- Email: [email protected]
88 The Hypophagia After Psychological Stress in Aged Male Mice Is Mediated by an Interaction of Activation of Estrogen Receptor α With Serotonin 2C Receptor
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Obese Patients Have Significantly Higher Motilin Plasma Levels Which Decrease After Roux-en-Y Gastric Bypass Surgery Eveline Deloose, Pieter Janssen, Matthias Lannoo, Bart Van der Schueren, Inge Depoortere, Jan F. Tack Objective: Changes in gut hormones are suggested to play an important role in the metabolic reprogramming that occurs after Roux-en-Y gastric bypass (RYGB) surgery. We have recently shown that gastric phases III of the migrating motor complex (MMC) are associated with hunger peaks in healthy lean volunteers and that both can be induced through the administration of erythromycin, a motilin receptor agonist. The aim of this study was to evaluate the role of motilin and phase III as hunger-inducing factors in obese patients and to study the effect of RYGB surgery on plasma motilin levels. Design: Blood samples for motilin and ghrelin (total and octanoylated) were taken during a complete MMC cycle in healthy lean controls (n=15, 28±2y, 22±0.5kg/m2) and obese patients (n=16, 40±3y, 41±0.8kg/m2) before surgery and 6 months (n=8, 39±4y, 29±1.3kg/m2) or 1 year after surgery (n=7, 40±4y, 25±1.1kg/m2). Gastrointestinal motility was measured using high-resolution manometry after a 12 hour overnight fast. Hunger was scored every 5 min on a visual analogue scale of 10 cm. An i.v. infusion of 40 mg erythromycin was given 20 min after the end of the second phase III in obese patients to evaluate the effect on hunger. Hedonic hunger was measured using the Power of Food scale in obese patients prior to, 6 months and 1 year after surgery. Results: A positive correlation was found between BMI and motilin (r=0.53; p=0.005) and a negative correlation between BMI and ghrelin (total (r=-0.52; p=0.007) and octanoylated (r=-0.43; p=0.02). Obese patients had significantly higher motilin plasma levels compared to lean controls (fig 1, p=0.0005) during the MMC cycle but tended to lack the motilin (-3±6% vs 15±6%; p=0.052) plasma peak prior to phase III. The obese patients had significantly less phases III that started in the antrum for both the first (44% vs 60%; p= 0.03) and second (22% vs 40%; p=0.0091) phase III compared to lean controls. Hunger scores during phase III were significantly lower in obese patients (p=0.0022) compared to lean controls, but could be increased through the administration of erythromycin (p=0.97) which induced a premature gastric phase III in all patients. After RYGB surgery the AUC of motilin plasma levels was significantly reduced (fig 2, p=0.018). Although hunger scores during phase III were not affected by RYGB (p=0.9), hedonic hunger was significantly decreased (p=0.0009). Conclusion: Our study is the first to describe a positive correlation between motilin plasma levels and BMI which could indicate that motilin plays an important role in the pathogenesis of obesity, moreover we have shown that motilin levels decrease after RYGB. The switch in origin of phase III contractions, due to the absence of a peak in plasma motilin levels, could represent a counter regulatory mechanism to decrease hunger in obese patients.
Figure 2. Area under the curve of motilin plasma levels measured between two consecutive phases III. Mean ± SEM. *: p<0.05.
87 Intraduodenal Infusion of a Combination of Tastants Decreases Food Intake Mark van Avesaat, Freddy Troost, Dina Ripken, Henk F. Hendriks, Ad Masclee Introduction: Taste receptors on the tongue are able to detect the tastants sweet, umami and bitter. The first two are detected by the taste 1 receptor family (TAS1R), while latter is detected by the taste 2 receptor family (TAS2R). Recently, it was found that taste receptors are also expressed in the intestine. Its functions are yet unknown although it has been hypothesized that they may initiate and transfer signals that originate from the gastrointestinal (GI) tract and modulate satiety and food intake. In the present study, we set out to investigate the effects of intraduodenal tastant infusion on food intake, hunger and fullness, GI symptoms and GI peptide release. Methods: This randomized, double blind, crossover study was performed in 15 healthy subjects (6 male; mean age 24.0 ± 2.0 yrs; mean BMI 22.4 ± 0.3 kg/m2) who participated in 5 experimental test days. Test days started with the insertion of a nasoduodenal catheter under fluoroscopy. After insertion of the catheter, subjects consumed a standardized liquid breakfast (250 mL, 153 kcal). At 150 min after breakfast an intraduodenal infusion, containing control (saline), bitter (quinine), sweet (rebaudioside A), umami (monosodium glutamate) or a combination of the tastants (quinine, rebaudioside A and MSG), over a period of 60 min was given. Food intake was measured during an ad libitum meal (standard meal, 15 min after the end of the infusion). VAS scales were used to monitor GI complains, hunger and fullness scores. Blood samples were drawn at regular intervals in order to measure the GI peptides CCK, GLP-1 and PYY. A mixed analysis of variance model was used to analyze ad libitum food intake, VAS scores and GI peptide release. Results: Intraduodenal infusion of the combination of tastants significantly decreased ad libitum food intake compared to control (422±97 vs 486±104 kcal, p<0.05). Food intake after infusion of the other tastants, separately, was not significantly different from control. However, umami and the combination of tastants significantly reduced hunger. No differences between tastant infusion and control were observed in the release of CCK, GLP-1 or PYY. Subjects did not report any gastrointestinal complaints during or after intraduodenal infusion with the tastants. Conclusion: Intraduodenal infusion of the combination of tastants, but not of single tastants, resulted in a decrease in food intake and feelings of hunger. This suggests that activation of multiple receptors (by several tastants) results in a synergistic effect with reduction of food intake. Furthermore, we could not find any differences in the release of CCK, GLP-1 and PYY, suggesting that the reduction in food intake seen in the infusion of the combination is not mediated via an endocrine pathway. This study shows the exciting potential of tastants receptor activation in the regulation of food intake.
Figure 1. Motilin plasma levels between consecutive phases III in lean and obese participants. Mean ± SEM. *: p<0.05, **: p<0.001. 88 The Hypophagia After Psychological Stress in Aged Male Mice Is Mediated by an Interaction of Activation of Estrogen Receptor a With Serotonin 2C Receptor Shunsuke Ohnishi, Koji Nakagawa, Naoto Okubo, Chiharu Sadakane, Yayoi Saegusa, Miwa Nahata, Chihiro Yamada, Tomohisa Hattori, Naoya Sakamoto, Hiroshi Takeda Background/Aim: Depressive symptoms in elderly people frequently lead to loss of appetite; ongoing malnutrition may greatly influence quality of life. In addition, it is known that there are differences between the sexes in onset of stress disorder or depression. However, the mechanism of loss of appetite caused by stress in elderly people and its association with sexes remains to be fully elucidated. It has recently been suggested that the central serotonin 2C receptor (5-HT2CR) is important for controlling feeding behavior. To clarify the mechanism of stress-induced feeding disorder and its association with sex hormones, we examined the role of 5-HT2CR and estrogen in stress-induced decrease in food intake in aged male and female mice using novelty stress. Methods: Young (6 weeks) and aged (79-80 weeks) male and female mice were placed in and acclimatized to group housing (n = 5/cage). After fasting for 20 h, mice were transferred to individual cages, which caused novelty stress. First, the effects of aromatase inhibitor or 5-HT2CR antagonists were examined to evaluate the association between estrogen and 5-HT2CR in stress-induced suppression of food intake. In addition, we measured plasma levels of estradiol and estrogen receptor (ER) a, ERb, and aromatase mRNA expression using RT-PCR. Furthermore, to evaluate the functional changes
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in ERa in aged mice, ERa agonist was administered and the effect on food intake was investigated. Finally, the effect of 5-HT2CR antagonist on ERa-agonist-induced decrease in food intake in aged mice was examined. Results: Novelty stress significantly and continuously suppressed food intake in aged male mice compared with young or female mice. The decreased food intake in aged male mice exposed to novelty stress was significantly ameliorated by the administration of the aromatase inhibitor letrozole or the 5-HT2CR antagonists SB242084 or rikkunshito, a Kampo medicine (Figure 1, 2). Compared with young male mice, hypothalamic ERa and aromatase mRNA expression levels were significantly increased in aged male mice, whereas ER b mRNA expression levels remained unaltered. There were no differences in plasma estradiol levels between groups. Administration of the ER aagonist significantly and dose-dependently suppressed food intake in aged male mice. Administration of the 5-HT2CR antagonist SB242084 caused marked recovery of the ER a-agonist-induced decrease in food intake in aged male mice. Conclusion: ERa activation occurred in aged male mice and caused 5-HT2CR activation. This may result in a persistent decrease in food intake under novelty stress.
90 Efficacy of a Novel Small Molecule RORgt Inverse Agonist in Mouse DSS and TNBS Models of Inflammatory Bowel Disease Leo R. Fitzpatrick, Jim Zapf, Elizabeth M. Flood, Satheesh B. Ravula, Laura K. Lingardo, Jeff Small, Fabio Tucci, Chris A. Buhr, Gordon Alton BACKGROUND: Retinoic acid-related Orphan nuclear Receptor gamma t (RORgt) is a nuclear receptor that is required to produce IL17A and IL-17F and to elicit IL-23/IL-17 dependent autoimmune disease. RORgt is expressed in a variety of inflammatory cells (Th17, innate lymphoid cells, gd T-cells, iNKT-cells and other lymphoid cells) that are implicated in the pathogenesis of inflammatory bowel disease (IBD). In IBD clinical trials, one biologic (antibody) approach targeting IL-23/IL-17 axis was moderately effective (Ustekinumab), while another was ineffective (Secukinumab). Therefore, there is significant interest in small molecule approaches for IBD. METHODS: A RORgt inverse agonist was evaluated in vitro and ex vivo. The in vivo activity was accessed in DSS (2% in drinking water for 6 days, male CB57BL/6 mice dosed IP at 10, 15, 25 mg/kg qd) and TNBS (20 mg/kg in 50% ethanol) models. In TNBS studies, female BALB/c mice were divided in 6 groups (PBS alone, 50 ethanol/PBS, TNBS alone, compound (15 or 25 mg/kg, IP) plus TNBS, or Dex (0.6 mg/kg, IP) plus TNBS. Test drugs were administered once daily for 6 days. On day 6, disease activity index (stool blood, stool form, and weight loss) (DAI), macroscopic colonic scores (combination of lesions, stool form, adhesions, thickness), IL-17 levels, IL-23 levels, and histology were assessed. Pharmacokinetic parameters were determined in mice, using oral (100 mg/kg), IV (3 mg/kg) and IP (10 mg/kg) dosing. RESULTS: A potent RORgt inverse agonist (VPR-7) was identified with transcriptional activity in a ROR gt GAL4 reporter assay (IC50 = 270 nM) and efficacy in reducing IL-17A production in human PBMC cultures (EC50 = 44 nM). The compound showed low oral bioavailability and moderate IP bioavailability in mice. VPR-7 dose dependently improved the DAI for DSS colitis, and the macroscopic colonic score for TNBS colitis, while not significantly affecting animal body or spleen weight. Compared to TNBS controls, colonic histopathology was significantly reduced (by 51%) in mice treated at 25 mg/kg. VPR-7 treated mice also significantly attenuated colonic ulcer scores and colonic weights, while improving colonic lengths compared those of mice treated with TNBS alone. Colonic tissue biomarker (IL-17 and IL-23) levels were returned to those seen in non-TNBS controls by compound treatment. Plasma concentrations of compound exceeded effective concentrations at least 2 hours post dose in study animals. SUMMARY: These results are consistent with 91decreased colonic inflammation with VPR-7 treatment compared to Vehicle/TNBS treatment. CONCLUSIONS: For the first time an RORgt inverse agonist (VPR-7) showed efficacy in models of IBD. Our studies further extend the results from RORgt knock out mice, and suggest that ROR gt inverse agonists may be effective in the treatment of IBD. Supported by NIH SBIRs 1R43DK098896 & 2R44DK098896
Figure 1 Effect of aromatase inhibitor on food intake in aged mice exposed to novelty stress.{BR}Letrozole (2.5 mg/kg) was orally administered to mice immediately after novelty stress exposure. ## p < 0.01 vs. control, *, ** p < 0.05, 0.01 vs. stress.
Figure 2 Effects of serotonin 2C receptor antagonists on food intake in aged mice exposed to novelty stress.{BR}SB242084 (6 mg/ kg) or rikkunshito (RKT; 1000 mg/kg) was orally administered to mice immediately after novelty stress exposure. ## p < 0.01 vs. control, *, *** p < 0.05, 0.001 vs. stress.
91 Losartan Abrogates Intestinal Fibrosis After Steroid Induced Remission in Experimental Ileitis Shuvra Ray, Carlo De Salvo, Loris Riccardo Lopetuso, Wei Xin, Jude Oben, Jeremy D. Sanderson, Theresa T. Pizarro
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Background. Intestinal fibrosis in Crohn's disease (CD) leads to significant morbidity. Despite the development of many novel anti-inflammatory strategies, anti-fibrotic effects have been disappointing with no significant reduction in fibrosis-related surgery rates. While inflammation is required to trigger fibrogenesis, the subsequent activation of fibrotic pathways promotes fibrosis even in the absence of significant inflammation. Specific anti-fibrotic agents are therefore an important unmet therapeutic need. Increasing evidence supports a role for the renin-angiotensin system in fibrogenesis in multiple organs. Angiotensin receptor blockers, such as losartan, reduce fibrosis in chronic human kidney and liver disease and diminish fibrosis in rodent models of colitis. We investigated the anti-fibrotic effects of losartan in a clinically-relevant model of CD after steroid induced remission using SAMP1/YitFc (SAMP) mice, which spontaneously develop ileitis from 6 weeks age and progressively severe fibrosis from 20 weeks onwards. Methods. SAMP mice at 20 weeks age were treated with dexamethasone (1mg/Kg, i.p.) for 7 days, after which they were then treated with losartan (0.6g/L) in drinking water or placebo. Mice were euthanized after 2 and 5 weeks and ilea harvested for histology and RNA. Tissues were stained with H&E for inflammation scoring and Masson's trichrome for fibrosis scoring. Slides were scored using a standardised inflammation (TIS) and fibrosis scoring system in a blinded fashion by a GI pathologist. Ileal RNA extracts were analysed by quantitative PCR for a panel of pro-fibrogenic genes: TGF-ß, IL-33, COL1A1, COL3A1, IGF-1 and CTGF. Results. Fibrosis scores were significantly lower in the losartan group at 2 (9.18 vs 16.2 p=0.0002) and 5 weeks (7.8 vs 13.4 p=0.0003) vs placebo. At 2 weeks, TIS was higher in the losartan group (17.5 vs 13.8 p=0.04) and not significant after 5 weeks (16.4 vs 13.2 p=0.0628). COL3A1 expression was significantly reduced at 5 vs 2 weeks in the losartan group (p=0.027) and unchanged in the placebo group. At 5 weeks, there was also a trend to reduction in COL3A1 in the losartan group compared to placebo (p=0.2). TGF-ß, COL1A1 and CTGF expression were unchanged in all groups at 2 and 5 weeks. At 5 weeks, interestingly, upregulation of IL-33 (1.8 fold p=0.03) and IGF-1 (1.5 fold p=0.04) expression was noted in the losartan group. Conclusion. Losartan significantly diminished the development of ileal fibrosis in steroid-treated SAMP mice. This effect appears independent of inflammation and does not occur through reduction in conventional fibrotic pathways. Losartan is known to act on muscle through enhancing the IGF-1 signalling system and this warrants further investigation in the gut. Losartan holds promise as a putative anti-fibrotic as an adjunct to conventional anti-inflammatory strategies.
Different Response to Chronic Isolation Stress Between Young and Aged Mice Hiroshi Takeda, Shunsuke Ohnishi, Koji Nakagawa, Naoto Okubo, Chihiro Yamada, Yayoi Saegusa, Miwa Nahata, Tomohisa Hattori, Naoya Sakamoto Background/Aim: It is well known that psychological stress causes abnormal feeding behavior. The number of elderly people with anxiety and depression is increasing as our society ages, and long-term loneliness causes several nutritional problems. It is unclear whether loneliness contributes to abnormal feeding behavior among the elderly. We have previously demonstrated that in mice acute isolation stress transiently decreased feeding behavior, and this tendency is particularly pronounced in aged mice (Saegusa et al. AJP 2011, Nahata et al. Psychoneuroendocrinology 2013). Chronic isolation stress causes abnormalities in social behavior (e.g., increased anxiety and aggressiveness); however, there are few reports about the changes in feeding behavior after the chronic isolation stress. Accordingly, to clarify the association of loneliness with feeding behavior, we assessed the changes in body weight, food intake, locomotor activity, plasma corticosterone, and ghrelin levels in young and aged mice under chronic isolation stress. Materials and Methods: Young (10-week-old) and aged (91-week-old) male mice were placed in and acclimatized to group housing cages (n = 3-4/cage). The mice were then transferred to small individual cages, which caused isolation stress for 2 weeks, and food intake and locomotor activity were evaluated. At the end of theis period, blood was collected to measure plasma corticosterone or acylated ghrelin levels using enzyme-linked immunosorbent assay (ELISA). Results: No significant differences in body weight were observed between the group-housed and isolated groups in young and aged mice during the experimental period; however, the body weight change in the aged male mice significantly decreased during isolation stress. In the young mice, a significant and sustained increase in 24-h food intake was observed during isolation stress. In contrast, food intake in the aged mice only increased slightly1 week after the start of isolation. A marked peak pattern of dark-phase locomotor activity was observed in the young grouphoused and isolated mice after two-week isolation; however, the peak pattern of locomotor activity was attenuated in the aged mice subjected to isolation. Although plasma corticosterone levels after the 2-week isolation did not change in any group, plasma acylated ghrelin levels tended to be elevated in the isolated mice compared with those in the controls (both in young and in aged mice). Conclusion: After chronic isolation stress, increased food intake was observed only in young mice; this effect may be a compensatory response to the stressinduced increase in energy consumption. It is also suggested that the stress response and/ or ghrelin sensitivity may be weakened in aged mice.
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Obese Patients Have Significantly Higher Motilin Plasma Levels Which Decrease After Roux-en-Y Gastric Bypass Surgery Eveline Deloose, Pieter Janssen, Matthias Lannoo, Bart Van der Schueren, Inge Depoortere, Jan F. Tack Objective: Changes in gut hormones are suggested to play an important role in the metabolic reprogramming that occurs after Roux-en-Y gastric bypass (RYGB) surgery. We have recently shown that gastric phases III of the migrating motor complex (MMC) are associated with hunger peaks in healthy lean volunteers and that both can be induced through the administration of erythromycin, a motilin receptor agonist. The aim of this study was to evaluate the role of motilin and phase III as hunger-inducing factors in obese patients and to study the effect of RYGB surgery on plasma motilin levels. Design: Blood samples for motilin and ghrelin (total and octanoylated) were taken during a complete MMC cycle in healthy lean controls (n=15, 28±2y, 22±0.5kg/m2) and obese patients (n=16, 40±3y, 41±0.8kg/m2) before surgery and 6 months (n=8, 39±4y, 29±1.3kg/m2) or 1 year after surgery (n=7, 40±4y, 25±1.1kg/m2). Gastrointestinal motility was measured using high-resolution manometry after a 12 hour overnight fast. Hunger was scored every 5 min on a visual analogue scale of 10 cm. An i.v. infusion of 40 mg erythromycin was given 20 min after the end of the second phase III in obese patients to evaluate the effect on hunger. Hedonic hunger was measured using the Power of Food scale in obese patients prior to, 6 months and 1 year after surgery. Results: A positive correlation was found between BMI and motilin (r=0.53; p=0.005) and a negative correlation between BMI and ghrelin (total (r=-0.52; p=0.007) and octanoylated (r=-0.43; p=0.02). Obese patients had significantly higher motilin plasma levels compared to lean controls (fig 1, p=0.0005) during the MMC cycle but tended to lack the motilin (-3±6% vs 15±6%; p=0.052) plasma peak prior to phase III. The obese patients had significantly less phases III that started in the antrum for both the first (44% vs 60%; p= 0.03) and second (22% vs 40%; p=0.0091) phase III compared to lean controls. Hunger scores during phase III were significantly lower in obese patients (p=0.0022) compared to lean controls, but could be increased through the administration of erythromycin (p=0.97) which induced a premature gastric phase III in all patients. After RYGB surgery the AUC of motilin plasma levels was significantly reduced (fig 2, p=0.018). Although hunger scores during phase III were not affected by RYGB (p=0.9), hedonic hunger was significantly decreased (p=0.0009). Conclusion: Our study is the first to describe a positive correlation between motilin plasma levels and BMI which could indicate that motilin plays an important role in the pathogenesis of obesity, moreover we have shown that motilin levels decrease after RYGB. The switch in origin of phase III contractions, due to the absence of a peak in plasma motilin levels, could represent a counter regulatory mechanism to decrease hunger in obese patients.
Figure 2. Area under the curve of motilin plasma levels measured between two consecutive phases III. Mean ± SEM. *: p<0.05.
87 Intraduodenal Infusion of a Combination of Tastants Decreases Food Intake Mark van Avesaat, Freddy Troost, Dina Ripken, Henk F. Hendriks, Ad Masclee Introduction: Taste receptors on the tongue are able to detect the tastants sweet, umami and bitter. The first two are detected by the taste 1 receptor family (TAS1R), while latter is detected by the taste 2 receptor family (TAS2R). Recently, it was found that taste receptors are also expressed in the intestine. Its functions are yet unknown although it has been hypothesized that they may initiate and transfer signals that originate from the gastrointestinal (GI) tract and modulate satiety and food intake. In the present study, we set out to investigate the effects of intraduodenal tastant infusion on food intake, hunger and fullness, GI symptoms and GI peptide release. Methods: This randomized, double blind, crossover study was performed in 15 healthy subjects (6 male; mean age 24.0 ± 2.0 yrs; mean BMI 22.4 ± 0.3 kg/m2) who participated in 5 experimental test days. Test days started with the insertion of a nasoduodenal catheter under fluoroscopy. After insertion of the catheter, subjects consumed a standardized liquid breakfast (250 mL, 153 kcal). At 150 min after breakfast an intraduodenal infusion, containing control (saline), bitter (quinine), sweet (rebaudioside A), umami (monosodium glutamate) or a combination of the tastants (quinine, rebaudioside A and MSG), over a period of 60 min was given. Food intake was measured during an ad libitum meal (standard meal, 15 min after the end of the infusion). VAS scales were used to monitor GI complains, hunger and fullness scores. Blood samples were drawn at regular intervals in order to measure the GI peptides CCK, GLP-1 and PYY. A mixed analysis of variance model was used to analyze ad libitum food intake, VAS scores and GI peptide release. Results: Intraduodenal infusion of the combination of tastants significantly decreased ad libitum food intake compared to control (422±97 vs 486±104 kcal, p<0.05). Food intake after infusion of the other tastants, separately, was not significantly different from control. However, umami and the combination of tastants significantly reduced hunger. No differences between tastant infusion and control were observed in the release of CCK, GLP-1 or PYY. Subjects did not report any gastrointestinal complaints during or after intraduodenal infusion with the tastants. Conclusion: Intraduodenal infusion of the combination of tastants, but not of single tastants, resulted in a decrease in food intake and feelings of hunger. This suggests that activation of multiple receptors (by several tastants) results in a synergistic effect with reduction of food intake. Furthermore, we could not find any differences in the release of CCK, GLP-1 and PYY, suggesting that the reduction in food intake seen in the infusion of the combination is not mediated via an endocrine pathway. This study shows the exciting potential of tastants receptor activation in the regulation of food intake.
Figure 1. Motilin plasma levels between consecutive phases III in lean and obese participants. Mean ± SEM. *: p<0.05, **: p<0.001. 88 The Hypophagia After Psychological Stress in Aged Male Mice Is Mediated by an Interaction of Activation of Estrogen Receptor a With Serotonin 2C Receptor Shunsuke Ohnishi, Koji Nakagawa, Naoto Okubo, Chiharu Sadakane, Yayoi Saegusa, Miwa Nahata, Chihiro Yamada, Tomohisa Hattori, Naoya Sakamoto, Hiroshi Takeda Background/Aim: Depressive symptoms in elderly people frequently lead to loss of appetite; ongoing malnutrition may greatly influence quality of life. In addition, it is known that there are differences between the sexes in onset of stress disorder or depression. However, the mechanism of loss of appetite caused by stress in elderly people and its association with sexes remains to be fully elucidated. It has recently been suggested that the central serotonin 2C receptor (5-HT2CR) is important for controlling feeding behavior. To clarify the mechanism of stress-induced feeding disorder and its association with sex hormones, we examined the role of 5-HT2CR and estrogen in stress-induced decrease in food intake in aged male and female mice using novelty stress. Methods: Young (6 weeks) and aged (79-80 weeks) male and female mice were placed in and acclimatized to group housing (n = 5/cage). After fasting for 20 h, mice were transferred to individual cages, which caused novelty stress. First, the effects of aromatase inhibitor or 5-HT2CR antagonists were examined to evaluate the association between estrogen and 5-HT2CR in stress-induced suppression of food intake. In addition, we measured plasma levels of estradiol and estrogen receptor (ER) a, ERb, and aromatase mRNA expression using RT-PCR. Furthermore, to evaluate the functional changes
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in ERa in aged mice, ERa agonist was administered and the effect on food intake was investigated. Finally, the effect of 5-HT2CR antagonist on ERa-agonist-induced decrease in food intake in aged mice was examined. Results: Novelty stress significantly and continuously suppressed food intake in aged male mice compared with young or female mice. The decreased food intake in aged male mice exposed to novelty stress was significantly ameliorated by the administration of the aromatase inhibitor letrozole or the 5-HT2CR antagonists SB242084 or rikkunshito, a Kampo medicine (Figure 1, 2). Compared with young male mice, hypothalamic ERa and aromatase mRNA expression levels were significantly increased in aged male mice, whereas ER b mRNA expression levels remained unaltered. There were no differences in plasma estradiol levels between groups. Administration of the ER aagonist significantly and dose-dependently suppressed food intake in aged male mice. Administration of the 5-HT2CR antagonist SB242084 caused marked recovery of the ER a-agonist-induced decrease in food intake in aged male mice. Conclusion: ERa activation occurred in aged male mice and caused 5-HT2CR activation. This may result in a persistent decrease in food intake under novelty stress.
90 Efficacy of a Novel Small Molecule RORgt Inverse Agonist in Mouse DSS and TNBS Models of Inflammatory Bowel Disease Leo R. Fitzpatrick, Jim Zapf, Elizabeth M. Flood, Satheesh B. Ravula, Laura K. Lingardo, Jeff Small, Fabio Tucci, Chris A. Buhr, Gordon Alton BACKGROUND: Retinoic acid-related Orphan nuclear Receptor gamma t (RORgt) is a nuclear receptor that is required to produce IL17A and IL-17F and to elicit IL-23/IL-17 dependent autoimmune disease. RORgt is expressed in a variety of inflammatory cells (Th17, innate lymphoid cells, gd T-cells, iNKT-cells and other lymphoid cells) that are implicated in the pathogenesis of inflammatory bowel disease (IBD). In IBD clinical trials, one biologic (antibody) approach targeting IL-23/IL-17 axis was moderately effective (Ustekinumab), while another was ineffective (Secukinumab). Therefore, there is significant interest in small molecule approaches for IBD. METHODS: A RORgt inverse agonist was evaluated in vitro and ex vivo. The in vivo activity was accessed in DSS (2% in drinking water for 6 days, male CB57BL/6 mice dosed IP at 10, 15, 25 mg/kg qd) and TNBS (20 mg/kg in 50% ethanol) models. In TNBS studies, female BALB/c mice were divided in 6 groups (PBS alone, 50 ethanol/PBS, TNBS alone, compound (15 or 25 mg/kg, IP) plus TNBS, or Dex (0.6 mg/kg, IP) plus TNBS. Test drugs were administered once daily for 6 days. On day 6, disease activity index (stool blood, stool form, and weight loss) (DAI), macroscopic colonic scores (combination of lesions, stool form, adhesions, thickness), IL-17 levels, IL-23 levels, and histology were assessed. Pharmacokinetic parameters were determined in mice, using oral (100 mg/kg), IV (3 mg/kg) and IP (10 mg/kg) dosing. RESULTS: A potent RORgt inverse agonist (VPR-7) was identified with transcriptional activity in a ROR gt GAL4 reporter assay (IC50 = 270 nM) and efficacy in reducing IL-17A production in human PBMC cultures (EC50 = 44 nM). The compound showed low oral bioavailability and moderate IP bioavailability in mice. VPR-7 dose dependently improved the DAI for DSS colitis, and the macroscopic colonic score for TNBS colitis, while not significantly affecting animal body or spleen weight. Compared to TNBS controls, colonic histopathology was significantly reduced (by 51%) in mice treated at 25 mg/kg. VPR-7 treated mice also significantly attenuated colonic ulcer scores and colonic weights, while improving colonic lengths compared those of mice treated with TNBS alone. Colonic tissue biomarker (IL-17 and IL-23) levels were returned to those seen in non-TNBS controls by compound treatment. Plasma concentrations of compound exceeded effective concentrations at least 2 hours post dose in study animals. SUMMARY: These results are consistent with 91decreased colonic inflammation with VPR-7 treatment compared to Vehicle/TNBS treatment. CONCLUSIONS: For the first time an RORgt inverse agonist (VPR-7) showed efficacy in models of IBD. Our studies further extend the results from RORgt knock out mice, and suggest that ROR gt inverse agonists may be effective in the treatment of IBD. Supported by NIH SBIRs 1R43DK098896 & 2R44DK098896
Figure 1 Effect of aromatase inhibitor on food intake in aged mice exposed to novelty stress.{BR}Letrozole (2.5 mg/kg) was orally administered to mice immediately after novelty stress exposure. ## p < 0.01 vs. control, *, ** p < 0.05, 0.01 vs. stress.
Figure 2 Effects of serotonin 2C receptor antagonists on food intake in aged mice exposed to novelty stress.{BR}SB242084 (6 mg/ kg) or rikkunshito (RKT; 1000 mg/kg) was orally administered to mice immediately after novelty stress exposure. ## p < 0.01 vs. control, *, *** p < 0.05, 0.001 vs. stress.
91 Losartan Abrogates Intestinal Fibrosis After Steroid Induced Remission in Experimental Ileitis Shuvra Ray, Carlo De Salvo, Loris Riccardo Lopetuso, Wei Xin, Jude Oben, Jeremy D. Sanderson, Theresa T. Pizarro
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Background. Intestinal fibrosis in Crohn's disease (CD) leads to significant morbidity. Despite the development of many novel anti-inflammatory strategies, anti-fibrotic effects have been disappointing with no significant reduction in fibrosis-related surgery rates. While inflammation is required to trigger fibrogenesis, the subsequent activation of fibrotic pathways promotes fibrosis even in the absence of significant inflammation. Specific anti-fibrotic agents are therefore an important unmet therapeutic need. Increasing evidence supports a role for the renin-angiotensin system in fibrogenesis in multiple organs. Angiotensin receptor blockers, such as losartan, reduce fibrosis in chronic human kidney and liver disease and diminish fibrosis in rodent models of colitis. We investigated the anti-fibrotic effects of losartan in a clinically-relevant model of CD after steroid induced remission using SAMP1/YitFc (SAMP) mice, which spontaneously develop ileitis from 6 weeks age and progressively severe fibrosis from 20 weeks onwards. Methods. SAMP mice at 20 weeks age were treated with dexamethasone (1mg/Kg, i.p.) for 7 days, after which they were then treated with losartan (0.6g/L) in drinking water or placebo. Mice were euthanized after 2 and 5 weeks and ilea harvested for histology and RNA. Tissues were stained with H&E for inflammation scoring and Masson's trichrome for fibrosis scoring. Slides were scored using a standardised inflammation (TIS) and fibrosis scoring system in a blinded fashion by a GI pathologist. Ileal RNA extracts were analysed by quantitative PCR for a panel of pro-fibrogenic genes: TGF-ß, IL-33, COL1A1, COL3A1, IGF-1 and CTGF. Results. Fibrosis scores were significantly lower in the losartan group at 2 (9.18 vs 16.2 p=0.0002) and 5 weeks (7.8 vs 13.4 p=0.0003) vs placebo. At 2 weeks, TIS was higher in the losartan group (17.5 vs 13.8 p=0.04) and not significant after 5 weeks (16.4 vs 13.2 p=0.0628). COL3A1 expression was significantly reduced at 5 vs 2 weeks in the losartan group (p=0.027) and unchanged in the placebo group. At 5 weeks, there was also a trend to reduction in COL3A1 in the losartan group compared to placebo (p=0.2). TGF-ß, COL1A1 and CTGF expression were unchanged in all groups at 2 and 5 weeks. At 5 weeks, interestingly, upregulation of IL-33 (1.8 fold p=0.03) and IGF-1 (1.5 fold p=0.04) expression was noted in the losartan group. Conclusion. Losartan significantly diminished the development of ileal fibrosis in steroid-treated SAMP mice. This effect appears independent of inflammation and does not occur through reduction in conventional fibrotic pathways. Losartan is known to act on muscle through enhancing the IGF-1 signalling system and this warrants further investigation in the gut. Losartan holds promise as a putative anti-fibrotic as an adjunct to conventional anti-inflammatory strategies.
Different Response to Chronic Isolation Stress Between Young and Aged Mice Hiroshi Takeda, Shunsuke Ohnishi, Koji Nakagawa, Naoto Okubo, Chihiro Yamada, Yayoi Saegusa, Miwa Nahata, Tomohisa Hattori, Naoya Sakamoto Background/Aim: It is well known that psychological stress causes abnormal feeding behavior. The number of elderly people with anxiety and depression is increasing as our society ages, and long-term loneliness causes several nutritional problems. It is unclear whether loneliness contributes to abnormal feeding behavior among the elderly. We have previously demonstrated that in mice acute isolation stress transiently decreased feeding behavior, and this tendency is particularly pronounced in aged mice (Saegusa et al. AJP 2011, Nahata et al. Psychoneuroendocrinology 2013). Chronic isolation stress causes abnormalities in social behavior (e.g., increased anxiety and aggressiveness); however, there are few reports about the changes in feeding behavior after the chronic isolation stress. Accordingly, to clarify the association of loneliness with feeding behavior, we assessed the changes in body weight, food intake, locomotor activity, plasma corticosterone, and ghrelin levels in young and aged mice under chronic isolation stress. Materials and Methods: Young (10-week-old) and aged (91-week-old) male mice were placed in and acclimatized to group housing cages (n = 3-4/cage). The mice were then transferred to small individual cages, which caused isolation stress for 2 weeks, and food intake and locomotor activity were evaluated. At the end of theis period, blood was collected to measure plasma corticosterone or acylated ghrelin levels using enzyme-linked immunosorbent assay (ELISA). Results: No significant differences in body weight were observed between the group-housed and isolated groups in young and aged mice during the experimental period; however, the body weight change in the aged male mice significantly decreased during isolation stress. In the young mice, a significant and sustained increase in 24-h food intake was observed during isolation stress. In contrast, food intake in the aged mice only increased slightly1 week after the start of isolation. A marked peak pattern of dark-phase locomotor activity was observed in the young grouphoused and isolated mice after two-week isolation; however, the peak pattern of locomotor activity was attenuated in the aged mice subjected to isolation. Although plasma corticosterone levels after the 2-week isolation did not change in any group, plasma acylated ghrelin levels tended to be elevated in the isolated mice compared with those in the controls (both in young and in aged mice). Conclusion: After chronic isolation stress, increased food intake was observed only in young mice; this effect may be a compensatory response to the stressinduced increase in energy consumption. It is also suggested that the stress response and/ or ghrelin sensitivity may be weakened in aged mice.
AGA Abstracts
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