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The threshold of amylin-induced anorexia is lower in chicks selected for low compared to high juvenile body weight
Behavioural Brain Research 208 (2010) 650–654
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
The threshold of amylin-induced anorexia is lower in chicks selected for low compared to high juvenile body weight Mark A. Cline a,∗ , Wint Nandar b , Christie Bowden a , Wendy Calchary c , Marissa L. Smith a,d , Brian Prall a , Brandon Newmyer a , J. Orion Rogers a , Paul B. Siegel d a
Department of Biology, Radford University, East Main Street, Radford, VA 24142, United States Department of Neurosurgery, Pennsylvania State University, Hershey, PA, United States Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, United States d Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States b c
a r t i c l e
i n f o
Article history: Received 1 December 2009 Received in revised form 17 December 2009 Accepted 21 December 2009 Available online 29 December 2009
a b s t r a c t Chicks that have undergone long-term selection for low body weight responded to intracerebroventricular amylin injection with reduced food intake at a dose considerably lower and with a greater magnitude suppression than those selected for high body weight. Behaviors unrelated to ingestion were not affected. These data support the thesis of correlated amylin system responses to selection for low or high body weight, with possible implications to other species. © 2009 Elsevier B.V. All rights reserved.
Keywords: Amylin Chick Hypothalamus Food intake Obesity Anorexia Behavior c-Fos
Amylin is receiving increasing attention in appetite biology. Cosecreted with insulin from pancreatic beta cells [15] in response to ingestion of a meal [2], it is well documented that amylin decreases food intake across a range of species [7,21,10]. Accordingly, because its basal concentration is higher in obese humans, it is primarily associated with the obese phenotype [16]. When centrally administered, amylin is more potent than other members of its family [22] such as calcitonin and calcitonin gene-related peptide, which also elicit anorexigenic responses [10,23]. Amylin decreases food intake in chicks when administered centrally or peripherally, and although it decreases c-Fos reactivity in the hunger-associated lateral hypothalamus, it does not affect c-Fos expression in the ventromedial hypothalamus, which is classically associated with satiety [7]. Furthermore, we demonstrated that central amylin increases transit time through the alimentary canal, anxious-like behavior, and plasma corticosterone concentration [7].
∗ Corresponding author. Tel.: +1 540 831 6431; fax: +1 540 831 5129. E-mail address: [email protected] (M.A. Cline). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.12.032
Rodent studies have also demonstrated roles for amylin in decreasing meal size [21] and frequency [24]. Diet-induced obese rats respond to amylin with decreased food intake and weight gain while increasing energy expenditure [25]. However, to our knowledge these effects have not been reported in a polygenic model of obesity, nor have the anorectic effects of amylin been studied simultaneously in animal models of anorexia and obesity. The study reported here aimed to determine if amylin differentially affects food intake and behavior in lines of chickens that have undergone long-term selection for high and low body weight and that are hypo- and hyperphagic. The chicks used in this study are the result of long-term divergent selection for low (LWS) or high (HWS) body weight at 56 days-of-age from a White Plymouth Rock founder population that consisted of crosses of 7 inbred lines and were maintained as closed populations [13,26,19]. Eggs obtained from age contemporary parents from S48 and S49 generation parental stocks were incubated in the same machine. After hatch, chicks were group-caged for 2 days, then individually in a room at 30 ± 2 ◦ C and 50 ± 5% relative humidity where they had ad libitum access to a mash diet (20% crude protein, 2685 kcal ME/kg) and tap water. The individual cages allowed visual and auditory contact with other chicks
M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654
(unless otherwise indicated). Chicks were handled twice daily to adapt to handling. All trials were conducted between 11:00 and 16:00 h using 5-day post-hatch chicks. Data in each experiment were recorded from both lines concurrently and injections were performed sequentially, LWS, HWS, LWS, HWS and so forth. Experimental procedures were performed according to the National Research Council publication, Guide for Care and Use of Laboratory Animals and were approved by the Radford University Institutional Animal Care and Use Committee. Chicks were injected using a method adapted from Davis et al. [9] that does not appear to induce physiological stress [14]. The head of the chick was briefly inserted into a restraining device that left the cranium exposed and allowed for free-hand injection. Injection coordinates were 3 mm anterior to the coronal suture, 1 mm lateral from the sagittal suture, and 2 mm deep, targeting the left lateral ventricle. Anatomical landmarks were determined visually and by palpation. Injection depth was controlled by placing a plastic tubing sheath over the needle. The needle remained at injection depth for 5 s post-injection to reduce backflow. Chicks were assigned to treatments at random. Amylin (American Peptide Co., Sunnyvale, CA, USA) was dissolved in artificial cerebrospinal fluid as a vehicle for a total injection volume of 5 L with 0.1% Evans Blue dye to facilitate injection site localization. The sequence of rat amylin injected (KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY) has 82% sequence identity to that of chicken [21]. After data collection, the chicks were decapitated and their heads sectioned coronally to determine site of injection. Any chick without dye present in the lateral ventricle system was eliminated from analysis. In Experiment 1, chicks from the S48 generation from each line were fasted for 180 min prior to injection to intensify the perception of hunger. They were randomly assigned to receive 0 (vehicle only), 128 (0.5 g), 255 (1.0 g) or 510 (2.0 g) pmol amylin by ICV injection. After injection, chicks were returned to their individual cages and given ad libitum access to both food and water, with individual food and water containers weighed (0.01 g) every 30 min for 180 min post-injection. Data were analyzed using analysis of variance (ANOVA) at each time point. The model included line, amylin dose and the line by amylin dose interaction. When the interaction was significant (P < 0.05), data were analyzed within each line for the effect of amylin dose using Tukey’s method of multiple comparisons. The LWS and HWS lines consume inherently different
651
amounts of food due to differences in body size. Therefore, food and water intake data were normalized per body weight at each time point. This conversion was made by dividing the amount of feed each chick consumed by its body weight (0.01 g) immediately prior to its ICV injection and multiplying by 100. In Experiment 2, chicks from the S49 generation from each line were kept in individual cages with auditory but not visual contact (to reduce isolation stress during behavior measurement) from 2 days post-hatch. They were randomly assigned to receive either vehicle or 255 pmol amylin, a dose that exceeded threshold in both lines in Experiment 1, by ICV injection. Following a 180 min fast they were injected and immediately placed in a 290 mm × 290 mm acrylic recording arena with food and water containers in diagonal corners. Chicks were simultaneously and automatically recorded from three angles for 30 min post-injection on DVD and data were analyzed in 10 min intervals using ANY-maze behavioral analysis software (Stoelting, Wood Dale, IL). Locomotion (m traveled), time spent (s) standing, sitting, preening, perching or in deep rest, and the number of jumps, food and exploratory pecks, drinks, defecations and escape attempts were recorded. Food pecks were defined as pecks within the food container, whereas all other pecks were considered as exploratory. Drinks were defined as the chick dipping its beak in water, then raising and extending its head to swallow. Preening was defined as trimming or dressing of down with the beak. Deep rest was defined as the eyes closed for greater than 3 s, starting 3 s after eye closure. The statistical model used was the same as that for Experiment 1. In Experiment 1, amylin reduced food intake in both lines (Fig. 1), and the line by amylin dose interaction was significant. The interaction occurred because the threshold of amylin-induced anorexia was lower for line LWS than line HWS chicks. That is, in line LWS all doses of amylin tested reduced food intake nearly 50%. In line HWS however, 128 pmol did not reduce food intake whereas 255 and 510 pmol of amylin caused only a 25% reduction in food intake. There were 12–14 chicks in the line LWS and 17–18 in HWS per amylin dose. These results demonstrate that line LWS has a lower threshold of amylin-induced anorexia than line HWS. The decreased food intake pattern in chicks from line HWS more closely resembles that of Cobb-500 chicks than that of line LWS [7] and the decreased food intake is consistent with that reported for rodents [21,10]. Therefore, selection for juvenile body weight may have favored a gain
Fig. 1. Cumulative food intake expressed as percent body weight following intracerebroventricular injection of amylin in low (LWS) and high (HWS) body weight long-term divergently selected lines of chicks (Experiment 1). Values are means ± SE; bars with different letters are different from each other within a time point (P < 0.05). n = 12–14 chicks in the line LWS and 17–18 in HWS per amylin dose.
0.3 ± 0.3 0.3 ± 0.2
0
ns 3.7 ± 2.6 0.5 ± 0.4 0 0 0.4 ± 0.3 0 ns * HWS
0.3 ± 0.3 0.3 ± 0.2
0.3 ± 0.3
14 ± 3.1 ns 37 ± 22 ns
7.7 ± 2.6 ns 4.1 ± 0.9
ns 3±2
3.7 ± 2.6 0
0.4 ± 0.3 0
0.2 ± 0.2 0.2 ± 0.2
0 0.4 ± 0.3
0 0.2 ± 0.2
304 ± 66 * 71 ± 19
0 ns
ns
293 ± 63 * 63 ± 20 112 ± 21 ns 30 ± 12 *
225 ± 79 ns 365 ± 112
* HWS
LWS 30
0.3 ± 0.3
0 0.3 ± 0.3
11 ± 2.4 * 15. ± 6.8
0.1 ± 0.1 ns
ns
6.2 ± 1.7 * 3.4 ± 0.6 3.3 ± 2.3
0.4 ± 0.4 0
0 0.2 ± 0.2
0 0
0.2 ± 0.2
0
0 0.2 ± 0.2
227 ± 40 * 62 ± 19
0 ns
ns
225 ± 49 * 58 ± 19 109 ± 20 ns 29 ± 12 *
165 ± 48 ns 261 ± 55
ns HWS
LWS 20
0 0.3 ± 0.3 LWS 10
ns
195 ± 49 * 44 ± 16
ns
82 ± 10 ns 23 ± 12 61 ± 28 ns 128 ± 51
156 ± 34 * 47 ± 17
0.2 ± 0.2
0
0.2 ± 0.2
0.2 ± 0.2
0
1.0 ± 0.8
3.7 ± 1.0 ns 1.9 ± 0.5
ns
5.4 ± 1.0 ns 4.8 ± 1.6
Amylin Vehicle Amylin
Locomotion (m)
Vehicle Amylin
Escape Attempts (n)
Vehicle Amylin
Jumps (n)
Vehicle Amylin
Drinks (n)
Vehicle Vehicle Vehicle
Amylin
Exploratory Pecks (n)
Amylin Food pecks (n)
Cumulative non-timed behaviors Line Time (min)
of sensitivity in the amylin anorexigenic system in line LWS but not in HWS. The lower threshold of anorexia in line LWS was consistent with that of other neuropeptides including alpha-MSH [6] and corticotrophin releasing factor [5] in these lines. Furthermore, the threshold of anorexia is at least two times higher in line LWS than HWS for neuropeptide S [8]. Therefore, selection may have affected several neurotransmitter systems concurrently, with some acquiring gain of function and others loss of function. These data suggest that modifications of several endogenous neurotransmitter systems including those aforementioned through selection may be responsible for the anorexia observed in LWS; altered response threshold for these peptides may additively elicit anorexia over time. It is also possible that the selection program has affected one or two systems that modulate the responses of these other neuropeptide systems. Testing these hypotheses is an ongoing process within our laboratory. Leptin, secreted from adipocytes in proportion to fat mass, is a well-known antiobesogenic signal that acts in concert with amylin. While both leptin and amylin have independent effects, when administered concurrently additive effects are observed on food intake, body weight, and adiposity [27]. Turek et al. [27] also reported that amylin increases central leptin receptor binding and decreases leptin receptor mRNA expression in amylin-deficient mice. These data suggest a key relationship between amylin and leptin, and that ultimate effects on food intake for humans may be related at least in part to adiposity. When the effect of leptin on food intake in these lines was measured [18], line LWS responded to central leptin as do broiler- and layer-type chicks [11] while there was no response in HWS chicks to the doses tested. Although beyond the scope of this study, line HWS’s lack of appetite-associated response to leptin, along with its increased threshold for amylin compared to line LWS, support the interplay demonstrated by Turek et al. [27] and a possible interdependency of leptin and amylin in food intake regulation, especially in animals in a positive energy state. Similarly to leptin resistance, obese humans may become amylin resistant [1], as may be the case in line HWS. In Experiment 2, amylin caused alterations in count-type behaviors (Table 1). Within 20 min post-injection, the number of amylin-induced feeding pecks was significantly reduced in both lines with the cumulative difference becoming greater over time. The line by amylin dose interaction was significant at both 20 and 30 min because the magnitude of amylin-induced peck suppression was greater in line HWS than line LWS (at time 30, a 50% vs 92% reduction in line LWS vs HWS, respectively). Other count-type behaviors including jumping, escape attempts, drinks, defecations and locomotion were not affected by amylin treatment (Table 1). A significant line by amylin interaction for preening at 30 min was the result of a cross-over effect; amylin tended to decrease the behavior in line LWS but increase it in line HWS (Table 2). There was a threshold response for time spent perching, sitting, and in deep rest; means were 0 for at least one group within each behavior. Thus, statistical analyses were not conducted for these three behaviors. There were 7 chicks in both line LWS treatment groups and 7 and 8 in control and amylin treatment groups, respectively, for line HWS. That amylin reduced feeding pecks, but did not change other behaviors that are not directly associated with food intake is inconsistent with our previous report [7] where Cobb-500 chicks treated with amylin had increased escape attempts, jumps and locomotion. None of the timed-type behaviors were affected except for preening, which is also inconsistent with our previous report with Cobb-500 chicks where standing and preening time were increased. Apparently, behavioral effects of amylin are stock dependant. Additionally, amylin decreases locomotor activity and does not affect grooming in rats [4]. Therefore, amylin’s behavioral effects are also species specific. Since the only behavior where
Defecations (n)
M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654 Table 1 Means ± standard error of cumulative non-timed behaviors following ICV injection of vehicle or of 255 pmol amylin in LWS and HWS lines of chicks. Means within a column and row within a time which are significantly different are indicated by *, and nonsignificance noted by ns. For threshold responses, statistical analyses were not performed; n = 7 in both line LWS treatment groups, 7 and 8 in control and amylin treatment groups for line HWS.
652
19 ± 11 19 ± 11
24 ± 14
0 318 ± 170
0
4.1 ± 1.7 1 ± 0.5 * 9 ± 2.1
4.8 ± 3.1 128 ± 83 0 137 ± 69
Amylin
1.3 ± 0.6 0.7 ± 0.5
82 ± 48
Vehicle
48 ± 47
Amylin
0.5 ± 0.3
Perching
653
amylin treatment effects were detected in the present study was food pecks, it appears that anorexia after central amylin in these lines is a primary effect and not secondary to other competitive behavior as is the case for some other neuropeptides that affect appetite [12,3]. In summary, lines divergently selected long term for body weight from in a common founder population, there was a lower threshold for the amylin-induced anorexia in line LWS than HWS. Although the source of these differential thresholds remains unclear, the effect of amylin appears to be primary on appetite. These results provide evidence of genetic variation in anorexigenic responses to central amylin in chicks selected for low and high juvenile body weight.
*
*
Acknowledgment
5±4 236 ± 212 199 ± 85
166 ± 73
1.9 ± 1.1 13 ± 7.6 * 2.6 ± 1.1 3.5 ± 3.5 0 156 ± 141 0 83 ± 45 136 ± 116
73 ± 36 26 ± 24
1.1 ± 0.9 11 ± 7 0 0 73 ± 73 0 4.8 ± 4.8 18 ± 18
25 ± 14 15 ± 12
5.5 ± 4
Vehicle Amylin
0 2±2
Vehicle Amylin
ns HWS
HWS LWS 30
ns ns
HWS LWS 20
ns ns
Vehicle Amylin
450 ± 64 ns 564 ± 20 1071 ± 83 ns 1021 ± 146 1621 ± 128 ns 1533 ± 61 391 ± 60 ns 585 ± 9 1008 ± 68 ns 1044 ± 69 1296 ± 174 ns 1425 ± 142 LWS 10
ns Vehicle
0
Deep resting Sitting Line
Cumulative timed behaviors (s)
Standing
0
Preening
This work was made possible by the Jeffress Memorial Trust.
Time (min)
Table 2 Means ± standard error of cumulative timed behaviors following ICV injection of vehicle or of 255 pmol amylin in LWS and HWS lines of chicks. Means within a column and row within a time which are significantly different are indicated by *, and nonsignificance noted by ns. For threshold responses, statistical analyses were not performed; n = 7 in both line LWS treatment groups, 7 and 8 in control and amylin treatment groups for line HWS.
M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654
References [1] Arora S, Anubhuti. Role of neuropeptides in appetite regulation and obesity—a review. Neuropeptides 2006;40:375–401. [2] Bhavsar S, Watkins J, Young A. Synergy between amylin and cholecystokinin for inhibition of food intake in mice. Physiol Behav 1998;64:557– 61. [3] Billington CJ, Levine AS, Morely JE. Are peptides truly satiety agents? A method of testing for neurohumoral satiety effects. Am J Physiol 1983;245:920–9. [4] Clementi G, Busa L, de Bernardis E, Prato E, Drago F. Effects of centrally injected amylin on sexual behavior of male rats. Peptides 1996;20:379–82. [5] Cline MA, Kuo AY, Smith ML, Nandar W, Prall BC. Differential feed intake responses to central corticotrophin releasing factor in lines of chickens divergently selected for low or high body weight. Comp Biochem Physiol 2009;152:130–4. [6] Cline MA, Nandar W, Bowden C, Hein PP, Denbow DM, Siegel PB. Differential feeding responses to central alpha-melanocyte stimulating hormone in genetically low and high body weight selected lines of chickens. Life Sci 2008;83:208–13. [7] Cline MA, Nandar W, Smith ML, Pittman BH, Kelly M, Rogers JO. Amylin causes anorexigenic effects via the hypothalamus and brain stem in chicks. Regul Peptides 2008;146:140–6. [8] Cline MA, Prall BC, Smith ML, Calchary WA, Siegel PB. Differential appetite-related responses to central neuropeptide S in lines of chickens divergently selected for low or high body weight. J Neuroendocrinol 2008;20: 904–8. [9] Davis JL, Masuoka DT, Gerbandt LK, Cherkin A. Autoradiographic distribution of l-proline in chicks after intracerebroventricular injection. Physiol Behav 1979;22:693–5. [10] Del Prete E, Schade B, Riediger T, Lutz TA, Scharrer E. Effects of amylin and salmon calcitonin on feeding and drinking behavior in pygmy goats. Physiol Behav 2002;75:593–9. [11] Denbow DM, Meade S, Robertson A, McMurtry JP, Richards M, Ashwell C. Leptin-induced decrease in food intake in chickens. Physiol Behav 2000;69:359–62. [12] Deutsch JA, Hardy TW. Cholecystokinin produces bait shyness in rats. Nature 1977;266:196. [13] Dunnington EA, Siegel PB. Long-term divergent selection for eight-week body weight in White Phymouth Rock chickens. Poult Sci 1996;75:1168– 79. [14] Furuse M, Ando R, Bungo T, Ao R, Shimojo M, Masuda Y. Intracerebroventricular injection of orexins does not stimulate food intake in neonatal chicks. Br Poult Sci 1999;40:698–700. [15] Kahn SE, D’Alessio DA, Schwartz MW, Fujimoto WY, Ensinck JW, Taborsky Jr GW, et al. Evidence of cosecretion of islet amyloid polypeptide and insulin by beta-cells. Diabetes 1990;39:634–8. [16] Kong MF, King P, Macdonald IA, Stubbs TA, Perkins AC, Blackshaw PE, et al. Infusion of pramlintide, a human amylin analogue, delays gastric emptying in men with IDDM. Diabetologia 1997;40:82–8. [18] Kuo AY, Cline MA, Werner E, Siegel PB, Denbow DM. Leptin effects on food and water intake in lines of chickens selected for high or low body weight. Physiol Behav 2005;84:459–64. [19] LeRouzic A, Siegel PB, Carlborg O. Phenotypic evolution from genetic polymorphism in a radial network architecture. BMC Biol 2007;5:50. [21] Lutz TA, Geary N, Szabady MM, Del Prete E, Scharrer E. Amylin decreases meal size in rats. Physiol Behav 1995;58:1197–202. [22] Lutz TA, Rossi R, Althaus J, Del Prete E, Scharrer E. Amylin reduces food intake more potently than calcitonin gene-related peptide (CGRP) when injected into the lateral brain ventricle in rats. Peptides 2009;19:1533–40. [23] Lutz TA, Rossi R, Althaus J, Del Prete E, Scharrer E. Amylin reduces food intake more potently than calcitonin gene-related peptide (CGRP) when injected into the lateral brain ventricle in rats. Peptides 1998;19:309– 17.
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M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654
[24] Olsson M, Herrington MK, Reidelberger RD, Permert J, Arnelo U. Comparison of the effects of chronic central administration and chronic peripheral administration of islet amyloid polypeptide on food intake and meal pattern in the rat. Peptides 2007;28:1416–23. [25] Roth JD, Hughes H, Kendall E, Baron AD, Anderson CM. Anti-obesity effects of the -cell hormone amylin in diet induced obese rats: effects on food intake, body
weight, composition, energy expenditure and gene expression. Endocrinology 2006;147:5855–64. [26] Siegel PB, Wolford JH. A review of some results of selection for juvenile body weight in chickens. Poult Sci 2003;40:81–91. [27] Turek VF, Trevaskis JL, Levin BE, Dunn-Meynell AA, Gu BIG, Wittmer C, et al. Mechanisms of amylin/leptin synergy in rodent models. Endocrinology 2009, online only.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
The threshold of amylin-induced anorexia is lower in chicks selected for low compared to high juvenile body weight Mark A. Cline a,∗ , Wint Nandar b , Christie Bowden a , Wendy Calchary c , Marissa L. Smith a,d , Brian Prall a , Brandon Newmyer a , J. Orion Rogers a , Paul B. Siegel d a
Department of Biology, Radford University, East Main Street, Radford, VA 24142, United States Department of Neurosurgery, Pennsylvania State University, Hershey, PA, United States Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, United States d Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States b c
a r t i c l e
i n f o
Article history: Received 1 December 2009 Received in revised form 17 December 2009 Accepted 21 December 2009 Available online 29 December 2009
a b s t r a c t Chicks that have undergone long-term selection for low body weight responded to intracerebroventricular amylin injection with reduced food intake at a dose considerably lower and with a greater magnitude suppression than those selected for high body weight. Behaviors unrelated to ingestion were not affected. These data support the thesis of correlated amylin system responses to selection for low or high body weight, with possible implications to other species. © 2009 Elsevier B.V. All rights reserved.
Keywords: Amylin Chick Hypothalamus Food intake Obesity Anorexia Behavior c-Fos
Amylin is receiving increasing attention in appetite biology. Cosecreted with insulin from pancreatic beta cells [15] in response to ingestion of a meal [2], it is well documented that amylin decreases food intake across a range of species [7,21,10]. Accordingly, because its basal concentration is higher in obese humans, it is primarily associated with the obese phenotype [16]. When centrally administered, amylin is more potent than other members of its family [22] such as calcitonin and calcitonin gene-related peptide, which also elicit anorexigenic responses [10,23]. Amylin decreases food intake in chicks when administered centrally or peripherally, and although it decreases c-Fos reactivity in the hunger-associated lateral hypothalamus, it does not affect c-Fos expression in the ventromedial hypothalamus, which is classically associated with satiety [7]. Furthermore, we demonstrated that central amylin increases transit time through the alimentary canal, anxious-like behavior, and plasma corticosterone concentration [7].
∗ Corresponding author. Tel.: +1 540 831 6431; fax: +1 540 831 5129. E-mail address: [email protected] (M.A. Cline). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.12.032
Rodent studies have also demonstrated roles for amylin in decreasing meal size [21] and frequency [24]. Diet-induced obese rats respond to amylin with decreased food intake and weight gain while increasing energy expenditure [25]. However, to our knowledge these effects have not been reported in a polygenic model of obesity, nor have the anorectic effects of amylin been studied simultaneously in animal models of anorexia and obesity. The study reported here aimed to determine if amylin differentially affects food intake and behavior in lines of chickens that have undergone long-term selection for high and low body weight and that are hypo- and hyperphagic. The chicks used in this study are the result of long-term divergent selection for low (LWS) or high (HWS) body weight at 56 days-of-age from a White Plymouth Rock founder population that consisted of crosses of 7 inbred lines and were maintained as closed populations [13,26,19]. Eggs obtained from age contemporary parents from S48 and S49 generation parental stocks were incubated in the same machine. After hatch, chicks were group-caged for 2 days, then individually in a room at 30 ± 2 ◦ C and 50 ± 5% relative humidity where they had ad libitum access to a mash diet (20% crude protein, 2685 kcal ME/kg) and tap water. The individual cages allowed visual and auditory contact with other chicks
M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654
(unless otherwise indicated). Chicks were handled twice daily to adapt to handling. All trials were conducted between 11:00 and 16:00 h using 5-day post-hatch chicks. Data in each experiment were recorded from both lines concurrently and injections were performed sequentially, LWS, HWS, LWS, HWS and so forth. Experimental procedures were performed according to the National Research Council publication, Guide for Care and Use of Laboratory Animals and were approved by the Radford University Institutional Animal Care and Use Committee. Chicks were injected using a method adapted from Davis et al. [9] that does not appear to induce physiological stress [14]. The head of the chick was briefly inserted into a restraining device that left the cranium exposed and allowed for free-hand injection. Injection coordinates were 3 mm anterior to the coronal suture, 1 mm lateral from the sagittal suture, and 2 mm deep, targeting the left lateral ventricle. Anatomical landmarks were determined visually and by palpation. Injection depth was controlled by placing a plastic tubing sheath over the needle. The needle remained at injection depth for 5 s post-injection to reduce backflow. Chicks were assigned to treatments at random. Amylin (American Peptide Co., Sunnyvale, CA, USA) was dissolved in artificial cerebrospinal fluid as a vehicle for a total injection volume of 5 L with 0.1% Evans Blue dye to facilitate injection site localization. The sequence of rat amylin injected (KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY) has 82% sequence identity to that of chicken [21]. After data collection, the chicks were decapitated and their heads sectioned coronally to determine site of injection. Any chick without dye present in the lateral ventricle system was eliminated from analysis. In Experiment 1, chicks from the S48 generation from each line were fasted for 180 min prior to injection to intensify the perception of hunger. They were randomly assigned to receive 0 (vehicle only), 128 (0.5 g), 255 (1.0 g) or 510 (2.0 g) pmol amylin by ICV injection. After injection, chicks were returned to their individual cages and given ad libitum access to both food and water, with individual food and water containers weighed (0.01 g) every 30 min for 180 min post-injection. Data were analyzed using analysis of variance (ANOVA) at each time point. The model included line, amylin dose and the line by amylin dose interaction. When the interaction was significant (P < 0.05), data were analyzed within each line for the effect of amylin dose using Tukey’s method of multiple comparisons. The LWS and HWS lines consume inherently different
651
amounts of food due to differences in body size. Therefore, food and water intake data were normalized per body weight at each time point. This conversion was made by dividing the amount of feed each chick consumed by its body weight (0.01 g) immediately prior to its ICV injection and multiplying by 100. In Experiment 2, chicks from the S49 generation from each line were kept in individual cages with auditory but not visual contact (to reduce isolation stress during behavior measurement) from 2 days post-hatch. They were randomly assigned to receive either vehicle or 255 pmol amylin, a dose that exceeded threshold in both lines in Experiment 1, by ICV injection. Following a 180 min fast they were injected and immediately placed in a 290 mm × 290 mm acrylic recording arena with food and water containers in diagonal corners. Chicks were simultaneously and automatically recorded from three angles for 30 min post-injection on DVD and data were analyzed in 10 min intervals using ANY-maze behavioral analysis software (Stoelting, Wood Dale, IL). Locomotion (m traveled), time spent (s) standing, sitting, preening, perching or in deep rest, and the number of jumps, food and exploratory pecks, drinks, defecations and escape attempts were recorded. Food pecks were defined as pecks within the food container, whereas all other pecks were considered as exploratory. Drinks were defined as the chick dipping its beak in water, then raising and extending its head to swallow. Preening was defined as trimming or dressing of down with the beak. Deep rest was defined as the eyes closed for greater than 3 s, starting 3 s after eye closure. The statistical model used was the same as that for Experiment 1. In Experiment 1, amylin reduced food intake in both lines (Fig. 1), and the line by amylin dose interaction was significant. The interaction occurred because the threshold of amylin-induced anorexia was lower for line LWS than line HWS chicks. That is, in line LWS all doses of amylin tested reduced food intake nearly 50%. In line HWS however, 128 pmol did not reduce food intake whereas 255 and 510 pmol of amylin caused only a 25% reduction in food intake. There were 12–14 chicks in the line LWS and 17–18 in HWS per amylin dose. These results demonstrate that line LWS has a lower threshold of amylin-induced anorexia than line HWS. The decreased food intake pattern in chicks from line HWS more closely resembles that of Cobb-500 chicks than that of line LWS [7] and the decreased food intake is consistent with that reported for rodents [21,10]. Therefore, selection for juvenile body weight may have favored a gain
Fig. 1. Cumulative food intake expressed as percent body weight following intracerebroventricular injection of amylin in low (LWS) and high (HWS) body weight long-term divergently selected lines of chicks (Experiment 1). Values are means ± SE; bars with different letters are different from each other within a time point (P < 0.05). n = 12–14 chicks in the line LWS and 17–18 in HWS per amylin dose.
0.3 ± 0.3 0.3 ± 0.2
0
ns 3.7 ± 2.6 0.5 ± 0.4 0 0 0.4 ± 0.3 0 ns * HWS
0.3 ± 0.3 0.3 ± 0.2
0.3 ± 0.3
14 ± 3.1 ns 37 ± 22 ns
7.7 ± 2.6 ns 4.1 ± 0.9
ns 3±2
3.7 ± 2.6 0
0.4 ± 0.3 0
0.2 ± 0.2 0.2 ± 0.2
0 0.4 ± 0.3
0 0.2 ± 0.2
304 ± 66 * 71 ± 19
0 ns
ns
293 ± 63 * 63 ± 20 112 ± 21 ns 30 ± 12 *
225 ± 79 ns 365 ± 112
* HWS
LWS 30
0.3 ± 0.3
0 0.3 ± 0.3
11 ± 2.4 * 15. ± 6.8
0.1 ± 0.1 ns
ns
6.2 ± 1.7 * 3.4 ± 0.6 3.3 ± 2.3
0.4 ± 0.4 0
0 0.2 ± 0.2
0 0
0.2 ± 0.2
0
0 0.2 ± 0.2
227 ± 40 * 62 ± 19
0 ns
ns
225 ± 49 * 58 ± 19 109 ± 20 ns 29 ± 12 *
165 ± 48 ns 261 ± 55
ns HWS
LWS 20
0 0.3 ± 0.3 LWS 10
ns
195 ± 49 * 44 ± 16
ns
82 ± 10 ns 23 ± 12 61 ± 28 ns 128 ± 51
156 ± 34 * 47 ± 17
0.2 ± 0.2
0
0.2 ± 0.2
0.2 ± 0.2
0
1.0 ± 0.8
3.7 ± 1.0 ns 1.9 ± 0.5
ns
5.4 ± 1.0 ns 4.8 ± 1.6
Amylin Vehicle Amylin
Locomotion (m)
Vehicle Amylin
Escape Attempts (n)
Vehicle Amylin
Jumps (n)
Vehicle Amylin
Drinks (n)
Vehicle Vehicle Vehicle
Amylin
Exploratory Pecks (n)
Amylin Food pecks (n)
Cumulative non-timed behaviors Line Time (min)
of sensitivity in the amylin anorexigenic system in line LWS but not in HWS. The lower threshold of anorexia in line LWS was consistent with that of other neuropeptides including alpha-MSH [6] and corticotrophin releasing factor [5] in these lines. Furthermore, the threshold of anorexia is at least two times higher in line LWS than HWS for neuropeptide S [8]. Therefore, selection may have affected several neurotransmitter systems concurrently, with some acquiring gain of function and others loss of function. These data suggest that modifications of several endogenous neurotransmitter systems including those aforementioned through selection may be responsible for the anorexia observed in LWS; altered response threshold for these peptides may additively elicit anorexia over time. It is also possible that the selection program has affected one or two systems that modulate the responses of these other neuropeptide systems. Testing these hypotheses is an ongoing process within our laboratory. Leptin, secreted from adipocytes in proportion to fat mass, is a well-known antiobesogenic signal that acts in concert with amylin. While both leptin and amylin have independent effects, when administered concurrently additive effects are observed on food intake, body weight, and adiposity [27]. Turek et al. [27] also reported that amylin increases central leptin receptor binding and decreases leptin receptor mRNA expression in amylin-deficient mice. These data suggest a key relationship between amylin and leptin, and that ultimate effects on food intake for humans may be related at least in part to adiposity. When the effect of leptin on food intake in these lines was measured [18], line LWS responded to central leptin as do broiler- and layer-type chicks [11] while there was no response in HWS chicks to the doses tested. Although beyond the scope of this study, line HWS’s lack of appetite-associated response to leptin, along with its increased threshold for amylin compared to line LWS, support the interplay demonstrated by Turek et al. [27] and a possible interdependency of leptin and amylin in food intake regulation, especially in animals in a positive energy state. Similarly to leptin resistance, obese humans may become amylin resistant [1], as may be the case in line HWS. In Experiment 2, amylin caused alterations in count-type behaviors (Table 1). Within 20 min post-injection, the number of amylin-induced feeding pecks was significantly reduced in both lines with the cumulative difference becoming greater over time. The line by amylin dose interaction was significant at both 20 and 30 min because the magnitude of amylin-induced peck suppression was greater in line HWS than line LWS (at time 30, a 50% vs 92% reduction in line LWS vs HWS, respectively). Other count-type behaviors including jumping, escape attempts, drinks, defecations and locomotion were not affected by amylin treatment (Table 1). A significant line by amylin interaction for preening at 30 min was the result of a cross-over effect; amylin tended to decrease the behavior in line LWS but increase it in line HWS (Table 2). There was a threshold response for time spent perching, sitting, and in deep rest; means were 0 for at least one group within each behavior. Thus, statistical analyses were not conducted for these three behaviors. There were 7 chicks in both line LWS treatment groups and 7 and 8 in control and amylin treatment groups, respectively, for line HWS. That amylin reduced feeding pecks, but did not change other behaviors that are not directly associated with food intake is inconsistent with our previous report [7] where Cobb-500 chicks treated with amylin had increased escape attempts, jumps and locomotion. None of the timed-type behaviors were affected except for preening, which is also inconsistent with our previous report with Cobb-500 chicks where standing and preening time were increased. Apparently, behavioral effects of amylin are stock dependant. Additionally, amylin decreases locomotor activity and does not affect grooming in rats [4]. Therefore, amylin’s behavioral effects are also species specific. Since the only behavior where
Defecations (n)
M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654 Table 1 Means ± standard error of cumulative non-timed behaviors following ICV injection of vehicle or of 255 pmol amylin in LWS and HWS lines of chicks. Means within a column and row within a time which are significantly different are indicated by *, and nonsignificance noted by ns. For threshold responses, statistical analyses were not performed; n = 7 in both line LWS treatment groups, 7 and 8 in control and amylin treatment groups for line HWS.
652
19 ± 11 19 ± 11
24 ± 14
0 318 ± 170
0
4.1 ± 1.7 1 ± 0.5 * 9 ± 2.1
4.8 ± 3.1 128 ± 83 0 137 ± 69
Amylin
1.3 ± 0.6 0.7 ± 0.5
82 ± 48
Vehicle
48 ± 47
Amylin
0.5 ± 0.3
Perching
653
amylin treatment effects were detected in the present study was food pecks, it appears that anorexia after central amylin in these lines is a primary effect and not secondary to other competitive behavior as is the case for some other neuropeptides that affect appetite [12,3]. In summary, lines divergently selected long term for body weight from in a common founder population, there was a lower threshold for the amylin-induced anorexia in line LWS than HWS. Although the source of these differential thresholds remains unclear, the effect of amylin appears to be primary on appetite. These results provide evidence of genetic variation in anorexigenic responses to central amylin in chicks selected for low and high juvenile body weight.
*
*
Acknowledgment
5±4 236 ± 212 199 ± 85
166 ± 73
1.9 ± 1.1 13 ± 7.6 * 2.6 ± 1.1 3.5 ± 3.5 0 156 ± 141 0 83 ± 45 136 ± 116
73 ± 36 26 ± 24
1.1 ± 0.9 11 ± 7 0 0 73 ± 73 0 4.8 ± 4.8 18 ± 18
25 ± 14 15 ± 12
5.5 ± 4
Vehicle Amylin
0 2±2
Vehicle Amylin
ns HWS
HWS LWS 30
ns ns
HWS LWS 20
ns ns
Vehicle Amylin
450 ± 64 ns 564 ± 20 1071 ± 83 ns 1021 ± 146 1621 ± 128 ns 1533 ± 61 391 ± 60 ns 585 ± 9 1008 ± 68 ns 1044 ± 69 1296 ± 174 ns 1425 ± 142 LWS 10
ns Vehicle
0
Deep resting Sitting Line
Cumulative timed behaviors (s)
Standing
0
Preening
This work was made possible by the Jeffress Memorial Trust.
Time (min)
Table 2 Means ± standard error of cumulative timed behaviors following ICV injection of vehicle or of 255 pmol amylin in LWS and HWS lines of chicks. Means within a column and row within a time which are significantly different are indicated by *, and nonsignificance noted by ns. For threshold responses, statistical analyses were not performed; n = 7 in both line LWS treatment groups, 7 and 8 in control and amylin treatment groups for line HWS.
M.A. Cline et al. / Behavioural Brain Research 208 (2010) 650–654
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