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Release of endogenous cholecystokinin in response to gastric preloads in rats on postnatal days 9–12
Physiology & Behavior 72 (2001) 1 ± 4
Release of endogenous cholecystokinin in response to gastric preloads in rats on postnatal days 9±12 Aron Wellera, Iris H. Gispana, Robert C. Ritterb, Lynne Brennerb, Gerard P. Smithc,* a
Developmental Psychobiology Laboratory, Department of Psychology, Bar Ilan University, Ramat Gan S2900, Israel Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Pullman, WA 99164-6520, USA c Department of Psychiatry, Joan and Sanford I. Weill Medical College of Cornell University and E.W. Bourne Research Laboratory, New York ± Presbyterian Hospital, Westchester Division, 21 Bloomingdale Road, White Plains, NY 10605, USA b
Received 10 January 2000; received in revised form 6 April 2000; accepted 7 April 2000
Abstract Release of endogenous cholecystokinin (CCK) from the small intestine by gastric loads was investigated in rats on postnatal days 9 ± 12 (P9 ± P12). After 5 ± 6 h of deprivation, pups received 5% b.wt. loads of water, 0.9% NaCl, 20% glucose, 20% maltose, 200 mg soybean trypsin inhibitor (SBTI) in 0.9% saline or sham load. Plasma was collected 15 min after the loads, and the concentration of bioactive CCK was measured by a specific and sensitive bioassay. Loads of SBTI and water significantly increased plasma CCK compared to sham loads, but loads of saline, glucose, and maltose did not. The efficacy of the water load was not demonstrated in adult rats. The results suggest that the previously reported reduction of intake during independent ingestion by hypertonic preloads of glucose and maltose was not mediated by the release of CCK sufficient to be detected in the plasma under these conditions. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Independent ingestion; Ontogeny; Inhibition of food intake; Glucose; Maltose; Soybean trypsin inhibitor; Water intake
1. Introduction Gastric preloads of glucose or maltose administered 5 min before an independent ingestion test in rat pups on postnatal day 12 (P12) significantly decreased intake during the 30-min test [1]. The magnitude of this inhibitory effect depended on the concentration of the carbohydrate preload. Preloads of isotonic solutions of glucose or maltose inhibited intake as much as the inhibition observed after preloads of water or an isotonic solution of sodium chloride. This result suggested that the adequate stimulus for the effects of isotonic carbohydrate preloads in rats of this age is volume, a stimulus that has been demonstrated to inhibit independent ingestion as early as P6 [2]. Hypertonic solutions (20% glucose and 20% maltose), however, decreased intake significantly more than isotonic solutions of glucose or maltose [1]. Thus, these preloads produced an inhibitory effect in addition to that produced by their volumes. This non-volumetric stimulus appears to be
* Corresponding author. Tel.: +1-914-997-5935. E-mail address: [email protected] (G.P. Smith).
due to the preabsorptive osmotic load of the hypertonic carbohydrate preloads because: (1) 20% 2-deoxy-D-glucose, an analogue of glucose that cannot be actively transported across the small intestine and cannot be utilized metabolically [3], produced the same inhibition as a preload of 20% glucose [4]; (2) maltose cannot be digested to glucose and absorbed at this age due to the lack of maltase in the mucosa of the small intestine [5]; and (3) preloads of mannitol, a non-absorbable sugar alcohol, decreased intake as a function of concentration Ð the more hypertonic preloads produced more inhibition [6]. In the experiments with glucose and maltose, we investigated the possibility that the inhibitory effects of the isotonic and hypertonic preloads were mediated by the release of cholecystokinin (CCK) from the small intestine. That possibility was based on the demonstration that a preload of soybean trypsin inhibitor (SBTI) released CCK [7] and that CCK inhibited intake during independent ingestion at this age [8,9]. Because devazepide, a specific antagonist of CCK at CCKA receptors, blocked the inhibitory effect of SBTI [7] or exogenous CCK-8 [9] on intake, we pretreated pups with devazepide prior to isotonic and hypertonic preloads as well as equivolmetric preloads of
0031-9384/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 0 ) 0 0 2 8 8 - 2
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A. Weller et al. / Physiology & Behavior 72 (2001) 1±4
water. The results were negative: devazepide did not decrease the inhibitory effect of any of the preloads under conditions in which it blocked the inhibitory effect of a preload of SBTI [4]. The lack of effect of devazepide suggests that preloads of water, isotonic saline, and hypertonic carbohydrates did not release CCK from the small intestine. We tested that inference in the experiments reported here by measuring plasma CCK with a sensitive and specific bioassay after equivolumetric gastric loads of water, 0.9% saline, 20% glucose, 20% maltose, and SBTI. The results demonstrate that loads of 0.9% saline, 20% glucose, and 20% maltose did not increase plasma CCK significantly, but loads of water and SBTI did.
stored at ÿ 80°C until the CCK assay was performed. Biologically active CCK was assayed using the method of Liddle et al. [10], as described previously [11]. The pups provided 1.2 ± 2.8 ml plasma per cartridge, attained by pooling together blood from 4± 10 littermates per cartridge, providing a total of 6± 10 cartridges per group (n = 55 to 65 rats/group). The study was performed in two separate years; all loads were studied in both years (minimum number of cartridges per load per year = 2). Because the n per year was small and the pattern of results was similar, the data were pooled and analyzed together. Concentration of CCK per ml plasma (pM), calculated separately for each cartridge, was the unit for statistical analysis. The data were analyzed by one-way analysis of variance (ANOVA) followed post hoc by Duncan's test.
2. Materials and methods
2.2. Experiments in adult rats
2.1. Experiment in pups
2.2.1. Animals The animals used in this experiment were adult male Sprague ± Dawley rats (Simonson Labs, CA), weighing 194 ± 223 g. The rats were individually housed in suspended stainless steel cages in an AAALAC-approved vivarium. The ambient temperature was thermostatically maintained at 23 1.5°C and a 12:12 light/dark cycle was maintained, with light on at 0730 h. Except as noted below, pelleted laboratory rodent chow (Harlan Tekclad Diet 8664, 3.3 kcal/ g) and tap water were available ad lib.
2.1.1. Animals Sprague ±Dawley rat pups of both sexes were tested on P9 ±P12. The pups were derived from our breeding colony at the Developmental Psychobiology laboratory in the Psychology Department at Bar Ilam University. Lights in the colony were on from 0500 to 1700 h, and temperature was maintained between 21°C and 25°C. Each litter was housed in a polycarbonate cage (38 21 18 cm) with a stainless steel wire lid and wood shavings as bedding material. Chow and tap water were continuously available in the cage top. Newborn litters found between 0800 and 1200 h were designated as born on that day (P0) and culled to 10 pups on P1 or P2. 2.1.2. Gastric loads The volume of all gastric loads was calculated to be 5% of body weight measured on the morning of the test day. The treatments were: sham load (tube insertion into stomach, but no load), or loads containing 0.9% NaCl, distilled water, 20% (w/v) glucose (Sigma, dissolved in distilled water), 20% maltose (Sigma, dissolved in distilled water) or SBTI (Sigma, 200 mg in 1 ml of 0.9% NaCl). 2.1.3. Procedure All 10 pups from each litter were taken from the nest and dam in the morning (0800 ± 0900 h). Each pup was weighed, marked, and then the pups were placed together in a small container in a humid and warm (33°C) incubator. After 5 ±6 h, each pup was weighed, excretion was stimulated by a cotton swab, and the pup received a specific load (see below) into the stomach via a polyethylene tube (Becton Dickinson, Franklin Lake, NJ; PE-50). After 15 min, the pup was decapitated and trunk blood was collected into heparinized tubes on ice and centrifuged at 4°C. CCK was extracted from the plasma by absorption on Sep-Pak C-18 cartridges (Millipore), which were rinsed, air-dried, and
2.2.2. Procedure The rats were deprived of food, but not water, for 16 h. Subsequently, each rat received one of the following gastric loads by gavage: 5 ml distilled water, 10 ml distilled water, 5 ml SBTI (Sigma, type II, 20 mg/ml), 5 ml 0.9% NaCl, and 10 ml 0.9% NaCl. The gastric loads of 5 and 10 ml were equivalent to approximately 2.5% and 5.0% of the body weight, respectively. Fifteen minutes after gavage, the rats were anesthetized with Metafane, a midline laparotomy was performed, and the rats were exsanguinated, the blood being drawn from the inferior vena cava into heparinized syringes. Plasma was separated by centrifugation and extracted on Sep-Pak C-18 cartridges. CCK from each sample was eluted and assayed as described above. Extraction of CCK from the relatively large plasma samples available through this Table 1 Plasma CCK (pM) in rat pups after gastric loads Preload Sham 0.9% NaCl Water 20% Glucose 20% Maltose SBTI
n 6 6 10 8 6 7
Mean SEM 2.4 0.3 3.8 0.6 12.5 2.7* 3.4 0.8 1.7 0.3 11.8 3.4*
* Significantly higher CCK concentration than after sham preloads, p < 0.05.
A. Weller et al. / Physiology & Behavior 72 (2001) 1±4 Table 2 Plasma CCK (pM) in adult male rats after gastric loads Preload Sham 0.9% NaCl (5 ml) 0.9% NaCl (10 ml) Water (5 ml) Water (10 ml) SBTI
n 6 5 3 5 11 6
Median 0.47 0.00 0.00 0.34 0.77 3.18
Range 0 ± 0.80 0 ± 0.44 0 ± 0.45 0.33 ± 0.36 0 ± 2.94 2.07 ± 5.56*
* Significantly higher CCK concentration than after sham preloads, p < 0.05.
method provides an assay detection threshold of 0.3 pM of biologically active CCK. Plasma CCK was not detected in four of five rats that received 5-ml loads of 0.9% NaCl and in two of three rats that received 10-ml loads of 0.9% NaCl. Therefore, for data analysis, these groups were pooled and the non-detectable concentrations of CCK were assigned the value of zero. Because the data were not normally distributed, they were analyzed nonparametrically using Kruskal ± Wallis one-way ANOVA on ranks, followed by Dunn's method for post hoc comparison of all groups with the sham preload as the control group. 3. Results Plasma concentration of CCK differed significantly among the treatments (F(6,42) = 4.41, p < 0.01; Table 1). Plasma CCK was significantly higher after loads of water or SBTI than after other loads (p < 0.05). Plasma CCK after loads of water did not differ significantly from that after loads of SBTI. In contrast to the increases of CCK produced by loads of water or SBTI, plasma CCK after loads of 0.9% saline, 20% glucose, and 20% maltose was not significantly different from plasma CCK after sham loads. To investigate whether water also increased plasma CCK in adult rats, we measured the plasma CCK response after sham loads and loads of 0.9% saline, water, and SBTI (Table 2). Plasma CCK varied significantly after the different loads (K ± W H = 24.3, df = 4, p = 0.001). Unlike the results in pups, however, neither 5 nor 10-ml loads of water increased plasma CCK significantly compared to sham loads. The lack of effect of water or saline loads was not an artifact of the bioassay because SBTI increased plasma CCK significantly compared to sham loads (p < 0.05; Table 2). 4. Discussion Isotonic and hypertonic gastric loads did not increase plasma CCK significantly more than sham loads did in pups on P9± P12 (Table 1). This is consistent with our previous report [4] that pretreatment with devazepide did not block
3
the inhibition of intake produced by the isotonic and hypertonic preloads. Of course, it is possible for a stimulus to release CCK from the small intestine in an amount that is sufficient to have a paracrine effect, but not sufficient to cause a detectable increase of plasma CCK. This appears to be the case for the inhibition of intake by intraduodenal infusions of maltose in adult rats. Devazepide, however, blocked the inhibitory effect of this apparently paracrine action of CCK [12], but devazepide had no effect on the inhibitory action of maltose or other loads in pups. Therefore, neither endocrine nor paracrine actions of CCK are necessary for the inhibitory effects of these loads in pups on P9 ±P12. The large increase of plasma CCK after loads of water in pups was surprising (Table 1). It appears to be an effect of water that does not continue throughout the life of the rat because it did not occur in adult rats (Table 2). The ontogeny of the release of CCK by water is unknown and requires further investigation. The function of the increased CCK after a load of water is also not clear. It does not appear to mediate the inhibition of intake produced by preloads of water because devazepide did not block it. The possibility that the circulating bioassayable CCK is inhibiting intake through a CCKB receptor mechanism in pups of this age is unlikely because the inhibition of intake produced by administration of CCK-8 at this age is not blocked by a CCKB antagonist, while it is blocked by a CCKA antagonist [9]. Note that this combination of increased plasma CCK that does not account for the inhibition of intake by water in pups is different from the effect of casein in adults Ð casein increased plasma CCK, but did not decrease intake [11]. In summary, the results demonstrate that isotonic and hypertonic loads of carbohydrates do not release sufficient CCK from the small intestine to be detected in the plasma under these experimental conditions. This is not due to immaturity of the CCK release mechanisms because loads of SBTI or water produced large increases of plasma CCK. Taken together with our previous results with devazepide [4], these data are strong evidence that the volumetric and hypertonic inhibition of intake by preloads in P9 ± P12 pups is not mediated by CCK. Thus, current evidence shows that during the preweaning period, endogenous CCK released from the small intestine only mediates the inhibition of intake produced by preloads of corn oil beginning on P15 [13]. In previous works, we showed that the CCK response to a preload of SBTI mediated the inhibitory effect of that preload on intake in pups on P9 ±P12 [7,14]. The results with preloads of water are a new phenomenon: CCK is released, but it apparently does not mediate the inhibitory effect on intake of water preloads because that inhibition was not blocked by the specific CCKA antagonist, devazepide [4].
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A. Weller et al. / Physiology & Behavior 72 (2001) 1±4
Acknowledgments The authors thank Ofra Schwartz for technical assistance and Ms. Laurel A. Torres for processing the manuscript. This research was supported by a grant from the U.S. ±Israel Binational Science Foundation. Dr. Ritter was supported by NS20561, and Dr. Smith was supported by MH40010. References [1] Weller A, Gispan IH, Smith GP. Postingestive inhibitory controls of independent ingestion in 12-day-old rats. Physiol Behav 1996;60: 361 ± 4. [2] Hall WG, Bruno JP. Inhibitory controls of ingestion in 6-day-old rat pups. Physiol Behav 1984;32:831 ± 41. [3] Kimmich GA. Intestinal absorption of sugar. In: Johnson LR, editor. Physiology of the gastrointestinal tract. New York: Raven Press, 1981. p. 1038. [4] Weller A, Gispan IH, Smith GP. Characteristics of glucose and maltose preloads that inhibit feeding in 12-day-old rats. Physiol Behav 1997;61:819 ± 22. [5] Galand G. Brush border membrane sucrose-isomaltase, maltaseglucoamylase and trehalase in mammals. Comp Biochem Physiol 1989;94B:1 ± 11.
[6] Weller A, Tsitolovskya L, Gispan IH, Smith GP. Ontogeny of hypertonic preabsorptive inhibitory control of intake in neonatal rats. Am J Physiol 2000;278:R44 ± 9. [7] Weller A, Smith GP, Gibbs J. Endogenous cholecystokinin reduces feeding in young rats. Science 1990;247:1589 ± 91. [8] Robinson PH, Moran TH, McHugh PR. Cholecystokinin inhibits independent ingestion in neonatal rats. Am J Physiol 1988;255:R14 ± 20. [9] Smith GP, Tyrka A, Gibbs J. Type-A CCK receptors mediate the inhibition of food intake and activity by CCK-8 in 9- to 12-day old rat pups. Pharmacol Biochem Behav 1991;38:207 ± 10. [10] Liddle RA, Goldfine ID, Williams JA. Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor, and alcohol. Gastroenterology 1984;87:542 ± 9. [11] Brenner L, Yox DP, Ritter RC. Suppression of sham feeding by intraintestinal nutrients is not correlated with plasma cholecystokinin elevation. Am J Physiol 1993;264:R972 ± 6. [12] Yox DP, Brenner L, Ritter RC. CCK-receptor antagonists attenuate suppression of sham feeding by intestinal nutrients. Am J Physiol 1992;262:R554 ± 61. [13] Weller A, Gispan IH, Armony-Sivan R, Ritter RC, Smith GP. Preloads of corn oil inhibit independent ingestion on postnatal day 15 in rats. Physiol Behav 1997;62:871 ± 4. [14] Weller A, Corp ES, Tyrka A, Ritter RC, Brenner L, Gibbs J, Smith GP. Trypsin inhibitor and maternal reunion increase plasma cholecystokinin in neonatal rats. Peptides 1992;13: 939 ± 41.
Release of endogenous cholecystokinin in response to gastric preloads in rats on postnatal days 9±12 Aron Wellera, Iris H. Gispana, Robert C. Ritterb, Lynne Brennerb, Gerard P. Smithc,* a
Developmental Psychobiology Laboratory, Department of Psychology, Bar Ilan University, Ramat Gan S2900, Israel Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Pullman, WA 99164-6520, USA c Department of Psychiatry, Joan and Sanford I. Weill Medical College of Cornell University and E.W. Bourne Research Laboratory, New York ± Presbyterian Hospital, Westchester Division, 21 Bloomingdale Road, White Plains, NY 10605, USA b
Received 10 January 2000; received in revised form 6 April 2000; accepted 7 April 2000
Abstract Release of endogenous cholecystokinin (CCK) from the small intestine by gastric loads was investigated in rats on postnatal days 9 ± 12 (P9 ± P12). After 5 ± 6 h of deprivation, pups received 5% b.wt. loads of water, 0.9% NaCl, 20% glucose, 20% maltose, 200 mg soybean trypsin inhibitor (SBTI) in 0.9% saline or sham load. Plasma was collected 15 min after the loads, and the concentration of bioactive CCK was measured by a specific and sensitive bioassay. Loads of SBTI and water significantly increased plasma CCK compared to sham loads, but loads of saline, glucose, and maltose did not. The efficacy of the water load was not demonstrated in adult rats. The results suggest that the previously reported reduction of intake during independent ingestion by hypertonic preloads of glucose and maltose was not mediated by the release of CCK sufficient to be detected in the plasma under these conditions. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Independent ingestion; Ontogeny; Inhibition of food intake; Glucose; Maltose; Soybean trypsin inhibitor; Water intake
1. Introduction Gastric preloads of glucose or maltose administered 5 min before an independent ingestion test in rat pups on postnatal day 12 (P12) significantly decreased intake during the 30-min test [1]. The magnitude of this inhibitory effect depended on the concentration of the carbohydrate preload. Preloads of isotonic solutions of glucose or maltose inhibited intake as much as the inhibition observed after preloads of water or an isotonic solution of sodium chloride. This result suggested that the adequate stimulus for the effects of isotonic carbohydrate preloads in rats of this age is volume, a stimulus that has been demonstrated to inhibit independent ingestion as early as P6 [2]. Hypertonic solutions (20% glucose and 20% maltose), however, decreased intake significantly more than isotonic solutions of glucose or maltose [1]. Thus, these preloads produced an inhibitory effect in addition to that produced by their volumes. This non-volumetric stimulus appears to be
* Corresponding author. Tel.: +1-914-997-5935. E-mail address: [email protected] (G.P. Smith).
due to the preabsorptive osmotic load of the hypertonic carbohydrate preloads because: (1) 20% 2-deoxy-D-glucose, an analogue of glucose that cannot be actively transported across the small intestine and cannot be utilized metabolically [3], produced the same inhibition as a preload of 20% glucose [4]; (2) maltose cannot be digested to glucose and absorbed at this age due to the lack of maltase in the mucosa of the small intestine [5]; and (3) preloads of mannitol, a non-absorbable sugar alcohol, decreased intake as a function of concentration Ð the more hypertonic preloads produced more inhibition [6]. In the experiments with glucose and maltose, we investigated the possibility that the inhibitory effects of the isotonic and hypertonic preloads were mediated by the release of cholecystokinin (CCK) from the small intestine. That possibility was based on the demonstration that a preload of soybean trypsin inhibitor (SBTI) released CCK [7] and that CCK inhibited intake during independent ingestion at this age [8,9]. Because devazepide, a specific antagonist of CCK at CCKA receptors, blocked the inhibitory effect of SBTI [7] or exogenous CCK-8 [9] on intake, we pretreated pups with devazepide prior to isotonic and hypertonic preloads as well as equivolmetric preloads of
0031-9384/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 0 ) 0 0 2 8 8 - 2
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A. Weller et al. / Physiology & Behavior 72 (2001) 1±4
water. The results were negative: devazepide did not decrease the inhibitory effect of any of the preloads under conditions in which it blocked the inhibitory effect of a preload of SBTI [4]. The lack of effect of devazepide suggests that preloads of water, isotonic saline, and hypertonic carbohydrates did not release CCK from the small intestine. We tested that inference in the experiments reported here by measuring plasma CCK with a sensitive and specific bioassay after equivolumetric gastric loads of water, 0.9% saline, 20% glucose, 20% maltose, and SBTI. The results demonstrate that loads of 0.9% saline, 20% glucose, and 20% maltose did not increase plasma CCK significantly, but loads of water and SBTI did.
stored at ÿ 80°C until the CCK assay was performed. Biologically active CCK was assayed using the method of Liddle et al. [10], as described previously [11]. The pups provided 1.2 ± 2.8 ml plasma per cartridge, attained by pooling together blood from 4± 10 littermates per cartridge, providing a total of 6± 10 cartridges per group (n = 55 to 65 rats/group). The study was performed in two separate years; all loads were studied in both years (minimum number of cartridges per load per year = 2). Because the n per year was small and the pattern of results was similar, the data were pooled and analyzed together. Concentration of CCK per ml plasma (pM), calculated separately for each cartridge, was the unit for statistical analysis. The data were analyzed by one-way analysis of variance (ANOVA) followed post hoc by Duncan's test.
2. Materials and methods
2.2. Experiments in adult rats
2.1. Experiment in pups
2.2.1. Animals The animals used in this experiment were adult male Sprague ± Dawley rats (Simonson Labs, CA), weighing 194 ± 223 g. The rats were individually housed in suspended stainless steel cages in an AAALAC-approved vivarium. The ambient temperature was thermostatically maintained at 23 1.5°C and a 12:12 light/dark cycle was maintained, with light on at 0730 h. Except as noted below, pelleted laboratory rodent chow (Harlan Tekclad Diet 8664, 3.3 kcal/ g) and tap water were available ad lib.
2.1.1. Animals Sprague ±Dawley rat pups of both sexes were tested on P9 ±P12. The pups were derived from our breeding colony at the Developmental Psychobiology laboratory in the Psychology Department at Bar Ilam University. Lights in the colony were on from 0500 to 1700 h, and temperature was maintained between 21°C and 25°C. Each litter was housed in a polycarbonate cage (38 21 18 cm) with a stainless steel wire lid and wood shavings as bedding material. Chow and tap water were continuously available in the cage top. Newborn litters found between 0800 and 1200 h were designated as born on that day (P0) and culled to 10 pups on P1 or P2. 2.1.2. Gastric loads The volume of all gastric loads was calculated to be 5% of body weight measured on the morning of the test day. The treatments were: sham load (tube insertion into stomach, but no load), or loads containing 0.9% NaCl, distilled water, 20% (w/v) glucose (Sigma, dissolved in distilled water), 20% maltose (Sigma, dissolved in distilled water) or SBTI (Sigma, 200 mg in 1 ml of 0.9% NaCl). 2.1.3. Procedure All 10 pups from each litter were taken from the nest and dam in the morning (0800 ± 0900 h). Each pup was weighed, marked, and then the pups were placed together in a small container in a humid and warm (33°C) incubator. After 5 ±6 h, each pup was weighed, excretion was stimulated by a cotton swab, and the pup received a specific load (see below) into the stomach via a polyethylene tube (Becton Dickinson, Franklin Lake, NJ; PE-50). After 15 min, the pup was decapitated and trunk blood was collected into heparinized tubes on ice and centrifuged at 4°C. CCK was extracted from the plasma by absorption on Sep-Pak C-18 cartridges (Millipore), which were rinsed, air-dried, and
2.2.2. Procedure The rats were deprived of food, but not water, for 16 h. Subsequently, each rat received one of the following gastric loads by gavage: 5 ml distilled water, 10 ml distilled water, 5 ml SBTI (Sigma, type II, 20 mg/ml), 5 ml 0.9% NaCl, and 10 ml 0.9% NaCl. The gastric loads of 5 and 10 ml were equivalent to approximately 2.5% and 5.0% of the body weight, respectively. Fifteen minutes after gavage, the rats were anesthetized with Metafane, a midline laparotomy was performed, and the rats were exsanguinated, the blood being drawn from the inferior vena cava into heparinized syringes. Plasma was separated by centrifugation and extracted on Sep-Pak C-18 cartridges. CCK from each sample was eluted and assayed as described above. Extraction of CCK from the relatively large plasma samples available through this Table 1 Plasma CCK (pM) in rat pups after gastric loads Preload Sham 0.9% NaCl Water 20% Glucose 20% Maltose SBTI
n 6 6 10 8 6 7
Mean SEM 2.4 0.3 3.8 0.6 12.5 2.7* 3.4 0.8 1.7 0.3 11.8 3.4*
* Significantly higher CCK concentration than after sham preloads, p < 0.05.
A. Weller et al. / Physiology & Behavior 72 (2001) 1±4 Table 2 Plasma CCK (pM) in adult male rats after gastric loads Preload Sham 0.9% NaCl (5 ml) 0.9% NaCl (10 ml) Water (5 ml) Water (10 ml) SBTI
n 6 5 3 5 11 6
Median 0.47 0.00 0.00 0.34 0.77 3.18
Range 0 ± 0.80 0 ± 0.44 0 ± 0.45 0.33 ± 0.36 0 ± 2.94 2.07 ± 5.56*
* Significantly higher CCK concentration than after sham preloads, p < 0.05.
method provides an assay detection threshold of 0.3 pM of biologically active CCK. Plasma CCK was not detected in four of five rats that received 5-ml loads of 0.9% NaCl and in two of three rats that received 10-ml loads of 0.9% NaCl. Therefore, for data analysis, these groups were pooled and the non-detectable concentrations of CCK were assigned the value of zero. Because the data were not normally distributed, they were analyzed nonparametrically using Kruskal ± Wallis one-way ANOVA on ranks, followed by Dunn's method for post hoc comparison of all groups with the sham preload as the control group. 3. Results Plasma concentration of CCK differed significantly among the treatments (F(6,42) = 4.41, p < 0.01; Table 1). Plasma CCK was significantly higher after loads of water or SBTI than after other loads (p < 0.05). Plasma CCK after loads of water did not differ significantly from that after loads of SBTI. In contrast to the increases of CCK produced by loads of water or SBTI, plasma CCK after loads of 0.9% saline, 20% glucose, and 20% maltose was not significantly different from plasma CCK after sham loads. To investigate whether water also increased plasma CCK in adult rats, we measured the plasma CCK response after sham loads and loads of 0.9% saline, water, and SBTI (Table 2). Plasma CCK varied significantly after the different loads (K ± W H = 24.3, df = 4, p = 0.001). Unlike the results in pups, however, neither 5 nor 10-ml loads of water increased plasma CCK significantly compared to sham loads. The lack of effect of water or saline loads was not an artifact of the bioassay because SBTI increased plasma CCK significantly compared to sham loads (p < 0.05; Table 2). 4. Discussion Isotonic and hypertonic gastric loads did not increase plasma CCK significantly more than sham loads did in pups on P9± P12 (Table 1). This is consistent with our previous report [4] that pretreatment with devazepide did not block
3
the inhibition of intake produced by the isotonic and hypertonic preloads. Of course, it is possible for a stimulus to release CCK from the small intestine in an amount that is sufficient to have a paracrine effect, but not sufficient to cause a detectable increase of plasma CCK. This appears to be the case for the inhibition of intake by intraduodenal infusions of maltose in adult rats. Devazepide, however, blocked the inhibitory effect of this apparently paracrine action of CCK [12], but devazepide had no effect on the inhibitory action of maltose or other loads in pups. Therefore, neither endocrine nor paracrine actions of CCK are necessary for the inhibitory effects of these loads in pups on P9 ±P12. The large increase of plasma CCK after loads of water in pups was surprising (Table 1). It appears to be an effect of water that does not continue throughout the life of the rat because it did not occur in adult rats (Table 2). The ontogeny of the release of CCK by water is unknown and requires further investigation. The function of the increased CCK after a load of water is also not clear. It does not appear to mediate the inhibition of intake produced by preloads of water because devazepide did not block it. The possibility that the circulating bioassayable CCK is inhibiting intake through a CCKB receptor mechanism in pups of this age is unlikely because the inhibition of intake produced by administration of CCK-8 at this age is not blocked by a CCKB antagonist, while it is blocked by a CCKA antagonist [9]. Note that this combination of increased plasma CCK that does not account for the inhibition of intake by water in pups is different from the effect of casein in adults Ð casein increased plasma CCK, but did not decrease intake [11]. In summary, the results demonstrate that isotonic and hypertonic loads of carbohydrates do not release sufficient CCK from the small intestine to be detected in the plasma under these experimental conditions. This is not due to immaturity of the CCK release mechanisms because loads of SBTI or water produced large increases of plasma CCK. Taken together with our previous results with devazepide [4], these data are strong evidence that the volumetric and hypertonic inhibition of intake by preloads in P9 ± P12 pups is not mediated by CCK. Thus, current evidence shows that during the preweaning period, endogenous CCK released from the small intestine only mediates the inhibition of intake produced by preloads of corn oil beginning on P15 [13]. In previous works, we showed that the CCK response to a preload of SBTI mediated the inhibitory effect of that preload on intake in pups on P9 ±P12 [7,14]. The results with preloads of water are a new phenomenon: CCK is released, but it apparently does not mediate the inhibitory effect on intake of water preloads because that inhibition was not blocked by the specific CCKA antagonist, devazepide [4].
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A. Weller et al. / Physiology & Behavior 72 (2001) 1±4
Acknowledgments The authors thank Ofra Schwartz for technical assistance and Ms. Laurel A. Torres for processing the manuscript. This research was supported by a grant from the U.S. ±Israel Binational Science Foundation. Dr. Ritter was supported by NS20561, and Dr. Smith was supported by MH40010. References [1] Weller A, Gispan IH, Smith GP. Postingestive inhibitory controls of independent ingestion in 12-day-old rats. Physiol Behav 1996;60: 361 ± 4. [2] Hall WG, Bruno JP. Inhibitory controls of ingestion in 6-day-old rat pups. Physiol Behav 1984;32:831 ± 41. [3] Kimmich GA. Intestinal absorption of sugar. In: Johnson LR, editor. Physiology of the gastrointestinal tract. New York: Raven Press, 1981. p. 1038. [4] Weller A, Gispan IH, Smith GP. Characteristics of glucose and maltose preloads that inhibit feeding in 12-day-old rats. Physiol Behav 1997;61:819 ± 22. [5] Galand G. Brush border membrane sucrose-isomaltase, maltaseglucoamylase and trehalase in mammals. Comp Biochem Physiol 1989;94B:1 ± 11.
[6] Weller A, Tsitolovskya L, Gispan IH, Smith GP. Ontogeny of hypertonic preabsorptive inhibitory control of intake in neonatal rats. Am J Physiol 2000;278:R44 ± 9. [7] Weller A, Smith GP, Gibbs J. Endogenous cholecystokinin reduces feeding in young rats. Science 1990;247:1589 ± 91. [8] Robinson PH, Moran TH, McHugh PR. Cholecystokinin inhibits independent ingestion in neonatal rats. Am J Physiol 1988;255:R14 ± 20. [9] Smith GP, Tyrka A, Gibbs J. Type-A CCK receptors mediate the inhibition of food intake and activity by CCK-8 in 9- to 12-day old rat pups. Pharmacol Biochem Behav 1991;38:207 ± 10. [10] Liddle RA, Goldfine ID, Williams JA. Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor, and alcohol. Gastroenterology 1984;87:542 ± 9. [11] Brenner L, Yox DP, Ritter RC. Suppression of sham feeding by intraintestinal nutrients is not correlated with plasma cholecystokinin elevation. Am J Physiol 1993;264:R972 ± 6. [12] Yox DP, Brenner L, Ritter RC. CCK-receptor antagonists attenuate suppression of sham feeding by intestinal nutrients. Am J Physiol 1992;262:R554 ± 61. [13] Weller A, Gispan IH, Armony-Sivan R, Ritter RC, Smith GP. Preloads of corn oil inhibit independent ingestion on postnatal day 15 in rats. Physiol Behav 1997;62:871 ± 4. [14] Weller A, Corp ES, Tyrka A, Ritter RC, Brenner L, Gibbs J, Smith GP. Trypsin inhibitor and maternal reunion increase plasma cholecystokinin in neonatal rats. Peptides 1992;13: 939 ± 41.