Peptides 31 (2010) 1055–1061
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Peripheral administration of pancreatic polypeptide inhibits components of food-intake behavior in dogs Helena Åkerberg a,∗ , Bengt Meyerson a , Marie Sallander b , Anne-Sofie Lagerstedt b , Åke Hedhammar b , Dan Larhammar a a b
Department of Neuroscience, Uppsala University, Box 593, SE-75124 Uppsala, Sweden Department of Clinical Sciences, Swedish University of Agricultural Sciences, Box 7070, SE-75007 Uppsala, Sweden
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Article history: Received 28 February 2010 Received in revised form 15 March 2010 Accepted 15 March 2010 Available online 23 March 2010 Keywords: Pancreatic polypeptide Appetite Satiety Feeding Food intake Dog
a b s t r a c t Pancreatic polypeptide (PP) belongs to the neuropeptide Y (NPY) family of peptides and is released from pancreatic F cells postprandially. PP functions as a peptide hormone and has been associated with decreased food intake in humans and rodents. Our study describes the effects of PP on feeding behavior in dogs, whose mammalian order (Carnivora) is more distantly related to primates and rodents than these are to each other. Furthermore, obesity is becoming more prevalent in dogs which makes knowledge about their appetite regulation highly relevant. Repeated peripheral administration of physiological doses of PP (three injections of 30 pmol/kg each that were administered within 30 min) to six male beagle dogs prolonged the median time spent eating three servings of food by 19% but resulted in no reduction of food intake. In addition, PP decreased the duration of food-seeking behavior after the first serving by 71%. Thus, a physiological dose of PP seems to decrease both the appetitive and the consummatory drive in dogs. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Food intake is one of the most crucial behaviors for survival in the vast majority of species and is an important component in disorders like obesity and anorexia nervosa. Species-specific behavioral strategies for food intake have evolved in line with ecological niches and show tremendous diversity. Whereas free-ranging cattle may graze for more than 11 h a day [21] the Burmese python eats as rarely as once a month and may fast for periods as long as 18 months [30]. Extensive research has identified a large number of neurotransmitters and hormones in the regulation of appetite and satiety, almost exclusively by studying rats and mice and to some extent humans [6,25]. One of the first compounds found to have prominent effects on appetite was neuropeptide Y (NPY) [9,31] and it is still considered to be the most potent endogenous stimulator of feeding [19]. NPY belongs to a family of peptides that also includes peptide YY (PYY)
∗ Corresponding author at: Department of Neuroscience, Unit of Pharmacology, Uppsala University, Box 593, SE-75124 Uppsala, Sweden. Tel.: +46 184714173; fax: +46 18511540. E-mail addresses:
[email protected] (H. Åkerberg),
[email protected] (B. Meyerson),
[email protected] (M. Sallander), anne-sofi
[email protected] (A.-S. Lagerstedt),
[email protected] (Å. Hedhammar),
[email protected] (D. Larhammar). 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.03.019
released from intestinal endocrine cells and pancreatic polypeptide (PP) from pancreatic F cells in mammals. Despite their shared evolutionary history with NPY, both PP and an endogenous truncated version of PYY, PYY3–36, have been found to decrease food intake in mice and rats [2,8] and humans [4,5,18]. The appetite-decreasing effect of PYY3–36 was questioned when several studies found no effect [35] but has now been confirmed in independent studies in both rats [7] and humans [11], albeit at supraphysiological doses in the latter. Considering the complexity of food-intake regulation as well as the complexity of feeding behavior, it is of interest to collect information from other species than human and rodents. The NPY system in dogs has so far been investigated mostly in terms of secretion of PYY and PP [12,13,16,24,27,33]. In the same way as in humans, dog PYY and PP are released postprandially from endocrine cells in the pancreas and intestine, respectively, and exert their effects via the Y2 and Y4 receptor, respectively. Studies of food intake after central administration of NPY in dogs have shown somewhat contradictory results. One study found no effect on appetite [17] while a later study found a stimulating effect on sham-feeding [13]. The effect on appetite of PYY3–36 has not been investigated in dogs to our knowledge. The only published study regarding effect of PP on food intake in dogs found no decrease after peripheral administration of a single high dose of PP [23]. A study of metabolic effects after peripheral infusion of physiological concentrations of PP indicated a weight-reducing
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effect, although it was unclear whether this was a consequence of decreased food intake or of metabolic factors [32]. If PP has a decreasing effect on food intake, this effect would probably be possible to detect by analyzing food-intake behavior. The aim of the present study was therefore to investigate the effect on both food intake and feeding behavior after peripheral administration of physiological doses of PP to beagle dogs. 2. Materials and methods The study was conducted at the Department of Clinical Sciences, Swedish University of Agricultural Sciences in Uppsala and consisted of two pilot studies and one main study. The experimental design was approved by the Ethical Committee in Uppsala. 2.1. Study subjects The studies involved eight dogs, born in and housed at the animal facility. The dogs were kept primarily for teaching of veterinary students and were used to a limited extent also in research but had not been used for that purpose during the preceding two weeks. Body condition score (BCS) was assessed for all dogs through a standardized validated physical examination [22] with a scale ranging from 1 to 9. A BCS of 4–6 indicated a normal body constitution, 1–3 indicated different degrees of underweight and 7–9 different degrees of overweight. Pilot study 1 involved two male beagles and pilot study 2 two females (one pure breed beagle and one mixed breed, beagle and ceskyterrier). The subjects in pilot study 1 were 7 and 8 years old with a body weight (BW) of 19.5 kg and 15.4 kg and a BCS of 6 and 5, respectively. In pilot study 2 the subjects were 2 and 8 years old, BW 11.5 kg and 13.2 kg and had a BCS score of 6 and 7, respectively. The two females in pilot study 2 were in anestrus, defined as the period of ovarian inactivity between two cycles. The main study was performed with six male pure breed beagles 2–9 years old, with a BW range of 11.5–19.5 kg and a BCS of 4–7. Two of the subjects in the main study had participated in pilot study 1. The group of dogs in the main study included two pair of full siblings. 2.2. Housing and routines The dogs were housed indoors and spent about 8 h daily in an outdoor exercise yard. At least once a week the dogs were walked in the neighborhood. The animal facility was closed to unauthorized people. There were six rooms in the dog facility with two to four dogs living in each room (23 m2 ) in constant constellations under constant and controlled conditions. The facility had daylight and the temperature regulated to 18–20 ◦ C. All rooms contained individual cages (1.6–1.9 m2 ) which were constantly open except during the feeding sessions. The dogs had daily access to blankets, a small cabin, toys and chewing bones. Time points for feeding, cleaning and for exercise yard were standardized in the daily routines. The dogs were fed twice a day and had free access to water. The dogs were fed with a dry food with a nutrient content corresponding to 26% protein, 15% fat, 2.5% fiber, 8.5% water and 7.5% minerals as fed which was allowed to swell in fresh water prior to the serving. 2.3. Factors changed for habituation and standardization In order to habituate the dogs to the trays used in the study, the food bowls were served on trays one week before pilot studies 1 and 2 and the main study. The trays were necessary to measure any spilled food during feeding. The dogs were used to individual feeding. At least one week before the studies the dogs were fed in the same cage every day.
2.4. Procedures in order to control for health and standardization All dogs were fasted with free access to tap water from 16 pm the day before the test day. In order to monitor any constipating effect of the peptides [2], the dogs were taken for a short walk after the feeding session in pilot study 2 and the main study where the incidence of defecation was registered. Furthermore, the time for walks and the time spent in the exercise yard were registered for each subject during the week before the start of the study. This was done to monitor for influences on appetite from extensive physical activities. Also, the BW and body constitution of the dogs was assessed before and after their participation in the study in order to monitor the BW during the test but also to enable analyses of correlation of body constitution with different parameters. In case a dog showed any side effects, the treatment was immediately terminated and the subject was examined by a veterinarian. 2.5. Injections Both saline and peptide solution were administered via a permanent intravenous blood collection needle in one foreleg. The injection volume was at maximum 5 mL per injection. 2.6. Blood sampling and radioimmunoassay A blood collection needle was inserted into a vein in one of the forelegs. Blood was collected in a lavender vacutainer with EDTA. In pilot study 1, a total of six blood samples of 2 mL were drawn per dog and test day. In pilot study 2 and in the main study a total of four samples of 4 mL per dog and test day were drawn. The needle was rinsed with saline before blood was collected. To prevent the dogs from interfering with the permanent blood collection needle they carried a plastic collar during the sampling period in pilot study 1. The collars were removed during feeding. In pilot study 2 and the main study the two last blood samples were drawn using a single use needle. Thereby the dogs had to wear the collar only between the first two blood samplings, thus minimizing stress caused by wearing the collar. Blood was transferred to new tubes pre-treated with 0.6 TIU kallikrein inhibitor units of aprotonin (GE Healthcare, Uppsala, Sweden) per mL blood. Plasma was separated immediately by centrifugation at 1600 × g for 15 min at 4 ◦ C and was transferred to new tubes and stored at −70 ◦ C until it was analyzed. Plasma concentration of PP was determined using radioimmunoassay (Phoenix Europe GmbH, Karlsruhe, Germany). The kit’s detection threshold was 55 pg/mL corresponding to 13 pmol/L, its intra-assay variation was 5–7%, and its inter-assay variation 12–15%. All samples were assayed in duplicate for pilot studies 1 and 2 and in triplicate for the main study. The assays were performed in a final volume of 1 mL for each sample. The determined concentrations of peptide were converted into pmol/L plasma. 2.7. PP Because canine PP is not commercially available human PP was used. It differs from canine PP in 2 of the 36 amino acid residues. Human PP was purchased from Bachem (Weil am Rhein, Germany) and dissolved in saline. The peptide solution was confirmed to be sterile on culture for both aerobic and anaerobic bacteria and fungi. The peptide was also negative in a quantitative limulus lysate test (
