High fat diet induced changes in gastric vagal afferent response to adiponectin

PHB-10910; No of Pages 9 Physiology & Behavior xxx (2015) xxx–xxx

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High fat diet induced changes in gastric vagal afferent response to adiponectin Stephen J. Kentish a,b,⁎, Kyle Ratcliff a, Hui Li a, Gary A. Wittert a,b,c, Amanda J. Page a,b,c a b c

Vagal Afferent Research Group, Centre for Nutrition and Gastrointestinal Disease, Discipline of Medicine, University of Adelaide, Frome Road, Adelaide, SA 5005, Australia Nutrition and Metabolism, South Australian Health and Medical Research Institute, North Terrace, SA 5000, Australia Royal Adelaide Hospital, North Terrace, Adelaide, SA 5000, Australia

H I G H L I G H T S • • • •

Adiponectin is expressed in the stomach. Adiponectin receptors are present in mucosal and muscular gastric vagal afferents. Adiponectin inhibits the mechanosensitivity of tension gastric vagal afferents. In obese mice adiponectin potentiates mucosal vagal afferent mechanosensitivity.

a r t i c l e

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Article history: Received 2 March 2015 Received in revised form 28 May 2015 Accepted 10 June 2015 Available online xxxx Keywords: Vagal afferents Obesity Adiponectin

a b s t r a c t Food intake is regulated by vagal afferent signals from the stomach. Adiponectin, secreted primarily from adipocytes, also has a role in regulating food intake. However, the involvement of vagal afferents in this effect remains to be established. We aimed to determine if adiponectin can modulate gastric vagal afferent (GVA) satiety signals and further whether this is altered in high fat diet (HFD)-induced obesity. Female C57BL/6J mice were fed either a standard laboratory diet (SLD) or a HFD for 12 weeks. Plasma adiponectin levels were assayed, and the expression of adiponectin in the gastric mucosa was assessed using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The location of adiponectin protein within the gastric mucosa was determined by immunohistochemistry. To evaluate the direct effect of adiponectin on vagal afferent endings we determined adiponectin receptor expression in whole nodose ganglia (NDG) and also specifically in GVA neurons using retrograde tracing and qRT-PCR. An in vitro preparation was used to determine the effect of adiponectin on GVA response to mechanical stimulation. HFD mice exhibited an increased body weight and adiposity and showed delayed gastric emptying relative to SLD mice. Plasma adiponectin levels were not significantly different in HFD compared to SLD mice. Adiponectin mRNA was detected in the gastric mucosa of both SLD and HFD mice and presence of protein was confirmed immunohistochemically by the detection of adiponectin immunoreactive cells in the mucosal layer of the stomach. Adiponectin receptor 1 (ADIPOR1) and 2 (ADIPOR2) mRNA was present in both the SLD and HFD whole NDG and also specifically traced gastric mucosal and muscular neurons. There was a reduction in ADIPOR1 mRNA in the mucosal afferents of the HFD mice relative to the SLD mice. In HFD mice adiponectin potentiated gastric mucosal afferent responses to mucosal stroking, an effect not observed in SLD mice. Adiponectin reduced the responses of tension receptors to circular stretch to a similar extent in both SLD and HFD mice. In conclusion, adiponectin modulates GVA satiety signals. This modulatory effect is altered in HFD-induced obesity. It remains to be conclusively determined whether this modulation is involved in the regulation of food intake and what the whole animal phenotypic consequence is. © 2015 Elsevier Inc. All rights reserved.

1. Introduction

⁎ Corresponding author at: Centre for Nutrition and Gastrointestinal Disease, Discipline of Medicine, University of Adelaide, Level 7 South Australian Health and Medical Research Institute, North Terrace, Adelaide, SA 5000, Australia. E-mail address: [email protected] (S.J. Kentish).

It is well established that vagal afferent signals generated in the gastrointestinal tract are able to influence food intake [1,2]. In the stomach, two mechanosensitive gastric vagal afferent (GVA) receptor subtypes, tension and mucosal receptors, have been identified based on their response to mechanical stimuli [3]. Although the role of gastric tension

http://dx.doi.org/10.1016/j.physbeh.2015.06.016 0031-9384/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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receptors in signaling distension to trigger satiety and accommodation has been well characterized [4], the role of mucosal receptors is less well understood. Andrews and Sanger suggested that mucosal receptors play a role in detecting chemical stimuli and generating sensations of nausea and triggering vomiting [5]. Mucosal receptors have also been proposed to be important in measuring food particle size and therefore digestion [6]. Particle size is a factor which influences the rate of gastric emptying, which relates inversely to satiety [7,8]. The mechanosensitivity of GVAs is modulated by peptides known to affect satiety and feeding behavior, released either locally in the gut or into the circulation [9–11]. Thus, vagal endings within the stomach wall are in an ideal position to detect not only mechanical stimuli but also locally released peptides/hormones from enteroendocrine cells in the gastric mucosa [12]. GVA satiety signals are influenced by nutritional status. For example, the sensitivity of tension sensitive GVA mechanoreceptors is reduced with fasting or chronic high fat diet (HFD) consumption [13]. Furthermore, the modulatory effect of appetite regulating hormones on GVAs changes with short-term food withdrawal or long-term HFD feeding [12]. Adiponectin is an adipokine that is abundant in the circulation, representing up to 0.05% of total circulating protein [14]. Adiponectin has a primary insulin sensitizing action but also has beneficial effects on metabolism and atherosclerosis [15–19]. Similar to other adipokines, adiponectin has been shown to affect satiety both centrally and peripherally, although there is a lack of consensus in rodent studies regarding the ability of adiponectin to regulate food intake. An intracerebroventricular (ICV) infusion of adiponectin can reduce body weight by either decreasing food intake and/or increasing energy expenditure [20,21]. However, in contrast, a central effect has been observed to increase food intake and cause weight gain by activation of adiponectin receptors in the hypothalamus [22]. Peripherally, overexpression of adiponectin in rats reduced food intake [23]. In contrast, adiponectin knockout mice have normal feeding behavior when fed standard chow [22,24], but reduced food intake in response to a high fat diet compared to wild type mice [22]. The effect of adiponectin on vagal afferents, has to date remained unexplored. Furthermore, the stomach has also been shown express leptin which acts in a paracrine manner on vagal afferents [25] but whether this also applies to adiponectin has not hitherto been explored. Two receptors for adiponectin have been identified, adiponectin receptor 1 (ADIPOR1) and adiponectin receptor 2 (ADIPOR2) [26], which appear to have opposite effects. Deletion of ADIPOR1 increases adiposity and decreases both glucose tolerance and locomotor activity, whilst deletion of ADIPOR2 results in a lean phenotype with increased locomotor activity and resistance to HFD-induced obesity [27]. The expression of adiponectin receptors is linked to metabolic status with abundance inversely proportional to circulating insulin levels [28]. The nodose ganglia (NDG) containing the cell bodies of vagal afferents incorporate a variety of receptors for peptides with food intake modulatory effects such as leptin, ghrelin and cholecystokinin [29]. The presence of adiponectin receptors in vagal afferents has not previously been reported and there are also no prior reports, as far as can be determined, as to whether a peripheral vagal pathway exists for adiponectin and whether any potential GVA signaling is disrupted in obesity where circulating adiponectin concentrations have been suggested to be decreased. The aim of the present study was to determine the expression of adiponectin receptors in the NDG, whether adiponectin is able to signal through GVAs and use an obese mouse model to determine whether such signals are disrupted in HFD induced obesity. 2. Materials and methods

University of Adelaide and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 2.2. High fat diet model Thirty-two, 7 week old female C57BL/6J mice (Animal Resource Centre, Perth, Australia) were acclimatized for 1 week before being randomly divided into two equal groups and fed either a high fat diet (HFD) comprising 60%, 20%, and 20% of energy from fat, protein, and carbohydrate (Adapted from Research Diets Inc., New Brunswick, USA) or a standard laboratory diet (SLD) comprising 12%, 23%, and 65% of energy from fat, protein and carbohydrate (Specialty Feeds, Glen Forest, Western Australia) for 12 weeks (N = 16/diet). Four mice were housed per cage and were kept under 12 h light:dark cycles with ad libitum access to food and water. 2.3. Quantification of plasma adiponectin and insulin Plasma samples extracted from the blood collected from the abdominal aorta during euthanasia were analyzed for circulating insulin and adiponectin using a Millipore Insulin ELISA kit (EZRMI-13K) and Millipore Adiponectin ELISA kit (EZMADP-60K) respectively as per the manufacturer's instructions (Millipore, Massachusetts, USA). Quantification was measured at 460 and 595 nm. The adiponectin assay has a detection threshold of 0.2 ng/mL with an intra-assay variation of 5.75% and an inter-assay variation of 5.97%. The insulin assay kit has a detection threshold of 0.1 ng/mL, with an intra-assay variation of 3.73% and an inter-assay variation of 6.03%. 2.4. Gastric emptying Gastric emptying was measured using a calibrated solid egg meal as previously described [30], the week prior to euthanasia for the GVA recordings. Briefly, after an overnight fast mice from both diet groups (N = 12/diet) were given 0.1 g of baked egg yolk containing 1 μg mL−1 of [13C]-labeled octanoic acid (99% enrichment, Cambridge Isotope Laboratories, MA, USA) to consume within 1 min. Breath samples were collected at regular intervals for solid meal emptying (0–150 min) and analyzed for [13CO2] content with an isotope ratio mass spectrometer (ABCA 20/20 Europa Scientific, Crewe, UK). The [13CO2] excretion data was analyzed by nonlinear regression analysis for curve fitting and for calculation of gastric half-emptying time (t½). 2.5. Retrograde tracing Cell bodies of GVAs innervating specific stomach layers were identified using differential tracing from the stomach as previously documented [31]. 2.5.1. Gastric muscle SLD (n = 8) and HFD (n = 8) mice were anesthetized with isoflurane (1–1.5% in oxygen), a laparotomy performed, and an Alexa Fluor® 555 conjugate of cholera toxin β-subunit (CTB-AF555 (0.5%); Invitrogen, Life Technologies, Mulgrave, Australia) injected subserosal into the muscularis externa of the proximal stomach using a 30 gauge Hamilton syringe. Multiple equally spaced injections of 2 μL were made parallel to and 1–2 mm from the lesser curvature on both dorsal and ventral surfaces of the stomach (total volume 10 μL). The injection sites were dried with a cotton tip to ensure no spillage of tracer, the laparotomy incision was closed, and antibiotic (Baytrill 10 mg 50 μL−1) and analgesic (Butorphenol; 5 mg kg−1) were administered.

2.1. Ethical approval All experimental protocols were approved by the animal ethics committees of the Institute of Medical and Veterinary Science and

2.5.2. Gastric mucosa SLD (n = 8) and HFD (n = 8) mice were anesthetized with isoflurane (1–1.5% in oxygen), a laparotomy performed and a mucolytic

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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(10% N-acetylcysteine; 200 μL, Bristol-Myers Squibb, Vic, Australia) was injected into the stomach lumen. The mucolytic was removed via syringe after 5 min and followed by two saline rinses (200 μL each). Subsequently, 10 μL of 0.5% CTB-AF555 was injected into the proximal gastric lumen via a 30 gauge Hamilton syringe and the proximal stomach walls gently pressed together to expose both the dorsal and ventral surfaces to the tracer. The laparotomy incision was then closed and antibiotic and analgesic administered as above. Food and water were withheld for 2 h postoperative to maximize exposure of tracer. 2.6. Laser capture microdissection Two days after the surgery mice were anesthetized with sodium pentobarbitone (0.2 mL IP, 60 mg mL−1) before decapitation and removal of the NDG. The retrogradely traced NDG were removed and dissociated. Briefly, to obtain optimal cell density, NDG from four mice were combined into a tube containing F12 (Invitrogen) complete nutrient medium +10% fetal calf serum and 1% penicillin/streptomycin and stored on ice. NDG were dissociated by incubating at 37 °C in a 4 mg mL− 1 solution of dispase and collagenase II (Invitrogen) made up in Hank's Balanced Salt Solution (HBSS; Invitrogen) with agitation at 5 min intervals. After 30 min the dispase/collagenase solution was removed, and NDG were further incubated in a 4 mg mL−1 solution of collagenase II in HBSS for 10 min. Then the cells were rinsed in cold HBSS and F12 and further dissociated by passing them through a fire polished Pasteur pipette until no cell clumps were visible. The cells were then pelleted, rinsed in HBSS, and resuspended in HBSS. A cell count was performed using Trypan blue exclusion, and then cells were diluted to 500– 1000 cells per 10 μL suspension and seeded to 50 mm duplex dishes (Carl Zeiss, Jena, Germany). The dishes were placed in a 5% CO2 incubator set at 37 °C for 2 h to allow cell adherence. Cells were then subject to laser-capture microdissection, performed on a P.A.L.M.® microbeam microdissection system (Carl Zeiss, Jena, Germany). Fluorescent labeled nodose neurons were microdissected and catapulted directly into a lysis and stabilization buffer (Buffer RLT, RNeasy Micro RNA extraction Kit, Qiagen) containing 0.14 M βMercaptoethanol (Sigma-Aldrich, Australia). Total RNA was extracted from these cells using the same protocol as for whole NDG. There was no significant difference in RNA yield between the diet groups. 2.7. Real-time quantitative reverse-transcription polymerase chain reaction The NDG of the mice used for in vitro gastroesophageal vagal afferent preparation were excised bilaterally. Total RNA was then extracted from the NDG using an RNeasy Micro RNA Extraction Kit, in accordance with the manufacturer's instructions (Qiagen, Hilden, Germany). Additionally, the mucosa was removed from the ventral side of the stomach and RNA was extracted using an RNeasy Mini RNA Extraction Kit according to the manufacturer's instructions (Qiagen). The RNA yield and quality were quantified on a NanoDrop™ Lite spectrophotometer (Thermo Fisher Scientific, Scoresby, Australia) with an OD260/280 of N1.8 accepted as sufficiently pure for downstream applications. For evaluation of adiponectin expression in SLD and HFD stomach mucosa and ADIPOR1 and ADIPOR2 expression in whole NDG and retrograde labeled neurons qRT-PCR reactions were performed as previously described [32], using a 7500 Fast Real-time PCR System (Applied Biosystems®, Life Technologies) and analyzed with DataAssist™ RealTime PCR Analysis Software (Applied Biosystems®, Life Technologies). Reactions were performed in triplicate according to the manufacturer's specifications with a QuantiTect SYBR®green RT-PCR one-step qRT-PCR kit (Qiagen), incorporating specific QuantiTect primers for detection of ADIPOR1, ADIPOR2, adiponectin, β-tubulin III and β-actin mRNA. Relative mRNA transcript expression was calculated as previously described

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[33]. No template controls for each gene expression assay were also performed. 2.8. Immunohistochemistry To determine the presence of adiponectin protein and the specific gastric cell type, dual labeling immunohistochemistry was performed. Separate age matched SLD fed mice were anesthetized with intraperitoneal IP injection of sodium pentobarbitone (0.2 mL, 60 mg mL−1) and transcardially perfused with heparinized saline at 40 °C, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PFA-PB) at 4 °C. The ventral side of the stomach was removed and placed in PFA-PB at room temperature 4 °C for 2 h. After post-fixation the stomach was cryoprotected in 30% sucrose-PB for a minimum of 18 h. The stomach was then frozen in optimal cutting temperature compound (O.C.T.; Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands) and 10 μm transverse section cut. Sections were air dried at room temperature on gelatin coated slides and rinsed in phosphate buffered saline (PBS) + 0.2% Triton X-100 (PBS-TX, pH 7.4; Sigma-Aldrich, NSW, Australia) to facilitate antibody penetration, and then blocked with 10% donkey serum diluted in PBS-TX for 40 min at room temperature. Sections were incubated with a goat polyclonal anti-adiponectin antibody (1:400; sc-26497; Santa Cruz Biotechnology, California, USA) and a gastric cell marker primary antibody, including rabbit antigastric intrinsic factor (GIF) (chief cell marker, 1:800; ab91322; Abcam, VIC, Australia), rabbit anti-5-HT (enterochromaffin cell (EC) marker, 1:200; ab8882; Abcam), rabbit anti-gastrin (G-cell marker, 1:200; sc-20729; Santa Cruz), rabbit anti-somatostatin (D-cell marker, 1:3200; sc-13099; Santa Cruz) or rabbit anti-histamine (enterochromaffin like cell (ECL) marker, 1:1600; H7403; Sigma-Aldrich, NSW, Australia). Unbound antibody was then removed with subsequent PBS-TX washes and sections incubated for 1 h at room temperature with secondary antibodies diluted 1:200 in PBS-TX (donkey anti-goat IgG conjugated to Alexa Fluor® 488 and donkey anti-rabbit IgG conjugated to Alexa Fluor® 568; Life Technologies). The sections were then given final PBS-TX washes, mounted on slides and cover slipped using ProLong antifade (Life Technologies). Slides where primary antibody was omitted showed no labeling and served as negative controls. Slide sections were visualized using an epifluorescence microscope (BX-51, Olympus, Australia) equipped with filters for Alexa Fluor® 488, with images acquired by a CoolSnapfx monochrome digital camera (Roper Scientific, Tucson, AZ, USA). 2.9. In vitro mouse gastroesophageal afferent preparation After the completion of the 12 week diet period mice fed ad libitum were euthanized between 0600 and 0900 h, via 4% isoflurane anesthetic and exsanguination through the abdominal aorta followed by decapitation. The stage of the estrous cycle was not determined and so the results obtained could be from mice at any stage in the cycle. The stomach and esophagus including both vagal nerves were dissected as previously described [3,13]. In short, the stomach and esophagus, with intact vagal nerves, were removed. The stomach and esophagus were opened out longitudinally mid-way between the two main vagal branches, either side of the esophagus, and along the greater curvature of the stomach. The ventral half of the stomach was removed. The tissue was then pinned down mucosa side up in an organ bath containing a modified Krebs solution composing of (in mM): 118.1 NaCl, 4.7 KCl, 25.1 NaHCO3, 1.3 NaH2PO4, 1.2 MgSO4·7H2O, 1.5 CaCl2, 1.0 citric acid, 11.1 glucose and 0.001 nifidipine, bubbled with 95% O2–5% CO2. The dissection process was carried out at 4 °C. 2.10. Characterization of gastroesophageal gastric vagal afferent properties The location of individual receptive fields corresponding to afferent fibers was identified by mechanical stimulation of the mucosa with a

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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soft brush. The sub-type of each afferent was then further determined by responses specific to each sub-type of GVA mechanoreceptor, as previously described [13]. Since the mucosal GVA receptive field can be very small (≈1 mm), the response of 10 stimulations was averaged to minimize error. For tension receptors, circular tension was applied by a hook, placed adjacent to the receptive field, attached to a cantilever via suture silk as previously described [3]. Weights were applied in a step-wise manner from 1 to 5 g for 1 min per weight and the response measured as mean discharge frequency over the 1 min period. A recovery period of 1 min was allowed between the applications of each tension stimulus.

mechanosensitivity was re-determined to ensure penetration of all layers of the gastric tissue by adiponectin. This procedure was repeated for higher doses of adiponectin (30 and 100 pM). Time control experiments were utilized to ensure that no significant change in mechanosensitivity occurred over a comparable duration independent of addition of adiponectin.

2.11. Effect of adiponectin on the mechanosensitivity of gastric vagal afferents

2.13. Statistical analysis

Following the establishment of the baseline mechanosensitivity of GVAs, the effect of adiponectin on the mechanosensitivity was determined. Adiponectin (10 pM), at a concentration previously reported in the literature [21], was added to the Krebs, superfused into the organ bath and allowed to equilibrate for 20 min before

2.12. Drugs Adiponectin (10 μM; Sigma, Castel Hill, Australia) was kept frozen (−80 °C) and used freshly diluted for each experiment.

All data in graphs are expressed as mean ± SEM with N = the number of individual animals used. Vagal afferent stimulus–response curves and weight change were analyzed using two-way analysis of variance (two-way ANOVA) and Bonferroni post hoc tests. The effect of diet on the modulatory action of adiponectin on responses to mechanical stimulation was determined by assessing the response to either stroking

Fig. 1. Parameters of high fat diet fed mice. Mice fed a high fat diet (HFD) gained more weight (A) and had greater adiposity (B) by the end of the 12 week diet period compared to the SLD mice. Mice fed a HFD ad libitum had increased blood glucose concentrations (C). There was no difference in the plasma level of insulin (D) or adiponectin (E) between HFD and SLD fed mice. HFD mice also exhibited delayed gastric emptying (F). A: **P b 0.01, ***P b 0.001 vs. SLD two-way ANOVA Bonferroni post-hoc test. B–F: *P b 0.05, **P b 0.01, ***P b 0.001 vs. SLD unpaired t-test.

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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Fig. 2. Diet-induced changes in adiponectin receptor expression in nodose ganglia (NDG). There was no difference in the abundance of either adiponectin receptor 1 (ADIPOR1) or 2 (ADIPOR2) in the whole NDG of SLD or HFD mice (A). ADIPOR1 was found to be more prevalent in the mucosal vagal cell bodies than muscular, but this was abrogated in HFD mice (B, ###P b 0.001 SLD muscular vs. SLD mucosal, **P b 0.01 SLD mucosal, HFD mucosal unpaired t-test). ADIPOR2 mRNA was found to be more prevalent in the mucosal cell bodies in SLD mice (C, #P b 0.05 SLD mucosal vs. SLD muscular unpaired t-test).

(200 mg von Frey hair; mucosal receptors) or tension (3 g; tension receptors) at different concentrations of adiponectin (10, 30 and 100 pM). Significant difference between diets and adiponectin concentration was assessed using two-way ANOVA to determine if diet caused a change in adiponectin effect. mRNA levels, fat mass, blood glucose, plasma insulin/adiponectin and gastric emptying rate were analyzed using unpaired t-tests. Significance was defined at P b 0.05.

3. Results 3.1. Effect of long-term alterations in diet on body composition HFD mice (N = 16) gained a greater amount of weight than the SLD mice (N = 16) over the 12 week diet period (11.04 ± 0.98 g vs. 4.78 ± 0.15 g respectively, P b 0.001; 2-way ANOVA, diet × time interaction,

Fig. 3. The stomach is a source of adiponectin. Adiponectin mRNA was detected in the gastric mucosa of both SLD and HFD mice. Protein expression was confirmed by the presence of adiponectin immunoreactive cells (white arrows) in gastric sections from SLD mice Bi–Fi (glandular region of antrum). 5-HT (Bii), gastrin (Cii), gastric intrinsic factor (GIF, Dii), histamine (Eii) and somatostatin (Fii) were detected in discrete cells within the glandular region of the stomach (arrow heads). Adiponectin was found to be co-localized (dashed arrows) with 5-HT (Biii), gastrin (Ciii), GIF (Diii) and histamine (Eiii), but not with somatostatin (Fiii). Negative control showed no adiponectin or cell marker immunopositive cells (Gi–iii). 20× magnification, scale bars = 20 μm.

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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Fig. 1A). Gonadal fat pad mass was significantly higher in HFD mice compared to SLD mice (1.41 ± 0.2 g vs. 0.23 ± 0.02 g respectively; N = 16: P b 0.001; unpaired t-test, Fig. 1B). Ad-libitum fed blood glucose levels were also significantly higher in HFD mice compared to SLD mice (12.07 ± 0.38 mM vs.9.4 ± 0.35 mM respectively; N = 16: P b 0.01; unpaired t-test, Fig. 1C). Plasma insulin was similar in both SLD and HFD mice (N = 12/diet, SLD 0.60 ± 0.02 ng mL− 1 vs. HFD 0.67 ± 0.03 ng mL−1, P N 0.05, unpaired t-test, Fig. 1D) as was plasma adiponectin (n = 12/diet, SLD 3.43 ± 0.12 μg mL− 1 vs. HFD 3.80 ± 0.25 μg mL− 1, P N 0.05, unpaired t-test, Fig. 1E). HFD feeding slowed the rate of gastric emptying as determined by an increased gastric emptying half time (N = 12/diet, P b 0.05, unpaired t-test, Fig. 1F).

mice (P N 0.05, unpaired t-test, Fig. 3A). The presence of adiponectin protein in the gastric mucosa was confirmed by adiponectin immunopositive cells being present in gastric antrum sections (Fig. 3Bi–Fi) but not in sections where primary antibody was omitted (Fig. 3G). Adiponectin cells were found to be co-localized with 5-HT (Fig. 3Biii), GIF (Fig. 3Ciii), gastrin (Fig. 3Diii) and histamine (Fig. 3Eiii) indicating that adiponectin is produced in EC cells, chief cells, G-cells and ECL cells respectively. There was no adiponectin co-localized with somatostatin (Fig. 3Fiii) indicating that adiponectin is not found in Dcells. 3.4. Effect of long-term alterations in diet on gastric vagal afferent response to adiponectin

3.2. Adiponectin receptor expression in vagal afferent pathways qRT-PCR was used to determine the expression of adiponectin receptors in vagal afferent neurons in the NDG, illustrated in Fig. 2. No significant difference was observed between SLD or HFD mice for either ADIPOR1 or ADIPOR2 expression (N = 6/diet, P N 0.05 SLD vs. HFD, unpaired t-test, Fig. 2A). There was no difference in the CT values of βtubulin III between the SLD and HFD mice (data not shown). When gastric mucosal and muscular neuron cell bodies were isolated, expression of ADIPOR1 mRNA was reduced in the mucosal cell bodies of the HFD mice (P b 0.01 SLD vs. HFD, unpaired t-test, Fig. 2B). There was no diet effect on the abundance of ADIPOR2 found in either muscular or mucosal neuron cell bodies (Fig. 2C). 3.3. Adiponectin expression in the gastric mucosal Adiponectin mRNA was detected in the gastric mucosa of both SLD (N = 10) and HFD (N = 9) mice. There was no significant difference in the relative levels of adiponectin mRNA between the two groups of

3.4.1. Mucosal receptors There was no difference in the baseline response of mucosal receptors to mucosal stroking with calibrated von Frey hairs (10–1000 mg) between SLD and HFD mice (N = 16/diet, P N 0.05, 2-way ANOVA, diet effect, Fig. 4A). Adiponectin (10–100 pM) had no effect on the mechanosensitivity of mucosal receptors from SLD mice (N = 16, P N 0.05 vs. control, 2-way ANOVA, adiponectin effect, Fig. 4B). In HFD mice adiponectin (10–100 pM) potentiated the response of mucosal receptors to stroking (N = 8, P b 0.05 vs. control, 2-way ANOVA, adiponectin effect, Fig. 4C). The potentiating ability of adiponectin (10–100 pM) was significantly different between the SLD and HFD mice (P b 0.05 SLD vs. HFD, 2-way ANOVA diet effect, Fig. 4D). 3.4.2. Tension receptors As previously observed the response of tension sensitive afferents to stretch was dampened in HFD mice (N = 16/diet, P b 0.001, SLD vs. HFD, 2-way ANOVA, diet effect, Fig. 5A). Adiponectin (30–100 pM) inhibited the mechanosensitivity of tension receptors in SLD mice (N = 11,

Fig. 4. High fat diet feeding induced a potentiating effect of adiponectin on gastric mucosal receptor mechanosensitivity. There was no difference in the baseline mechanosensitivity of mucosal receptors between the SLD and HFD mice (A). (B and C) The responses of gastric mucosal receptors to mucosal stroking with calibrated von Frey hairs (10–1000 mg) in the absence (●,○) and presence of adiponectin (10 (■,□), 30 (▲, Δ) and 100 pM (♦,◊) from mice fed a SLD (B—filled symbols) or a HFD (C—open symbols). Adiponectin significantly increased the mechanical response of mucosal receptors from HFD mice (**P b 0.01, ***P b 0.001 vs. ○, adiponectin effect, two-way ANOVA). Diet significantly enhanced the potentiating action of adiponectin to the response of mucosal stroking with a 200 mg von Frey hair (D, *P b 0.05; diet effect, two-way ANOVA).

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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30 pM, P b 0.05, 100 pM P b 0.001 vs. control, 2-way ANOVA, adiponectin effect, Fig. 5B). Adiponectin (10–100 pM) also reduced the response of tension receptors to stretch in HFD mice (N = 9, P b 0.001 vs. control, 2-way ANOVA, adiponectin effect, Fig. 5C). There was no difference in the degree of inhibition caused by adiponectin (10–100 pM) on the response to 3 g stretch between the SLD and HFD mice (P N 0.05 SLD vs. HFD, 2-way ANOVA diet effect, Fig. 5D). 4. Discussion We have shown that the NDG and specific GVAs express adiponectin receptors, adiponectin is expressed in specific gastric cells and that adiponectin inhibits tension sensitive GVA mechanosensitivity. In HFD induced obesity the inhibitory effect of adiponectin on tension sensitive GVAs is maintained. However, adiponectin potentiates the mechanosensitivity of mucosal GVAs; an effect not observed in lean mice. Plasma adiponectin did not decrease in the HFD fed mice, contrary to previous findings in human and rodent obesity [15,34–36]. Such a discrepancy may be caused by subtle differences in the state of adipocyte growth (i.e. balance between hypertrophy and hyperplasia), with a greater number of smaller adipocytes linked with increased release of adiponectin [37]. Adiponectin has also been reported to have an active role in adipocyte differentiation in vitro, where hormonally induced adipocyte differentiation resulted in a 100-fold increased expression of adiponectin [14]. The absence of a decreased in adiponectin may reflect the dynamically increasing weights of the HFD mice. As we have previously shown, long-term HFD feeding attenuated the mechanosensitivity of gastric tension receptors [13] a consequence of which is likely to be delayed satiety signaling leading to higher food intake. This is consistent with the finding that obese humans have been shown to possess increased gastric capacity [38,39] and a reduced

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ability to detect gastric distension [38]. The mechanism responsible for this reduced mechanosensitivity remains to be determined, but it likely involves altered ion channel expression or function leading to changes in neuron excitability with similar results observed in jejunal afferents from diet induced obese mice [40,41]. There was a concentration-dependent effect of adiponectin to reduce the mechanosensitivity of tension receptors to circular stretch that was similar in the SLD and HFD mice. The action of adiponectin on tension receptors may constitute an orexigenic effect, decreasing the satiety signal in response to distension and promoting feeding [42–44]. Acute fasting for 12 h has been reported to increase circulating adiponectin concentrations in mice [22], which may allow for increased food intake when food access is restored. This may be a beneficial evolutionary adaption enabling maximal feeding when food availability is sporadic and unreliable. Such an effect would be detrimental in obesity when there are excessive energy stores available. The potential orexigenic action of adiponectin on GVA tension receptors is consistent with the observation of Kobuta et al. that food intake increased during the 6 h following adiponectin administration [22]. Although Kobuta et al. were investigating the central action of adiponectin and attributed the effect to a central pathway, the protocol utilized a jugular vein infusion and relied on the increased peripherally circulating adiponectin to be transported across the blood–brain-barrier to achieve an increased central concentration and elicit an effect. However, when infused ICV adiponectin has been shown to have an anorexigenic effect [20,21] in the absence of increased peripheral adiponectin concentration [20]. Different peripheral and central effects may explain these discrepant findings and explanation that would be consistent with the inhibitory effect of adiponectin on GVAs although this still needs to be conclusively determined. The mechanosensitivity of mucosal receptors was not altered by HFD feeding, consistent with our previous studies [13,45]. Mucosal

Fig. 5. Tension receptor response to adiponectin is unaffected by HFD feeding. There was a substantial reduction in the mechanical sensitivity of tension receptors in HFD mice compared to the SLD mice (A, ***P b 0.001, diet effect, two-way ANOVA). (B and C) The responses of gastric tension receptors to tension (1–5 g) in the absence (●,○) and presence of adiponectin (10 (■,□), 30 (▲,Δ) and 100 pM (♦,◊) from mice fed a SLD (B—filled symbols) or a HFD (C—open symbols) (*P b 0.05, **P b 0.01, ***P b 0.001 vs. ● or ○, adiponectin effect, two-way ANOVA). Diet had no effect of the degree of inhibition adiponectin caused on tension receptors (D).

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

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receptors have been proposed to sense particle size within the stomach contents and therefore indicate the progress of digestion [46]. Increased particle size of stomach contents is a factor that has been shown to slow gastric emptying [7]. The lack of effect of adiponectin on mucosal receptors in SLD mice suggests that adiponectin would not regulate gastric emptying in SLD mice through mucosal GVA pathways. Conversely, adiponectin potentiated the response of mucosal receptor mechanosensitivity in HFD mice suggesting that in these mice adiponectin would lead to a heightened response to food particles in the stomach possibly delaying gastric emptying and further work is required to confirm this. Although the rate of gastric emptying is generally considered to be increased in obesity, that is not always the case [47]. The HFD used to induce obesity in the current study could also induce small intestinal gut peptide inhibition of gastric motility [48,49] again delaying gastric emptying. Thus further investigation is required to determine the mechanisms controlling gastric emptying. Adiponectin is not the first adipokine to be found to be produced in the stomach. Apelin [50] and leptin [25] have also been detected in discrete gastric cells. The co-localization of adiponectin with 5-HT, gastrin and histamine suggests, at least potentially that stimuli, such as nutrient exposure, which trigger the release of 5-HT, gastrin and histamine may also release adiponectin [51,52] but this remains to be determined The presence of adiponectin in chief cells also suggests that adiponectin may be secreted into the gastric lumen potentially acting on receptors along the gastrointestinal epithelium [53]. However, this requires specific further investigation. In the current study the plasma concentration of adiponectin was significantly higher (≈110 nM) than the concentration of adiponectin used (10–100 pM) to modulate GVA mechanosensitivity in the isolated organ bath preparation. In humans, the concentration of adiponectin in gastric tissue is similar to plasma levels [54]. Therefore, with plasma levels exceeding the concentration of adiponectin observed to affect vagal afferent mechanosensitivity, the role that adiponectin from gastric cells plays in modulating vagal afferent mechanosensitivity is unclear. Additionally, adiponectin exists in multiple isoforms in the circulation [55] and the assay utilized detects all full length variants. Thus, it remains to be determined whether the gastric source of adiponectin preferentially produces a particular isoform which may have enhanced local activity [55]. The existence of the adiponectin receptors, ADIPOR1 and ADIPOR2 in both the gastric mucosal and muscular vagal afferents provides support for a stomach–vagal–brain pathway for adiponectin signaling. Given that there was a gain of function in the mucosal receptors of HFD mice we would expect an increase in the expression of mRNA in this subpopulation of neurons. Neither the levels of ADIPOR1 or ADIPOR2 showed such a change. However, mRNA changes have been shown to not always accurately represent protein levels [56] or GVA responses to peptide modulators [45]. Determining the receptor subtype involved in the GVA modulatory effect of adiponectin was not possible due to a lack of selective antagonists. However some insight can be gained from examining the phenotypes of adiponectin receptor knockout mice. ADIPOR1 knockout mice have increased body weight, whereas, ADIPOR2 knockout mice have decreased body weight compared to controls [57]. If we speculate that the knockout phenotype is due, at least in part, to disruption of adiponectin effects on GVAs then ADIPOR2 is likely the receptor subtype which adiponectin activates on GVAs. However, this requires more detailed investigation. 5. Conclusion In conclusion, long-term HFD feeding reduced the basal mechanosensitivity of GVA tension receptors, a consequence of which may be decreased satiety and increased food intake. Adiponectin further reduced mechanosensitivity of gastric tension receptors in both

SLD and HFD mice. Adiponectin also acquired the ability to potentiate gastric mucosal receptors. The change in response to adiponectin could not be explained by altered mRNA expression of adiponectin receptors in vagal afferents. Thus, whilst we have reported a distinct ability for adiponectin to modulate GVAs it remains to be conclusively determined what the functional consequence of this modulation is. Acknowledgments SJK is funded by an NH&MRC Peter Doherty Fellowship #1091586. This project was supported by NH&RMC project grant #1023972. The NH&MRC had no involvement in the design, execution or analysis of the study. References [1] G.A. Bray, Afferent signals regulating food intake, Proc. Nutr. Soc. 59 (2000) 373–384. [2] D.E. Cummings, J. Overduin, Gastrointestinal regulation of food intake, J. Clin. Invest. 117 (2007) 13–23. [3] A.J. Page, C.M. Martin, L.A. Blackshaw, Vagal mechanoreceptors and chemoreceptors in mouse stomach and esophagus, J. Neurophysiol. 87 (2002) 2095–2103. [4] C. de Graaf, W.A. Blom, P.A. Smeets, A. Stafleu, H.F. Hendriks, Biomarkers of satiation and satiety, Am. J. Clin. Nutr. 79 (2004) 946–961. [5] P.L.R. Andrews, G.J. Sanger, Abdominal vagal afferent neurones: an important target for the treatment of gastrointestinal dysfunction, Curr. Opin. Pharmacol. 2 (2002) 650–656. [6] Becker, J. M., Kelly, K. A. Antral control of canine gastric emptying of solids. Am. J. Physiol. Gastrointest. Liver Physiol. 1983,8:334–8. [7] A. McIntyre, R.M. Vincent, A.C. Perkins, R.C. Spiller, Effect of bran, ispaghula, and inert plastic particles on gastric emptying and small bowel transit in humans: the role of physical factors, Gut 40 (1997) 223–227. [8] C. Tuleu, C. Andrieux, P. Boy, J.C. Chaumeil, Gastrointestinal transit of pellets in rats: effect of size and density, Int. J. Pharm. 180 (1999) 123–131. [9] H.E. Raybould, Y. Tache, Cholecystokinin inhibits gastric motility and emptying via a capsaicin-sensitive vagal pathway in rats, Am. J. Physiol. Gastrointest. Liver Physiol. 255 (1988) G242–G246. [10] K. Toshinai, M.S. Mondal, M. Nakazato, Y. Date, N. Murakami, M. Kojima, et al., Upregulation of ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration, Biochem. Biophys. Res. Commun. 281 (2001) 1220–1225. [11] G.J. Dockray, G. Burdyga, Plasticity in vagal afferent neurones during feeding and fasting: mechanisms and significance*, Acta Physiol. 201 (2011) 313–321. [12] S.J. Kentish, A.J. Page, Plasticity of gastro-intestinal vagal afferent endings, Physiol. Behav. 136 (2014) 170–178. [13] S. Kentish, H. Li, L.K. Philp, T.A. O'Donnell, N.J. Isaacs, R.L. Young, et al., Diet-induced adaptation of vagal afferent function, J. Physiol. 590 (2012) 209–221. [14] P.E. Scherer, S. Williams, M. Fogliano, G. Baldini, H.F. Lodish, A novel serum protein similar to C1q, produced exclusively in adipocytes, The Journal of Biological Chemistry 270 (1995) 26746–26749. [15] T. Yamauchi, J. Kamon, H. Waki, Y. Terauchi, N. Kubota, K. Hara, et al., The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity, Nat. Med. 7 (2001) 941–946. [16] A.H. Berg, T.P. Combs, X. Du, M. Brownlee, P.E. Scherer, The adipocyte-secreted protein Acrp30 enhances hepatic insulin action, Nat. Med. 7 (2001) 947–953. [17] J. Fruebis, T.S. Tsao, S. Javorschi, D. Ebbets-Reed, M.R. Erickson, F.T. Yen, et al., Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 2005–2010. [18] N. Ouchi, S. Kihara, Y. Arita, Y. Okamoto, K. Maeda, H. Kuriyama, et al., Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway, Circulation 102 (2000) 1296–1301. [19] N. Ouchi, S. Kihara, Y. Arita, K. Maeda, H. Kuriyama, Y. Okamoto, et al., Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin, Circulation 100 (1999) 2473–2476. [20] Y. Qi, N. Takahashi, S.M. Hileman, H.R. Patel, A.H. Berg, U.B. Pajvani, et al., Adiponectin acts in the brain to decrease body weight, Nat. Med. 10 (2004) 524–529. [21] A. Coope, M. Milanski, E.P. Araujo, M. Tambascia, M.J. Saad, B. Geloneze, et al., AdipoR1 mediates the anorexigenic and insulin/leptin-like actions of adiponectin in the hypothalamus, FEBS Lett. 582 (2008) 1471–1476. [22] N. Kubota, W. Yano, T. Kubota, T. Yamauchi, S. Itoh, H. Kumagai, et al., Adiponectin stimulates AMP-activated protein kinase in the hypothalamus and increases food intake, Cell Metab. 6 (2007) 55–68. [23] S. Shklyaev, G. Aslanidi, M. Tennant, V. Prima, E. Kohlbrenner, V. Kroutov, et al., Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 14217–14222. [24] N. Maeda, I. Shimomura, K. Kishida, H. Nishizawa, M. Matsuda, H. Nagaretani, et al., Diet-induced insulin resistance in mice lacking adiponectin/ACRP30, Nat. Med. 8 (2002) 731–737. [25] A. Bado, S. Levasseur, S. Attoub, S. Kermorgant, J. Laigneau, M.I. Bortoluzzi, et al., The stomach is a source of leptin, Nature 394 (1998) 790–793.

Please cite this article as: S.J. Kentish, et al., High fat diet induced changes in gastric vagal afferent response to adiponectin, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.06.016

S.J. Kentish et al. / Physiology & Behavior xxx (2015) xxx–xxx [26] T. Yamauchi, J. Kamon, Y. Ito, A. Tsuchida, T. Yokomizo, S. Kita, et al., Cloning of adiponectin receptors that mediate antidiabetic metabolic effects, Nature 423 (2003) 762–769. [27] M. Bjursell, A. Ahnmark, M. Bohlooly-Y, L. William-Olsson, M. Rhedin, X.-R. Peng, et al., Opposing effects of adiponectin receptors 1 and 2 on energy metabolism, Diabetes 56 (2007) 583–593. [28] A. Tsuchida, T. Yamauchi, Y. Ito, Y. Hada, T. Maki, S. Takekawa, et al., Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity, The Journal of Biological Chemistry 279 (2004) 30817–30822. [29] S.J. Kentish, G.A. Wittert, L.A. Blackshaw, A.J. Page, A chronic high fat diet alters the homologous and heterologous control of appetite regulating peptide receptor expression, Peptides 46 (2013) 150–158. [30] E.L. Symonds, R.N. Butler, T.I. Omari, Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test, Clin. Exp. Pharmacol. Physiol. 27 (2000) 671–675. [31] S.J. Kentish, T.A. O'Donnell, C.L. Frisby, H. Li, G.A. Wittert, A.J. Page, Altered gastric vagal mechanosensitivity in diet-induced obesity persists on return to normal chow and is accompanied by increased food intake, Int. J. Obes. 38 (2014) 636–642 (2005). [32] H. Li, S.J. Kentish, S. Kritas, R.L. Young, N.J. Isaacs, T.A. O'Donnell, et al., Modulation of murine gastric vagal afferent mechanosensitivity by neuropeptide W, Acta Physiol (Oxf.) 209 (2013) 179–191. [33] M.W. Pfaffl, G.W. Horgan, L. Dempfle, Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR, Nucleic Acids Res. 30 (2002) e36. [34] Y. Arita, S. Kihara, N. Ouchi, M. Takahashi, K. Maeda, J. Miyagawa, et al., Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity, Biochem. Biophys. Res. Commun. 257 (1999) 79–83. [35] A. Gavrila, J.L. Chan, N. Yiannakouris, M. Kontogianni, L.C. Miller, C. Orlova, et al., Serum adiponectin levels are inversely associated with overall and central fat distribution but are not directly regulated by acute fasting or leptin administration in humans: cross-sectional and interventional studies, The Journal of Clinical Endocrinology and Metabolism 88 (2003) 4823–4831. [36] P.A. Kern, G.B. Di Gregorio, T. Lu, N. Rassouli, G. Ranganathan, Adiponectin expression from human adipose tissue: relation to obesity, insulin resistance, and tumor necrosis factor-alpha expression, Diabetes 52 (2003) 1779–1785. [37] R. Drolet, C. Belanger, M. Fortier, C. Huot, J. Mailloux, D. Legare, et al., Fat depotspecific impact of visceral obesity on adipocyte adiponectin release in women, Obesity (Silver Spring, Md.) 17 (2009) 424–430. [38] A. Geliebter, Gastric distension and gastric capacity in relation to food intake in humans, Physiol. Behav. 44 (1988) 665–668. [39] D.Y. Kim, M. Camilleri, J.A. Murray, D.A. Stephens, J.A. Levine, D.D. Burton, Is there a role for gastric accommodation and satiety in asymptomatic obese people? Obes. Res. 9 (2001) 655–661. [40] D.M. Daly, S.J. Park, W.C. Valinsky, M.J. Beyak, Impaired intestinal afferent nerve satiety signalling and vagal afferent excitability in diet induced obesity in the mouse, J. Physiol. 589 (2011) 2857–2870. [41] A.J. Page, E. Symonds, M. Peiris, L.A. Blackshaw, R.L. Young, Peripheral neural targets in obesity, Br. J. Pharmacol. 166 (2012) 1537–1558.

9

[42] H.R. Berthoud, W.L. Neuhuber, Functional and chemical anatomy of the afferent vagal system, Auton. Neurosci. 85 (2000) 1–17. [43] H.-R. Berthoud, The vagus nerve, food intake and obesity, Regul. Pept. 149 (2008) 15–25. [44] R.J. Phillips, T.L. Powley, Tension and stretch receptors in gastrointestinal smooth muscle: re-evaluating vagal mechanoreceptor electrophysiology, Brain Res. Brain Res. Rev. 34 (2000) 1–26. [45] S.J. Kentish, T.A. O'Donnell, N.J. Isaacs, R.L. Young, H. Li, A.M. Harrington, et al., Gastric vagal afferent modulation by leptin is influenced by food intake status, J. Physiol. 591 (2013) 1921–1934. [46] J.M. Becker, K.A. Kelly, Antral control of canine gastric emptying of solids, The American Journal of Physiology 245 (1983) G334–G338. [47] M. Al Mushref, S. Srinivasan, Effect of high fat-diet and obesity on gastrointestinal motility, Annals of Translational Medicine 1 (2013) 14. [48] R. Heddle, P.J. Collins, J. Dent, M. Horowitz, N.W. Read, B. Chatterton, et al., Motor mechanisms associated with slowing of the gastric emptying of a solid meal by an intraduodenal lipid infusion, J. Gastroenterol. Hepatol. 4 (1989) 437–447. [49] T.J. Little, C. Feinle-Bisset, Effects of dietary fat on appetite and energy intake in health and obesity—oral and gastrointestinal sensory contributions, Physiol. Behav. 104 (2011) 613–620. [50] E. Susaki, G. Wang, G. Cao, H.Q. Wang, E.W. Englander, G.H. Greeley Jr., Apelin cells in the rat stomach, Regul. Pept. 129 (2005) 37–41. [51] M.G. Korman, C. Soveny, J. Hansky, Effect of food on serum gastrin evaluated by radioimmunoassay, Gut 12 (1971) 619–624. [52] M. Kidd, I.M. Modlin, B.I. Gustafsson, I. Drozdov, O. Hauso, R. Pfragner, Luminal regulation of normal and neoplastic human EC cell serotonin release is mediated by bile salts, amines, tastants, and olfactants, Am. J. Physiol. Gastrointest. Liver Physiol. 295 (2008) G260–G272. [53] K. Yoneda, A. Tomimoto, H. Endo, H. Iida, M. Sugiyama, H. Takahashi, et al., Expression of adiponectin receptors, AdipoR1 and AdipoR2, in normal colon epithelium and colon cancer tissue, Oncol. Rep. 20 (2008) 479–483. [54] M. Seker, A. Bilici, D.O. Guler, B.B. Ustaalioglu, H. Gozu, F.Y. Erkal, et al., Tissue and serum adiponectin levels in patients with gastric cancer: are there any correlations between adiponectin levels and histopathological variables? Hepato-Gastroenterology 58 (2011) 1841–1846. [55] K. Dadson, Y. Liu, G. Sweeney, Adiponectin action: a combination of endocrine and autocrine/paracrine effects, Front. Endocrinol. 2 (2011). [56] F.M. Shebl, L.A. Pinto, A. Garcia-Pineres, R. Lempicki, M. Williams, C. Harro, et al., Comparison of mRNA and protein measures of cytokines following vaccination with human papillomavirus-16L1 virus-like particles, Cancer Epidemiology, Biomarkers & Prevention: A Publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 19 (2010) 978–981. [57] J.L. Parker-Duffen, K. Nakamura, M. Silver, M.A. Zuriaga, S. MacLauchlan, T.R. Aprahamian, et al., Divergent roles for adiponectin receptor 1 (AdipoR1) and AdipoR2 in mediating revascularization and metabolic dysfunction in vivo, The Journal of Biological Chemistry 289 (2014) 16200–16213.

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