maze [6] and thirst [30] test, maybe warranted in the future. Peripheral administration of obestatin is reported able to alter gastroduodenal motility via CRF receptors, while immunohistochemical analysis reveals that intravenous obestatin elicits CRF and urocortin 2-containing neurons in the forebrain [1]. Therefore, we
used h/rCRF as a stimulatory control for comparison in this study. From our thorough literature review, the current study is the first to simultaneously investigate effects of ICV h/rCRF on colonic motility and secretion. We found that ICV injection of h/rCRF at the low dose (0.3 nmol) potently accelerated colonic transit time, enhanced fecal pellet output, as well as increased total fecal weight, dried solid weight and fluid weight during the first hour post-injection, but not the frequency of watery diarrhea, compared to ICV saline-injected controls. As the same in our previous study, we tried to extend our study to observe the longer temporal effects of the peptides [10]. We found that both agents did not change any parameters of colonic motility and secretion, including the fecal pellet output, frequency of watery diarrhea, fecal total weight, dried weight and fluid weight, during the second hour post-injection. The stimulatory effects on colonic motor and secretory functions induced by ICV h/rCRF did not persist into the second hour post-injection, the same like its IV injection on colon motor and secretory functions [10]. It is very likely that h/r CRF in the cerebrospinal fluid of rats has already been degraded in the second hour after single ICV injection, because the half-life of h/rCRF has been reported less than 10 min in the human cerebrospinal fluid [18], which is much shorter than that in the plasma [25]. The shorter half-life of h/rCRF in the cerebrospinal fluid than in the plasma could explain the fact that intravenous, but not ICV, injection of h/rCRF induced significant frequency of water diarrhea during the first hour after injection [10]. In summary, we successfully applied our novel in vivo animal model to simultaneously investigate the ICV effects of obestatin and h/rCRF on colonic motor and secretory functions in conscious fed rats. ICV injection of h/rCRF at the low dose stimulates colon motor and secretory functions, whereas acutely intracerebroventricular administration of obestatin has no influence on colonic motility and secretion in rats. Acknowledgments This work was supported by grants from Yen Tjing Ling Medical Foundation (CI-97-15 to C.Y.C.), as well as in part from Veterans General Hospitals University System of Taiwan Joint Research Program (by Tsou’s Foundation, VGHUST98-P6-22 to C.Y.C. and C.P.L.) and the intramural grants from Taipei Veterans General Hospitals (V96C1-112), Taiwan. The authors thank the help from Chi ChinWen, Ph.D., Hung Mei-Whey, MS, and the Clinical Research Core Laboratory, Taipei Veterans General Hospital. References [1] Ataka K, Inui A, Asakawa A, Kato I, Fujimiya M. Obestatin inhibits motor activity in the antrum and duodenum in the fed state of conscious rats. Am J Physiol Gastrointest Liver Physiol 2008;294:G1210–8. [2] Ataka K, Kuge T, Fujino K, Takahashi T, Fujimiya M. Wood creosote prevents CRF-induced motility via 5-HT3 receptors in proximal and 5-HT4 receptors in distal colon in rats. Auton Neurosci 2007;133:136–45. [3] Atsuchi K, Asakawa A, Ushikai M, Ataka K, Tanaka R, Kato I, et al. Centrally administered neuromedin S inhibits feeding behavior and gastroduodenal motility in mice. Horm Metab Res, PMID: 20352600, in press. [4] Bassil AK, Häglund Y, Brown J, Rudholm T, Hellström PM, Näslund E, et al. Little or no ability of obestatin to interact with ghrelin or modify motility in the rat gastrointestinal tract. Br J Pharmacol 2007;150:58–64. ˜ JP, Campos JF, Caminos JE, Dieguez C, Casanueva FF. Obestatin[5] Camina mediated proliferation of human retinal pigment epithelial cells: regulatory mechanisms. J Cell Physiol 2007;211:1–9.
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117 [6] Carlini VP, Schiöth HB, Debarioglio SR. Obestatin improves memory performance and causes anxiolytic effects in rats. Biochem Biophys Res Commun 2007;352:907–12. [7] Chanoine JP, Wong AC, Barrios V. Obestatin, acylated and total ghrelin concentrations in the perinatal rat pancreas. Horm Res 2006;66:81–8. [8] Chartrel N, Alvear-Perez R, Leprince J, Iturrioz X, Reaux-Le Goazigo A, Audinot V, et al. Comment on “obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake”. Science 2007;315:766c. [9] Chen CY, Asakawa A, Fujimiya M, Lee SD, Inui A. Ghrelin gene products and the regulation of food intake and gut motility. Pharmacol Rev 2009;61:430–81. [10] Chen CY, Chien EJ, Chang FY, Lu CL, Luo JC, Lee SD. Impacts of peripheral obestatin on colonic motility and secretion in conscious fed rats. Peptides 2008;29:1603–8. [11] Chen CY, Inui A, Asakawa A, Fujino K, Kato I, Chen CC, et al. Des-acyl ghrelin acts by CRF type 2 receptors to disrupt fasted stomach motility in conscious rats. Gastroenterology 2005;129:8–25. [12] Chen CY, Fujimiya M, Asakawa A, Chang FY, Cheng JT, Lee SD, et al. At the cutting edge: ghrelin gene products in food intake and gut motility. Neuroendocrinology 2009;89:9–17. [13] Chen CY, Fujimiya M, Laviano A, Chang FY, Lin HC, Lee SD. Mini-review: Modulation of ingestive behavior and gastrointestinal motility by ghrelin in diabetic animals and humans. J Chin Med Assoc 2010;73:225–30. [14] De Smet B, Thijs T, Peeters TL, Depoortere I. Effect of peripheral obestatin on gastric emptying and intestinal contractility in rodents. Neurogastroenterol Motil 2007;19:211–7. [15] Dun SL, Brailoiu GC, Brailoiu E, Yang J, Chang JK, Dun NJ. Distribution and biological activity of obestatin in the rat. J Endocrinol 2006;191:481–9. [16] Fujimiya M, Asakawa A, Ataka K, Chen CY, Kato I, Inui A. Ghrelin, des-acyl ghrelin and obestatin: regulatory roles on the gastrointestinal motility. Int J Pept 2010;2010. Article ID 305192. [17] Fraser GL, Venkova K, Hoveyda HR, Thomas H, Greenwood-Van Meerveld B. Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus. Eur J Pharmacol 2009;604:132–7. [18] Geracioti Jr TD, Orth DN, Ekhator NN, Blumenkopf B, Loosen PT. Serial cerebrospinal fluid corticotropin-releasing hormone concentrations in healthy and depressed humans. J Clin Endocrinol Metab 1992;74:1325–30. [19] Gourcerol G, Coskun T, Craft LS, Mayer JP, Heiman ML, Wang L, et al. Preproghrelin-derived peptide, obestatin, fails to influence food intake in lean or obese rodents. Obesity 2007;15:2643–52. [20] Gourcerol G, Million M, Adelson DW, Wang Y, Wang L, Rivier J, et al. Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signaling in rodents. Peptides 2006;27:2811–9. [21] Gülpinar MA, Bozkurt A, Cos¸kun T, Ulusoy NB, Yegen BC. Glucagon-like peptide (GLP-1) is involved in the central modulation of fecal output in rats. Am J Physiol Gastrointest Liver Physiol 2000;278:G924–9.
1117
[22] Holst B, Egerod KL, Schild E, Vickers SP, Cheetham S, Gerlach LO, et al. GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 2007;148:13–20. [23] Lauwers E, Landuyt B, Arckens L, Schoofs L, Luyten W. Obestatin does not activate orphan G protein-coupled receptor GPR39. Biochem Biophys Res Commun 2006;351:21–5. [24] Nakade Y, Mantyh C, Pappas TN, Takahashi T. Fecal pellet output does not always correlate with colonic transit in response to restraint stress and corticotropin-releasing factor in rats. J Gastroenterol 2007;42:279–82. [25] Saphier PW, Faria M, Grossman A, Coy DH, Besser GM, Hodson B, et al. A comparison of the clearance of ovine and human corticotrophin-releasing hormone (CRH) in man and sheep: a possible role for CRH-binding protein. J Endocrinol 1992;133:487–95. [26] Saunders PR, Maillot C, Million M, Taché Y. Peripheral corticotropin-releasing factor induces diarrhea in rats: role of CRF1 receptor in fecal watery excretion. Eur J Pharmacol 2002;435:231–5. [27] Shafton AD, Sanger GJ, Witherington J, Brown JD, Muir A, Butler S, et al. Oral administration of a centrally acting ghrelin receptor agonist to conscious rats triggers defecation. Oral administration of a centrally acting ghrelin receptor agonist to conscious rats triggers defecation. Neurogastroenterol Motil 2009;21:71–7. [28] Szentirmai E, Kapás L, Sun Y, Smith RG, Krueger JM. The preproghrelin gene is required for the normal integration of thermoregulation and sleep in mice. Proc Natl Acad Sci USA 2009;106:14069–74. [29] Szentirmai E, Krueger JM. Obestatin alters sleep in rats. Neurosci Lett 2006;404:222–6. [30] Samson WK, White MM, Price C, Ferguson AV. Obestatin acts in brain to inhibit thirst. Am J Physiol Regul Integr Comp Physiol 2007;292:R637–43. [31] Tebbe JJ, Mronga S, Tebbe CG, Ortmann E, Arnold R, Schäfer MK. Ghrelininduced stimulation of colonic propulsion is dependent on hypothalamic neuropeptide Y1- and corticotrophin-releasing factor 1 receptor activation. J Neuroendocrinol 2005;17:570–6. [32] Tebbe JJ, Tebbe CG, Mronga S, Ritter M, Schäfer MK. Central neuropeptide Y receptors are involved in 3rd ventricular ghrelin induced alteration of colonic transit time in conscious fed rats. BMC Gastroenterol 2005;5:5. [33] Venkova K, Mann W, Nelson R, Greenwood-Van Meerveld B. Efficacy of ipamorelin, a novel ghrelin mimetic, in a rodent model of postoperative ileus. J Pharmacol Exp Ther 2009;329:1110–6. [34] Zhang JV, Klein C, Ren PG, Kass S, Donck LV, Moechars D, et al. Response to comment on ‘obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake’. Science 2007;315:766d. [35] Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, et al. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310:996–9.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
A novel simultaneous measurement method to assess the influence of intracerebroventricular obestatin on colonic motility and secretion in conscious rats Chih-Yen Chen a,b,∗ , Ming-Luen Doong c,1 , Chung-Pin Li a,b,1 , Wen-Jinn Liaw d,1 , Hsing-Feng Lee a,b , Full-Young Chang a,b , Han-Chieh Lin a,b , Shou-Dong Lee a,b a
Faculty of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taiwan c Department and Institute of Physiology, National Yang-Ming University School of Medicine, Taipei, Taiwan d Department of Anesthesiology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 22 February 2010 Received in revised form 17 March 2010 Accepted 17 March 2010 Available online 23 March 2010 Keywords: Obestatin Colonic transit time Fecal pellet output Colonic secretion Intracerebroventricular
a b s t r a c t Obestatin, a novel putative 23-amino acid peptide, is derived from mammalian preproghrelin gene via a bioinformatics approach. Although obestatin regulates thirst, sleep, memory, anxiety, activates cortical neurons in the brain and stimulate proliferation of retinal pigment epithelial cells, there is no study to explore its central impacts on the lower gut motility and secretion. We investigated the influence of intracerebroventricular (ICV) injection of obestatin on rat colonic motor and secretory functions. Colonic transit time, fecal pellet output and fecal content were assessed in freely fed, conscious rats, which were implanted with ICV and colonic catheters chronically. Human/rat corticotropin-releasing factor (h/rCRF) was applied as a stimulatory inducer of colonic motility and secretion. ICV injection of obestatin (0.1, 0.3, 1.0 nmol/rat) did not modify the colonic transit time, whereas ICV injection of h/rCRF (0.3 nmol/rat) significantly shortened colonic transit time. ICV obestatin in any dose we tested did not affect the fecal pellet output, frequency of watery diarrhea, total fecal weight, fecal dried solid weight, or fecal fluid weight in the first hour post-injection, either. In contrast, ICV injection of h/rCRF effectively stimulated fecal pellet output, as well as increased total fecal weight, fecal dried solid weight and fecal fluid weight during the first hour post-injection, compared to ICV saline controls. In conclusion, using our novel simultaneous measurement method, acutely central administration of obestatin exhibits no influence on colonic motility and secretion in conscious rats. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Obestatin, a novel 23-amino acid peptide recently identified from rat stomach, is derived from C-terminal part of mammalian preproghrelin gene that also encodes ghrelin, by comparative genomic analyses [35]. It was originally projected that acyl ghrelin and obestatin exhibit opposite influence on ingestive behaviors, e.g. acyl ghrelin elicits food intake whereas obestatin inhibits it [35], although the effects of obestatin on food intake are debatable [12,13]. Acyl ghrelin binds to growth hormone secretagogue receptor 1a, and, then, signals via a Gq/11 ␣-subunit that results in the release of inositol trisphosphate and Ca2+ [9]. Obestatin
∗ Corresponding author at: Division of Gastroenterology, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Tel.: +886 2 28712121x3341; fax: +886 2 28711058. E-mail address: [email protected] (C.-Y. Chen). 1 These authors contributed equally to this work.
was suggested binding to an orphan G protein-coupled receptor (GPR), termed GPR39 [7,35], and induced the increase of intracellular cAMP [35]. GPR39 expression has been detected in peripheral organs such as the duodenum and kidney but not in the pituitary or hypothalamus [22]. But recent studies reveal that obestatin is not the endogenous cognate ligand for GPR39 [8,22,23,34]. Obestatin manifested various biological functions at the central nervous system, such as inhibition of thirst [30], increase of nonrapid eye movement sleep episodes and decrease of sleep latency [29], as well as improvement of memory retention and reduction of anxiety [6] in rats. Besides, obestatin has also been reported to activate cortical neurons [15], and stimulate proliferation of retinal pigment epithelial cells [5]. In addition, a recent study indicates that mice lacking the preproghrelin gene have impaired abilities to manifest and integrate normal sleep and thermoregulatory responses to metabolic challenges [28]. When considered together, these results imply that obestatin may have some unknown but important novel roles on the central nervous system in the uninvestigated field, which deserve further exploration.
0196-9781/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.03.024
1114
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117
Regarding gastrointestinal motility, many studies of obestatin focus on its effects on the upper gut [1,4,14,20]. Peripheral obestatin inhibited gastroduodenal motility in the fed state but not in the fasted state of conscious rats through activating corticotropinreleasing factor (CRF) receptor 1 and 2 in the brain, while vagal afferent pathways might be partially involved [1,16]. On the other hand, intracisternal injection of obestatin did not affect gastric phasic contraction of anesthetized fasted rats [19]. Our previous paper showed that peripheral administration of obestatin has no impact on colonic motility and secretion in rats [10]. From our PubMed search, there is no study to address the influence of centrally administered obestatin on rat colonic motor and secretory functions. As the above-mentioned multifaceted functions of obestatin in the brain, it is very interesting to investigate the potential, central effects of obestatin on colon motility and secretion. In the present study, we used our newly established animal model to simultaneously measure colonic transit time, fecal pellet output and fecal content in conscious fed rats [10]. Corticotropin-releasing factor (CRF), a well-known colonoprokinetic peptide, was applied to serve as a stimulatory agent to test the validity of this in vivo model. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (National Laboratory Animal Center, Taipei, Taiwan) weighing 250–320 g at the initial period of experiment were used and housed in group cages under controlled illumination (light cycle: 08:00–20:00), humidity and temperature (22.5 ± 1.5 ◦ C) with free access to water and laboratory chow pellets (LabDiet® , Brentwood, MO, USA). All experiments were performed since 9 a.m. in freely moving conscious fed rats, in accordance to guidelines, which have been approved by the Institutional Animal Care and Use Committee (IACUC), Taipei Veterans General Hospital. 2.2. Surgery 2.2.1. Implantation of intracerebroventricular (ICV) catheter Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg, Nembutal; Abbott Laboratories, Abbott Park, IL, USA), placed in a stereotaxic apparatus, and implanted with a guide cannula (25-gauge; Eicom, Kyoto, Japan), which reached the right lateral ventricle. Stereotaxic coordinates were 0.8 mm posterior to bregma, 1.4 mm right lateral to the midline, and 4.5 mm below the outer surface of the skull using a stereotaxic frame (BenchmarkTM , myNeuroLab, St. Louis, MO, USA) with the incisor bar set at the horizontal plane passing through bregma and lambda. The guide cannule was secured with dental cement anchored by two stainless steel screws fixed on the dorsal surface of the skull. After surgery, a dummy cannula (Eicom) was inserted into each guide cannula, and a screw cap (Eicom) was put on the guide cannula to prevent blockade [11]. The correctness of ICV cannula placement was verified by injection of 100 l dye (0.05% cresyl violet, Sigma) into the right lateral ventricle by the brain sections at the end of experiments after euthanasia [11]. Before beginning all colonic motor and secretory function tests, those animals receiving implantation of colonic tubes with ICV catheters were allowed 7 days for recovery. All ICV injections were performed over 60 s in 5 l using the AMI-5 (Eicom). 2.2.2. Implantation of colonic catheter Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg, Nembutal; Abbott). After the lower abdomen laparatomy, a catheter (3 Fr, 1-mm diameter; ATOM) was implanted into the proximal colon 2 cm distal from the ceco-colonic junction chronically. The catheter was fixed at the colonic wall by a
purse-string suture and routed subcutaneously to the interscapular region, where it was exteriorized through the skin using for intracolonic administration of dye marker [10]. Before simultaneous measurement of colonic motor and secretory functions, rats were allowed 7 days to recovery. 2.3. Preparation of drugs Rat obestatin (Peptides International, Inc., Louisville, KY, USA) and human/rat CRF (h/r CRF) (American Peptide Company, Sunnyvale, CA, USA) were kept in powder form at −20 ◦ C, and dissolved in sterile, pyrogen-free 0.9% saline (Otsuka, Tokyo, Japan) immediately before use. The doses of obestatin, 0.1, 0.3, and 1.0 nmol/rat, were selected similar to those effective in elevated plus maze test [6] and thirst study [30], while the dose of human/rat CRF (0.3 nmol/rat) was chosen mimicking the effective one in manometric recording [2]. 2.4. Colonic motor and secretory function tests 2.4.1. Measurement of colonic transit time Colonic transit time was calculated using an enteral dye marker, trypan blue (Sigma Chemical Co, St. Louis, MO, USA), a nonabsorbable dye. The dye was injected in 0.2 ml volume through the catheter positioned in the proximal colon, and followed by a 0.2 ml saline flush 10 min after the ICV injection of either saline, obestatin, or h/rCRF [10]. Colonic transit time was defined as the time interval between the dye injection and the discharge of the first blue pellet. 2.4.2. Measurement of fecal pellet output Rats were accustomed to single housing cage individually for 7 days before the experiment. The total number of pellets was recorded every hour after an intracolonic injection of trypan blue for 2 h. 2.4.3. Measurements of characteristics of fecal content The frequency of watery diarrhea was assessed by the number of rats expelling loose watery stool. The total fecal material was collected every hour after the intracolonic injection of trypan blue for 2 h, and its content was weighed then desiccated overnight at 50 ◦ C, and the fecal fluid and solid output were calculated from the total and dry weights [10,26]. 2.5. Statistical analysis All results are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post hoc test was used to analyze difference among groups. Differences were considered statistically significant with p < 0.05. 3. Results 3.1. Colonic transit time ICV injection of obestatin at 0.1, 0.3, and 1.0 nmol/rat did not change the colonic transit time (290.7 ± 29.9, 265.6 ± 29.4, and 292.9 ± 19.8 min vs. 300.3 ± 21.0 min, p > 0.05), compared to ICV saline controls (Fig. 1). Contrary, ICV injection of h/rCRF at 0.3 nmol/rat significantly shortened colonic transit time (190.3 ± 23.5 vs. 300.3 ± 21.0 min, p < 0.05) in conscious fed rats. 3.2. Fecal pellet output ICV obestatin (0.1, 0.3, and 1.0 nmol/rat) did not affect the number of fecal pellet output (1.17 ± 1.08, 0.92 ± 0.47, and 0.58 ± 0.29 vs. 1.00 ± 0.48 per hour), whereas ICV injection of h/rCRF at 0.3
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117
1115
Fig. 1. The influence of intracerebroventricular (ICV) injection of obestatin and human/rat corticotropin-releasing factor (h/rCRF) on colonic transit time in conscious rats. ICV obestatin did not modify colonic transit time, whereas h/rCRF significantly speeded colonic transit time. *p < 0.05, compared to all the other groups, n = 12 rats in each group.
nmol/rat effectively increased the number of fecal pellet output (2.75 ± 0.58 vs. 1.00 ± 0.48 per hour, p < 0.05) during the first hour post-injection in conscious fed rats (Fig. 2). However, neither any dose of ICV obestatin nor h/rCRF injection modified the number of fecal pellet output (0.33 ± 0.26, 0.58 ± 0.50, 0.67 ± 0.58, and 1.33 ± 0.62 vs. 0.33 ± 0.33 per hour), though h/rCRF treatment showed a tendency to enhance the number of fecal pellet output during the second hour post-injection (Fig. 2). 3.3. Fecal content ICV obestatin (0.1, 0.3, and 1.0 nmol/rat) did not have any impact on the total fecal weight (0.10 ± 0.07, 0.23 ± 0.12, and 0.18 ± 0.09 g/h), dried solid weight (0.05 ± 0.05, 0.09 ± 0.05, and 0.07 ± 0.03 g/h) and fecal fluid weight (0.05 ± 0.03, 0.14 ± 0.08, and 0.11 ± 0.05 g/h) during the first hour in conscious rats (Fig. 3A). In contrary, ICV injection of h/rCRF at 0.3 nmol/rat significantly increased total fecal weight (0.95 ± 0.23 and 0.18 ± 0.08 g/h, p < 0.001), dried solid weight (0.30 ± 0.06 g/h vs. 0.06 ± 0.03 g/h, p < 0.01) and fecal fluid weight (0.65 ± 0.17 g/h vs. 0.12 ± 0.05 g/h, p < 0.001) during
Fig. 3. The influence of intracerebroventricular (ICV) injection of obestatin and human/rat corticotropin-releasing factor (h/rCRF) on colonic secretion 1 h (A) and 2 h (B) after the injection. (A) ICV obestatin did not modify any colonic secretory parameters, whereas h/rCRF significantly enhanced total fecal weight, fecal dried weight, and fecal fluid weight during the first hour after injection. **p < 0.01, ***p < 0.001, compared to all the other groups, n = 12 rats in each group. (B) Neither ICV obestatin nor h/rCRF treatment affected any parameters of colonic secretion during the second hour after injection; n = 12 rats in each group.
the first hour post-injection, compared to ICV saline controls (Fig. 3A). Neither any dose of ICV obestatin nor h/rCRF injection influenced total fecal weight (0.11 ± 0.09 g/h, 0.14 ± 0.12 g/h, 0.18 ± 0.15 g/h, and 0.35 ± 0.14 g/h vs. 0.08 ± 0.08 g/h), dried solid weight (0.04 ± 0.03 g/h, 0.04 ± 0.03 g/h, 0.08 ± 0.06 g/h, and 0.12 ± 0.05 g/h vs. 0.03 ± 0.03 g/h) and fecal fluid weight (0.07 ± 0.06 g/h, 0.10 ± 0.08 g/h, 0.10 ± 0.08 g/h, and 0.23 ± 0.09 g/h vs. 0.04 ± 0.04 g/h), though h/rCRF treatment showed a tendency to increase these fecal parameters during the second hour postinjection (Fig. 3B). Besides, ICV obestatin (0.1, 0.3, and 1.0 nmol/rat) did not change the frequency of watery diarrhea during the first hour post-injection, while ICV injection of h/rCRF at 0.3 nmol/rat exhibited a mild tendency to enhance the frequency of watery diarrhea during the first hour post-injection (Table 1). Similarly, neither any dose of ICV obestatin nor h/rCRF treatment modified the frequency of watery diarrhea during the second hour post-injection (Table 1). 4. Discussion
Fig. 2. The influence of intracerebroventricular (ICV) injection of obestatin and human/rat corticotropin-releasing factor (h/rCRF) on fecal pellet output in conscious rats. ICV obestatin did not affect the fecal pellet output during the first and second hour after injection. However, ICV h/rCRF significantly increased fecal pellet output during the first but not the second hour after injection. *p < 0.05, compared to all the other groups, n = 12 rats in each group.
Previous studies could only just explore either colonic motor or secretory functions in rodents. Our newly established animal model makes a breakthrough over this limitation and is capable of simultaneously measuring both functions [10]. In the present study, we tried to investigate the central, potential effects of obestatin on colonic transit and secretion in rats. The mean obtained colonic
1116
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117
Table 1 Effects of ICV injection of saline, obestatin and h/rCRF on the frequency of watery diarrhea in conscious fed rats; n = 12 rats in each group.
The first hour post-injection The second hour post-injection
Saline (5 l/rat)
Obestatin (0.1 nmol/rat)
Obestatin (0.3 nmol/rat)
Obestatin (1.0 nmol/rat)
h/rCRF (0.3 nmol/rat)
0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00
0.25 ± 0.25 0.25 ± 0.25
transit time of ICV saline-injected control rats as 300 min in our study was comparable to the previous study showing 320 min using the similar trypan blue dye method [31]. The data of fecal pellet output in our ICV saline-injected controls were also comparable with those obtained by the other study group (1.33 vs. 1.28 n/2 h) [21]. In addition, our study could be the first to investigate ICV injections of peptides on fecal content in rats, because no previous literature is found. Moreover, none of our controls demonstrated watery diarrhea, implying that these animals had acclimated and been well handled before and during experiments without any stressful factors. Thus, our measurement for ICV injections of peptides on colonic motor and secretory functions in conscious fed rats should be valid. The first preproghrelin gene product, acyl ghrelin, poses prokinetic activity along the entire gastrointestinal tract [9]. Acyl ghrelin, when ICV administered as well as microinjected inside, but not outside the paraventricular nucleus, does accelerate colonic transit time in rats [31,32]. Acyl ghrelin acts in the central nervous system to modulate colonic motility via hypothalamic neuropeptide Y1 and CRF1 receptors pathways [31]. In addition to stimulating colonic motor functions, oral administration of a centrally acting ghrelin receptor agonist, GSK894281, significantly increases fecal weight in rats [27]. Moreover, ghrelin receptor agonists have been shown to improve colonic functions in rats with post-operative ileus. Intravenous administration of TZP-101 and ipamorelin, another two ghrelin receptor agonists, were demonstrated to effectively reverse laparotomy-induced delay of colonic transit time [14] and inhibition of fecal pellet output [17,33] in rats. In the current study, the influence of the third preproghrelin gene product, obestatin, on colonic motility and secretion in rats were examined. On the contrary, our study indicated that ICV injection of obestatin at low doses (0.1–1.0 nmol), different from ghrelin and/or ghrelin receptor agonists, did not modify colonic transit time. ICV obestatin did not change the fecal pellet output, either. In addition, ICV obestatin did not impact any fecal content parameters during the first hour post-injection, such as the frequency of watery diarrhea, fecal total weight, dried weight and fluid weight. Though both colonic transit time and fecal pellet output belong to colonic motor functions, some differences of physiological significance between the measurement of colonic transit time and fecal pellet output exist. The fecal pellet output is considered as the distal colonic motor function, whereas the colonic transit time corresponds to the motor function of the entire colon [24]. Although central obestatin has important roles in influencing thirst [30], sleep [29], memory and anxiety [6], our study suggested that obestatin does not act in the forebrain to affect colonic motor and secretory functions in conscious rats. However, higher sensitivity of manometric method than solid gastric emptying in detecting the inhibitory effects of anorexigenic peptides, such as neuromedin S, on the upper gut motility has been clearly demonstrated by a recent report [3]. Further studies using the potentially more sensitive manometric method to investigate central effects of obestatin on gastric and colonic motility, including applying the higher effective dose (3 nmol/rat) shown in the elevated plus maze [6] and thirst [30] test, maybe warranted in the future. Peripheral administration of obestatin is reported able to alter gastroduodenal motility via CRF receptors, while immunohistochemical analysis reveals that intravenous obestatin elicits CRF and urocortin 2-containing neurons in the forebrain [1]. Therefore, we
used h/rCRF as a stimulatory control for comparison in this study. From our thorough literature review, the current study is the first to simultaneously investigate effects of ICV h/rCRF on colonic motility and secretion. We found that ICV injection of h/rCRF at the low dose (0.3 nmol) potently accelerated colonic transit time, enhanced fecal pellet output, as well as increased total fecal weight, dried solid weight and fluid weight during the first hour post-injection, but not the frequency of watery diarrhea, compared to ICV saline-injected controls. As the same in our previous study, we tried to extend our study to observe the longer temporal effects of the peptides [10]. We found that both agents did not change any parameters of colonic motility and secretion, including the fecal pellet output, frequency of watery diarrhea, fecal total weight, dried weight and fluid weight, during the second hour post-injection. The stimulatory effects on colonic motor and secretory functions induced by ICV h/rCRF did not persist into the second hour post-injection, the same like its IV injection on colon motor and secretory functions [10]. It is very likely that h/r CRF in the cerebrospinal fluid of rats has already been degraded in the second hour after single ICV injection, because the half-life of h/rCRF has been reported less than 10 min in the human cerebrospinal fluid [18], which is much shorter than that in the plasma [25]. The shorter half-life of h/rCRF in the cerebrospinal fluid than in the plasma could explain the fact that intravenous, but not ICV, injection of h/rCRF induced significant frequency of water diarrhea during the first hour after injection [10]. In summary, we successfully applied our novel in vivo animal model to simultaneously investigate the ICV effects of obestatin and h/rCRF on colonic motor and secretory functions in conscious fed rats. ICV injection of h/rCRF at the low dose stimulates colon motor and secretory functions, whereas acutely intracerebroventricular administration of obestatin has no influence on colonic motility and secretion in rats. Acknowledgments This work was supported by grants from Yen Tjing Ling Medical Foundation (CI-97-15 to C.Y.C.), as well as in part from Veterans General Hospitals University System of Taiwan Joint Research Program (by Tsou’s Foundation, VGHUST98-P6-22 to C.Y.C. and C.P.L.) and the intramural grants from Taipei Veterans General Hospitals (V96C1-112), Taiwan. The authors thank the help from Chi ChinWen, Ph.D., Hung Mei-Whey, MS, and the Clinical Research Core Laboratory, Taipei Veterans General Hospital. References [1] Ataka K, Inui A, Asakawa A, Kato I, Fujimiya M. Obestatin inhibits motor activity in the antrum and duodenum in the fed state of conscious rats. Am J Physiol Gastrointest Liver Physiol 2008;294:G1210–8. [2] Ataka K, Kuge T, Fujino K, Takahashi T, Fujimiya M. Wood creosote prevents CRF-induced motility via 5-HT3 receptors in proximal and 5-HT4 receptors in distal colon in rats. Auton Neurosci 2007;133:136–45. [3] Atsuchi K, Asakawa A, Ushikai M, Ataka K, Tanaka R, Kato I, et al. Centrally administered neuromedin S inhibits feeding behavior and gastroduodenal motility in mice. Horm Metab Res, PMID: 20352600, in press. [4] Bassil AK, Häglund Y, Brown J, Rudholm T, Hellström PM, Näslund E, et al. Little or no ability of obestatin to interact with ghrelin or modify motility in the rat gastrointestinal tract. Br J Pharmacol 2007;150:58–64. ˜ JP, Campos JF, Caminos JE, Dieguez C, Casanueva FF. Obestatin[5] Camina mediated proliferation of human retinal pigment epithelial cells: regulatory mechanisms. J Cell Physiol 2007;211:1–9.
C.-Y. Chen et al. / Peptides 31 (2010) 1113–1117 [6] Carlini VP, Schiöth HB, Debarioglio SR. Obestatin improves memory performance and causes anxiolytic effects in rats. Biochem Biophys Res Commun 2007;352:907–12. [7] Chanoine JP, Wong AC, Barrios V. Obestatin, acylated and total ghrelin concentrations in the perinatal rat pancreas. Horm Res 2006;66:81–8. [8] Chartrel N, Alvear-Perez R, Leprince J, Iturrioz X, Reaux-Le Goazigo A, Audinot V, et al. Comment on “obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake”. Science 2007;315:766c. [9] Chen CY, Asakawa A, Fujimiya M, Lee SD, Inui A. Ghrelin gene products and the regulation of food intake and gut motility. Pharmacol Rev 2009;61:430–81. [10] Chen CY, Chien EJ, Chang FY, Lu CL, Luo JC, Lee SD. Impacts of peripheral obestatin on colonic motility and secretion in conscious fed rats. Peptides 2008;29:1603–8. [11] Chen CY, Inui A, Asakawa A, Fujino K, Kato I, Chen CC, et al. Des-acyl ghrelin acts by CRF type 2 receptors to disrupt fasted stomach motility in conscious rats. Gastroenterology 2005;129:8–25. [12] Chen CY, Fujimiya M, Asakawa A, Chang FY, Cheng JT, Lee SD, et al. At the cutting edge: ghrelin gene products in food intake and gut motility. Neuroendocrinology 2009;89:9–17. [13] Chen CY, Fujimiya M, Laviano A, Chang FY, Lin HC, Lee SD. Mini-review: Modulation of ingestive behavior and gastrointestinal motility by ghrelin in diabetic animals and humans. J Chin Med Assoc 2010;73:225–30. [14] De Smet B, Thijs T, Peeters TL, Depoortere I. Effect of peripheral obestatin on gastric emptying and intestinal contractility in rodents. Neurogastroenterol Motil 2007;19:211–7. [15] Dun SL, Brailoiu GC, Brailoiu E, Yang J, Chang JK, Dun NJ. Distribution and biological activity of obestatin in the rat. J Endocrinol 2006;191:481–9. [16] Fujimiya M, Asakawa A, Ataka K, Chen CY, Kato I, Inui A. Ghrelin, des-acyl ghrelin and obestatin: regulatory roles on the gastrointestinal motility. Int J Pept 2010;2010. Article ID 305192. [17] Fraser GL, Venkova K, Hoveyda HR, Thomas H, Greenwood-Van Meerveld B. Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus. Eur J Pharmacol 2009;604:132–7. [18] Geracioti Jr TD, Orth DN, Ekhator NN, Blumenkopf B, Loosen PT. Serial cerebrospinal fluid corticotropin-releasing hormone concentrations in healthy and depressed humans. J Clin Endocrinol Metab 1992;74:1325–30. [19] Gourcerol G, Coskun T, Craft LS, Mayer JP, Heiman ML, Wang L, et al. Preproghrelin-derived peptide, obestatin, fails to influence food intake in lean or obese rodents. Obesity 2007;15:2643–52. [20] Gourcerol G, Million M, Adelson DW, Wang Y, Wang L, Rivier J, et al. Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signaling in rodents. Peptides 2006;27:2811–9. [21] Gülpinar MA, Bozkurt A, Cos¸kun T, Ulusoy NB, Yegen BC. Glucagon-like peptide (GLP-1) is involved in the central modulation of fecal output in rats. Am J Physiol Gastrointest Liver Physiol 2000;278:G924–9.
1117
[22] Holst B, Egerod KL, Schild E, Vickers SP, Cheetham S, Gerlach LO, et al. GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 2007;148:13–20. [23] Lauwers E, Landuyt B, Arckens L, Schoofs L, Luyten W. Obestatin does not activate orphan G protein-coupled receptor GPR39. Biochem Biophys Res Commun 2006;351:21–5. [24] Nakade Y, Mantyh C, Pappas TN, Takahashi T. Fecal pellet output does not always correlate with colonic transit in response to restraint stress and corticotropin-releasing factor in rats. J Gastroenterol 2007;42:279–82. [25] Saphier PW, Faria M, Grossman A, Coy DH, Besser GM, Hodson B, et al. A comparison of the clearance of ovine and human corticotrophin-releasing hormone (CRH) in man and sheep: a possible role for CRH-binding protein. J Endocrinol 1992;133:487–95. [26] Saunders PR, Maillot C, Million M, Taché Y. Peripheral corticotropin-releasing factor induces diarrhea in rats: role of CRF1 receptor in fecal watery excretion. Eur J Pharmacol 2002;435:231–5. [27] Shafton AD, Sanger GJ, Witherington J, Brown JD, Muir A, Butler S, et al. Oral administration of a centrally acting ghrelin receptor agonist to conscious rats triggers defecation. Oral administration of a centrally acting ghrelin receptor agonist to conscious rats triggers defecation. Neurogastroenterol Motil 2009;21:71–7. [28] Szentirmai E, Kapás L, Sun Y, Smith RG, Krueger JM. The preproghrelin gene is required for the normal integration of thermoregulation and sleep in mice. Proc Natl Acad Sci USA 2009;106:14069–74. [29] Szentirmai E, Krueger JM. Obestatin alters sleep in rats. Neurosci Lett 2006;404:222–6. [30] Samson WK, White MM, Price C, Ferguson AV. Obestatin acts in brain to inhibit thirst. Am J Physiol Regul Integr Comp Physiol 2007;292:R637–43. [31] Tebbe JJ, Mronga S, Tebbe CG, Ortmann E, Arnold R, Schäfer MK. Ghrelininduced stimulation of colonic propulsion is dependent on hypothalamic neuropeptide Y1- and corticotrophin-releasing factor 1 receptor activation. J Neuroendocrinol 2005;17:570–6. [32] Tebbe JJ, Tebbe CG, Mronga S, Ritter M, Schäfer MK. Central neuropeptide Y receptors are involved in 3rd ventricular ghrelin induced alteration of colonic transit time in conscious fed rats. BMC Gastroenterol 2005;5:5. [33] Venkova K, Mann W, Nelson R, Greenwood-Van Meerveld B. Efficacy of ipamorelin, a novel ghrelin mimetic, in a rodent model of postoperative ileus. J Pharmacol Exp Ther 2009;329:1110–6. [34] Zhang JV, Klein C, Ren PG, Kass S, Donck LV, Moechars D, et al. Response to comment on ‘obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake’. Science 2007;315:766d. [35] Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, et al. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310:996–9.