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Endogenous cholecystokinin’s satiating action increases during estrus in female rats
Peptides 20 (1999) 451– 456
Endogenous cholecystokinin’s satiating action increases during estrus in female rats Lisa A. Eckel*, Nori Geary Joan and Sanford I. Weill Medical College of Cornell University, and the Edward W. Bourne Laboratory, New York Presbyterian Hospital-Westchester Division, White Plains, NY 10605, USA Received 24 August 1998; accepted 5 November 1998
Abstract Food intake and meal size are reduced in female Long–Evans rats during estrus. To investigate the contribution of the satiating action of endogenous cholecystokinin (CCK) to this, rats were injected with 1 mg/kg of the potent, selective CCKA receptor antagonist, devazepide, during diestrus, when meal size is maximal, and during estrus, when it is minimal. Devazepide increased spontaneous food intake and meal size during estrus, but not during diestrus. Meal frequency was not affected by devazepide treatment. These results indicate that the potency of the CCK satiety-signaling system increases during estrus. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Devazepide; Estradiol; Meal size; Meal patterns; Satiety; Ovarian cycle
1. Introduction Ovarian cycles are associated with numerous physiological and behavioral changes. For example, the increased sexual receptivity and locomotor activity that occur during estrus are accompanied by decreased food intake under many conditions in rats [2,4,9,28,33]. The decreased feeding occurs because rats eat smaller spontaneous meals during estrus, not because they change meal frequency [3,10, 15]. This suggests that the decreased feeding during estrus results from a selective change in the neurobiological controls of meal size rather than being secondary to an increase in competing behaviors, such as increased sexual receptivity and locomotor activity, that could divert the rats from initiating meals. The orosensory and postingestive consequences of feeding stimulate physiological feedback mechanisms that are integrated by central neural networks to control meal size [29]. Positive feedback signals arise primarily from the mouth and tend to sustain the meal. Negative feedback signals arise primarily from the stomach and small intestine and promote meal termination. Extensive research in male rats indicates that cholecystokinin (CCK) is one of the
* Corresponding author. Tel.: ⫹1-914-997-5935; fax: ⫹1-914-682-3793. E-mail address: [email protected] (L.A. Eckel)
negative feedback controls of meal size [23,27,30]. During a meal, CCK is released from the intestine in response to nutrient stimulation, acts on low affinity CCKA receptors in the gut, and initiates a vagal afferent satiety signal to the brain. Exogenous CCK produces a dose-dependent reduction in meal size, and antagonism of endogenous CCK increases meal size. The clearest antagonist effects come from work with devazepide, the most potent and selective CCKA receptor antagonist available [6,18]. In contrast to the extensive work in male rats, much less is known about the role of endogenous CCK in the control of meal size in female rats. There have been two studies of devazepide’s effect on feeding in intact adult female rats. Salorio et al. [26] reported that devazepide increased the size of scheduled test meals of a solution of 12.5% glucose that was presented to female rats after 5-h chow deprivation, but they did not monitor phase of the ovarian cycle. Huang et al. [17] detected no effects of devazepide on the size of chow meals induced by 18-h deprivation in female rats at any phase of the ovarian cycle. Therefore, we investigated the effects of devazepide-induced CCKA receptor blockade on spontaneous feeding at different phases of the ovarian cycle: diestrus, when meal size and food intake are maximal, and estrus, when they are minimal. Devazepide increased meal size and food intake during estrus, but not during diestrus. Thus, an enhancement in the potency of the peripheral CCK satiety-signaling system appears to contrib-
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ute to the decrease in meal size and food intake during estrus in female rats.
2. Method 2.1. Animals and housing Fourteen adult female Long–Evans hooded rats (Charles River Breeding Laboratories, Wilmington, MA) were housed individually in Plexiglas cages (floor area 475 cm2; height 50 cm) connected to stainless steel running wheels (35 cm in diameter) by a 5-cm Plexiglas tube (7 cm in diameter). A feeding niche (8 ⫻ 9 ⫻ 13 cm) protruded from each cage about 4 cm above the wire mesh floor. A circular opening (4.5 cm in diameter) in the floor of each niche allowed access to a spill-resistant food bowl that was mounted on an electronic balance (EW 300, A&D, Tokyo, Japan; ⫾ 0.1 g). Rats were given continual access to ground rat chow (#5001, Ralston Purina, St. Louis, MO) and tap water. The room was maintained at 20 ⫾ 2°C with a 12 light:12 dark cycle (lights off, 1:00 p.m. to 1:00 a.m.). A moderate-intensity white noise generator (Lafayette Instruments, Lafayette, IN) masked extraneous noise except from 8:30 to 9:30 a.m., when daily maintenance was done. The rats were adapted to the housing conditions and daily maintenance for at least 8 days before tests began. 2.2. Meal patterns The electronic balances were connected via an interface (Plus 8, Stargate Technologies, Solon, OH) into a computer. Custom-designed software recorded the weight of each balance at 30 s intervals (VZM Software, Entwicklung Kru¨gel, Munich, Germany), and converted these data into spontaneous feeding patterns (Cafe´ Mahlzeit, T.A. Houpt, Florida State University, Tallahassee, FL). A meal was defined as any feeding bout of at least 0.2 g that was separated from other bouts by at least 15 min as described previously [12]. With these criteria, rats consumed an average of 7–12 meals per day. Recorded meals accounted for 98% of total daily food intake. 2.3. Ovarian cycles Phase of the ovarian cycle was monitored by examination of vaginal smears taken daily 4 h prior to dark onset. A cotton swab moistened with warm physiological saline was inserted about 1.5 cm into the vagina, making gentle contact with the vaginal walls. The sample was transferred to a glass slide, fixed with alcohol (Surgipath Cytology Spray, Richmond, IL), and stained with hematoxylin and eosin (HHS-32 and HT40 –2-32, Sigma Diagnostics, St. Louis, MO). Microscopic examination of the sample was used to identify the phases of the ovarian cycle using standard criteria [20]. The first day of diestrus is characterized by
leukocytes interspersed with small clusters of non-nucleated cornified cells or nucleated epithelial cells. The second day of diestrus is characterized by leukocytes and nucleated epithelial cells. Proestrus is characterized by large clumps of round nucleated epithelial cells, the absence of leukocytes, and, occasionally, a few small clusters of cornified cells. Estrus is characterized by large clumps of non-nucleated squamous cornified cells. Because the luteinizing hormone surge, ovulation, increased sexual receptivity, and increased locomotor activity occur during the nocturnal period that precedes a fully cornified (estrus) smear, cycle phase labels were assigned to the 24-h period ending at the time of sampling. Using this scheme, metestrus, which lasts 8 –10 h in rats, was included in the first day of diestrus. Throughout the experiment, twelve rats had regular 4-day cycles and two rats had regular 5-day cycles, in which they spent an additional day in diestrus. Body weight, food intake, water intake, meal patterns, and activity levels did not differ between rats with 4- and 5-day cycles. 2.4. Procedure Each day at 8:30 a.m. the VZM system monitoring spontaneous meal patterns was halted for 0.5 h. During this time, activity (number of revolutions in the running wheels) and food intake (⫾ 0.1 g) for the previous 23.5 h, body weight (⫾ 1 g), and phase of the ovarian cycle were recorded. Beginning on the first day of diestrus, rats were adapted to a daily intraperitoneal (IP) injection of vehicle (0.5% carboxymethylcellulose dissolved in distilled water) in a volume of 1 ml/kg for one ovarian cycle. Because rats feed primarily during the nocturnal period (about 90% during adaptation here), injections were given at 12:00 p.m., 1 h prior to dark onset. The operation of the CCK satiety-signaling system was probed with devazepide (Merck, Sharpe, & Dohme Research Laboratories, West Point, PA). Devazepide is the most potent antagonist yet developed for CCKA receptors and is highly selective, with about 1000 fold less affinity for CCKB receptors than CCKA receptors and even less affinity for other receptors [6,18]. The use of devazepide to antagonize the actions of endogenous or exogenous CCK has produced crucial evidence for CCK’s physiological roles in feeding and other functions including gallbladder contraction, pancreatic exocrine secretion, gastric emptying, and gastric acid secretion [18,22,23,27,30,37]. We used a dose of 1 mg/kg devazepide because it has consistently attenuated or completely blocked the inhibitory effects of exogenous CCK on feeding and has dramatically increased feeding under several conditions when administered to male rats before meals [21,25]. Furthermore, 1 mg/kg devazepide produced a sustained increase in food intake for 21 h [25], the approximate duration of our test periods. During 2 consecutive ovarian cycles, rats received single IP injections of devazepide (1 mg/kg) suspended in 0.5% carboxymethylcellulose and vehicle alone (1 ml/kg) during the
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second day of diestrus, when meal size is maximal, and during estrus, when meal size is minimal. Cycle phases were verified by the appearance of the vaginal mucosa preceding and subsequent to the injections. A within-subjects design was used, and the order of devazepide and vehicle injections was randomized for each rat. On the intervening days (the first day of diestrus and proestrus of 4-day cycles; the first and third days of diestrus and proestrus of 5-day cycles), rats received vehicle injections. 2.5. Data analysis Changes in food intake, meal patterns, body weight and activity during the second day of diestrus and estrus were analyzed by the two-factor (cycle phase by drug treatment) repeated measures ANOVA. Food intake and meal patterns during the diurnal and nocturnal periods were analyzed separately because about 90% of total daily food intake occurred during the nocturnal period. Mean meal size during 3-h quartiles through the nocturnal period was analyzed by two-factor (nocturnal quartile by drug treatment) repeated measures ANOVA during diestrus and estrus. When significant effects were detected, differences between individual means were tested with Tukey’s honestly significant difference test. Differences were considered significant when P ⬍ 0.05. The standard error of the difference (SED) is presented as a measure of residual experiment-wide variability. Data were analyzed with SAS (Cary, NC) and BMDP (SOLO V6.0, SPSS, Chicago, IL) statistical packages.
3. Results 3.1. Food intake Devazepide increased nocturnal food intake during estrus, but not during diestrus, F(1,12) ⫽ 10.81, P ⬍ 0.005, SED ⫽ 0.5 g (Fig. 1A). The difference in nocturnal food intake between devazepide and vehicle treatment was significantly larger during estrus (5.3 ⫾ 1.5 g) than during diestrus (0.8 ⫾ 0.6 g), P ⬍ 0.01. Both devazepide- and vehicle-injected rats consumed less food during estrus than they did during diestrus (Ps ⬍ 0.05). Diurnal food intake accounted for only 5–15% of total daily food intake and was not affected by devazepide at either cycle phase, F(1,12) ⫽ 0.10, n.s., SED ⫽ 0.4 g (Fig. 1B).
Fig. 1. Nocturnal and diurnal food intake in female rats after injection of devazepide during diestrus and estrus. Devazepide (DEV) and vehicle (VEH) injections were administered 1 h prior to dark onset. Data are mean ⫾ SE. A. Devazepide increased nocturnal food intake during estrus, but not during diestrus. B. Devazepide did not alter diurnal food intake. *Nocturnal food intake after DEV larger than after VEH during estrus (P ⬍ 0.01), and DEV-VEH difference larger during estrus than diestrus (P ⬍ 0.01).
larger during estrus (0.7 ⫾ 0.2 g) than during diestrus (0.2 ⫾ 0.2 g), P ⬍ 0.05. It should be noted, however, that the typical reduction in meal size during estrus was observed in both devazepide- and vehicle-injected rats. That is, although devazepide-injected rats ate larger meals than vehicle-injected rats during estrus, these meals were still smaller than those during diestrus (see Fig. 2A). Because spontaneous meal size increases during the nocturnal period when rats have access to running wheels [15], we examined the effect of devazepide treatment on meal size during each 3 h quartile of the nocturnal period. During estrus, the effect of devazepide treatment varied by nocturnal quartile, F(3,34) ⫽ 42.00, P ⬍ 0.0001, SED ⫽ 0.2 g (Fig. 3, bottom). That is, devazepide significantly increased meal size during estrus both early in the dark (quartiles 1 and 2), when meals were very small, and late in the dark
3.2. Meal patterns Devazepide increased nocturnal feeding by increasing meal size, F(1,11) ⫽ 5.06, P ⬍ 0.05, SED ⫽ 0.1 g (Fig. 2A), not by changing meal frequency, F(1,11) ⫽ 0.44, NS, SED ⫽ 0.4 (Fig. 2B). The difference in nocturnal meal size between devazepide and vehicle treatment was significantly
Fig. 2. Devazepide increased nocturnal food intake by selectively increasing meal size. Data are mean ⫾ SE. A. Devazepide increased nocturnal meal size during estrus, but not during diestrus. B. Devazepide did not alter nocturnal meal number at either cycle phase. *Nocturnal meal size after DEV larger than after VEH during estrus (P ⬍ 0.05), and DEV-VEH difference larger during estrus than diestrus (P ⬍ 0.05).
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Fig. 4. Effects of devazepide on body weight and daily running wheel activity. Data are mean ⫾ SE. A. Devazepide increased body weight during estrus, but not during diestrus. B. Devazepide did not affect running wheel activity during either cycle phase. *Body weight after DEV larger than after VEH during estrus (P ⬍ 0.05). ‡Larger than same condition during diestrus (Ps ⬍ 0.01). Fig. 3. Devazepide increased meal size throughout the nocturnal period during estrus, but not during diestrus. Data are mean meal size per 3-h nocturnal quartile ⫾ SE. During diestrus (upper panel), devazepide failed to alter meal size during each nocturnal period. During estrus (lower panel), devazepide increased nocturnal meal size during three of the four nocturnal quartiles. Note that control meal size during the fourth nocturnal quartile of estrus, when devazepide increased meal size, was as large as control meal size during nocturnal quartiles 1 and 2 of diestrus, when devazepide was ineffective. *Nocturnal meal size after DEV larger than after VEH during estrus (Ps ⬍ 0.05).
(quartile 4), when meals approached the size of diestrus meals (Ps ⬍ 0.05). During diestrus (Fig. 3, top), devazepide did not significantly affect meal size during any 3-h nocturnal quartile. 3.3. Body weight Devazepide increased body weight during estrus, but not during diestrus, F(1,13) ⫽ 10.45, P ⬍ 0.01, SED ⫽ 0.6 g (Fig. 4A). The difference in body weight between devazepide and vehicle treatment was significantly larger during estrus (5 ⫾ 1 g) than during diestrus (1 ⫾ 1 g), P ⬍ 0.01. Body weight often decreases during estrus [3,4,15,32,34]. This occurred here in vehicle-treated rats (P ⬍ 0.05), but not in devazepide-treated rats. 3.4. Running wheel activity Devazepide did not alter activity levels during diestrus or estrus, F(1,10) ⫽ 1.48, NS, SED ⫽ 570 revolutions (Fig. 4B). Activity was increased during estrus as compared to diestrus in both groups (Ps ⬍ 0.001).
4. Discussion During estrus, when spontaneous meal size and food intake are typically smallest in Long–Evans and other
strains of rats [3,10,15], devazepide significantly increased food intake by increasing meal size, not by changing meal frequency. During diestrus, when meal size and food intake are largest [3,10,15], devazepide did not significantly increase nocturnal food intake, meal size, or meal frequency. Because devazepide is the most selective and potent antagonist of physiological functions mediated by CCKA receptors [6,12,18,27,30], these results indicate that the potency of the endogenous CCK satiety-signaling system varies across the ovarian cycle. That is, endogenous CCK contributes more to the control of meal size during estrus than during diestrus. We believe this to be the first direct indication that the satiating potency of CCK, or any other endogenous satiety mechanism, varies across the ovarian cycle. Extensive evidence indicates that CCK released from the small intestine during meals acts on low affinity CCKA receptors on vagal afferents in the pylorus or proximal duodenum to initiate a satiety signal [27,30]. This evidence includes demonstrations that infusions of CCK and devazepide into the superior pancreatic-duodenal artery are sufficient to affect feeding in doses that are inactive when infused into the general circulation [7,8] and that abdominal vagotomy blocks the effects of CCK and devazepide on feeding [13,24,31]. In view of these data, we conclude that devazepide increased meal size in female rats during estrus by acting on pyloric or proximal duodenal vagal CCKA receptors rather than on CCKA receptors at other peripheral or central sites. This change in the potency of the CCK satiety-signaling system may be due to changes in CCK secretion, CCK receptor function, transduction of receptor activation into a neural signal, or central processing of the peripheral CCK satiety signal. Devazepide’s stimulatory effect on meal size appeared physiologically and behaviorally specific. The appearance of the vaginal smears was normal during each phase of the ovarian cycle and each rat’s cycle length remained constant.
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Devazepide did not alter the ovarian rhythm of running wheel activity. Finally, the typical decreases in food intake and meal size during estrus compared to diestrus were still evident, although smaller in magnitude, after devazepide treatment. That devazepide-treated rats consumed smaller meals during estrus than diestrus indicates that CCK is not the only factor involved in mediating the reduction of meal size during estrus in female rats. For example, pancreatic glucagon [14] also appears to be involved. Because average nocturnal meal size here was almost twice as large during diestrus than estrus (3.3 ⫾ 0.2 g vs. 1.9 ⫾ 0.2 g, P ⬍ 0.05), it is possible that a ceiling masked devazepide’s effect during diestrus. That is, the rats may not have been able to increase meal size further during diestrus. Inspection of devazepide’s effects during the 3-h quartiles of the nocturnal period, however, discounts this possibility. This is because devazepide increased meal size during the fourth nocturnal quartile of estrus, but not during the first and second nocturnal quartiles of diestrus, despite that meal sizes were similar in each of these periods. Thus, the selective ability of devazepide to increase nocturnal meal size during estrus does not appear to be an artifact of the normal differences in meal size across the phases of the ovarian cycle. Rather, our findings suggest that endogenous CCK plays a greater role in the control of meal size during estrus than diestrus, presumably due to some neuroendocrine change across the ovarian cycle. We hypothesize that rising plasma estradiol levels early in the ovarian cycle enhance the potency of the CCK satiety-signaling system during estrus, and that this effect wanes by the subsequent diestrus. The results of ovariectomy indicate that the inhibitory influence of the hypothalamic-pituitary-gonadal (HPG) axis on feeding has two components: a phasic inhibition, revealed by the disappearance of the cyclic decrease in spontaneous meal size and food intake associated with estrus, and a tonic inhibition, revealed by an increase in the basal level of meal size and food intake over that occurring during any phase of the ovarian cycle in intact rats [10]. The effects of devazepide that we report here are consistent with the idea that endogenous CCK contributes to the phasic inhibitory effect of the HPG axis, but not to its tonic inhibitory effect. Devazepide increased meal size more during estrus than diestrus, indicating an involvement of endogenous CCK in the phasic inhibition of feeding during estrus. Because devazepide failed to increase food intake or meal size during diestrus, endogenous CCK does not appear to play an important role in the tonic inhibition of feeding. This is the first evidence that the phasic and tonic components of the inhibitory action of the HPG axis on feeding may be mediated by separate physiological mechanisms. Several lines of evidence link the influence of the HPG axis on meal size to variations in plasma estradiol concentration [34,35], and estradiol’s effects on meal size have been linked to CCK. In three studies, the satiating potency of exogenous CCK was increased by chronic estradiol treatment in ovariectomized rats [5,16,19]. The enhancement of
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the CCK satiety-signaling system during estrus that we report here extends these pharmacological studies by demonstrating that the normally functioning HPG axis can modulate the physiological action of endogenous CCK in the control of meal size in intact, spontaneously feeding rats. This effect of the HPG axis may be mediated by estradiol because devazepide increased meal size more in estradioltreated ovariectomized rats than in oil-treated ovariectomized rats [1,5]. An important aspect of the putative effect of estradiol on feeding is the phase lag between the changes in plasma estradiol concentration and in food intake during the ovarian cycle. This, like the similar phase lags of ovulation, sexual receptivity and increased locomotor activity, is presumed to be due to the time required by the activation of the physiological cascades initiated by the effects of estrogen receptor binding on gene expression. In three studies of exogenous CCK’s satiating effect across the ovarian cycle [11,17,36], CCK tended to decrease feeding more during diestrus than other phases of the ovarian cycle. That is, the modulation of exogenous CCK’s satiating potency was opposite to that of endogenous CCK reported here. However, it is possible that the larger amounts of baseline food intake during diestrus may permit the expression of larger inhibitory effects of exogenous CCK. Alternatively, the enhancement of the endogenous CCK satiety-signaling system during estrus that we observed here may reduce its accessibility to exogenous CCK. For example, if the enhanced satiating potency of the endogenous CCK-signaling system during estrus is due to increased CCK secretion during meals, exogenous CCK may compete less effectively for the CCKA receptors mediating satiety. In summary, devazepide produced a significantly larger increase in spontaneous meal size and food intake during estrus than diestrus in naturally cycling female Long–Evans rats. We believe this is the first report of a variation in the potency of an endogenous satiety signal across the ovarian cycle. The selective increase in meal size by devazepide during estrus, and the lack of effect of devazepide during diestrus, indicate that endogenous CCK is involved in the phasic, but not the tonic, inhibitory influence of the HPG axis on meal size. Because estradiol appears to link the HPG axis with the neural mechanisms controlling feeding, these results suggest that estradiol may act directly on interneuronal networks to produce a phasic increase in the potency of the CCK satiety-signaling system during the estrus phase of the ovarian cycle.
Acknowledgments This research was supported by a National Institute of Health Grant MH51135 (N.G.) and a Natural Sciences and Engineering Research Council of Canada Fellowship
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(L.A.E.). We thank L. Asarian, B.S., for helpful comments on a previous version of the manuscript.
References [1] Asarian L, Geary N. Cyclic estradiol treatment phasically potentiates endogenous cholecystokinin’s satiating action in ovariectomized rats. Peptides 1999;20:445–50. [2] Bartness TJ, Waldbillig RJ. Dietary self-selection in intact, ovariectomized, and estradiol-treated female rats. Behav Neurosci 1984;98: 125–37. [3] Blaustein JD, Wade GN. Ovarian influences on the meal patterns of female rats. Physiol Behav 1976;17:201– 8. [4] Brobeck JR, Wheatland M, Strominger JL. Variations in regulation of energy exchange associated with estrus, diestrus and pseudopregnancy in rats. Endocrinology 1947;40(2):65–72. [5] Butera PC. CNS steroids and the control of feeding. In: Stone TW, ed. CNS neurotransmitters and neuromodulators: neuroactive steroids. New York: CRC Press; 1996:211–35. [6] Chang RSL, Lotti VJ. L-364,718: a review of the biochemical and pharmacological characterization as a highly potent peripherally selective CCK antagonist. In: Wang RY, Schoenfeld R, eds. Cholecystokinin Antagonists. New York: Alan R. Liss, Inc.; 1988:13–28. [7] Cox JE. Cholecystokinin satiety involves CCKA receptors perfused by the superior pancreaticoduodenal artery. Am J Physiol 1998;274: R1390 – 6. [8] Cox JE, McCown SM, Bridges JM, Tyler WJ. Inhibition of sucrose intake by continuous celiac, superior mesenteric and intravenous CCK-8 infusions. Am J Physiol 1996;270:R319 –25. [9] Drewett RF. Oestrous and dioestrous components of the ovarian inhibition on hunger in the rat. Anim Behav 1973;21:772– 80. [10] Drewett RF. The meal patterns of the oestrous cycle and their motivational significance. Quart J Exp Psychol 1974;26:489 –94. [11] Dulawa SC, VanderWeele DA. Cholecystokinin and estradiol synergistically potentiate satiety in rats. Peptides 1994;15(5):913– 8. [12] Eckel LA, Langhans W, Kahler A, Campfield LA, Smith FJ, Geary N. Chronic administration of OB protein decreases food intake by selectively reducing meal size in female rats. Am J Physiol 1998;275: R186 –93. [13] Garlicki J, Konturek PK, Majka J, Kwiecien N, Konturek SJ. Cholecystokinin receptors and vagal nerves in control of food intake in rats. Am J Physiol 1990;258:E40 –5. [14] Geary N, Asarian L. Estradiol increases the satiating potency of endogenous glucagon in ovariectomized rats (Abstract). Appetite 31:273. [15] Geary N, Eckel LA. Decreased meal size and increased activity during estrus in rats (abstract). Int Behavioral Neurosci Soc 1997;6: 23. [16] Geary N, Trace D, McEwen B, Smith GP. Cyclic estradiol replacement increases the satiety effect of CCK-8 in ovariectomized rats. Physiol Behav 1994;56:281–9. [17] Huang YS, Doi R, Chowdhury P, Pasley JN, Nishikawa M, Huang TJ, Rayford PL. Effect of cholecystokinin on food intake at different stages of the estrous cycle in female rats. J Assoc Acad Minor Phys 1993;4:56 – 8. [18] Liddle RA. Cholecystokinin. In: Walsh JH, Dockray GJ, eds. Gut Peptides: Biochemistry and Physiology. New York: Raven Press, Ltd.; 1994:175–216.
[19] Linde´n A, Uvna¨s–Moberg K, Forsberg G, Bednar I, So¨dersten P. Involvement of cholecystokinin in food intake: III. Oestradiol potentiates the inhibitory effect of cholecystokinin octapeptide on food intake in ovariectomized rats. J Neuroendocrinol 1990;2:797– 801. [20] Long JA, Evans HM. The oestrous cycle in the rat and its associated phenomena. In: Leuschner AO, ed. Memoirs of the University of California, vol. 6. Berkely, CA: University of California Press; 1922: 1–148. [21] Moran TH, Ameglio PJ, Schwartz GJ, McHugh PR. Blockade of type A, not type B, CCK receptors attenuates satiety actions of exogenous and endogenous CCK. Am J Physiol 1992;262:R46 –50. [22] Moran TH, Field DG, Knipp S, Carrigan TS, Schwartz GJ. Endogenous CCK in the control of gastric emptying of glucose and maltose. Peptides 1997;18:547–50. [23] Moran TH, Katz LF, Plata–Salaman CR, Schwartz GJ. Disordered food intake and obesity in rats lacking cholecystokinin A receptors. Am J Physiol 1998;274:R618 –25. [24] Reidelberger RD. Abdominal vagal mediation of the satiety effects of exogenous and endogenous cholecystokinin in rats. Am J Physiol 1992;263:R1354 – 8. [25] Reidelberger RD, O’Rourke MF. Potent cholecystokinin antagonist L-364,718 stimulates food intake in rats. Am J Physiol 1989;257: R1512– 8. [26] Salorio CF, Hammond PB, Schwartz GJ, McHugh PR, Moran TH. Age-dependent effects of CCK and devazepide in male and female rats. Physiol Behav 1994;56:645– 8. [27] Schwartz GJ, Moran TH. Progress in psychobiology, and physiological psychology. In: Fluharty S, Morrison A, eds. Integrative gastrointestinal actions of the brain-gut peptide cholecystokinin in satiety. New York: Academic Press; 1998:1–34. [28] Slonaker JR. The effect of copulation, pregnancy, pseudopregnancy and lactation on the voluntary activity and food consumption of the albino rat. Am J Physiol 1925;71:362–94. [29] Smith GP. The direct and indirect controls of meal size. Neurosci Biobehav Rev 1996;20:41– 6. [30] Smith GP, Gibbs J. The development and proof of the CCK hypothesis of satiety. In: Dourish CT, Cooper SJ, Iversen SD, Iversen LL, eds. Multiple cholecystokinin receptors in the CNS. Oxford: Oxford University Press; 1992:166 – 82. [31] Smith GP, Jerome C, Norgren R. Afferent axons in abdominal vagus mediate satiety effect of cholecystokinin in rats. Am J Physiol 1985; 249:R638 – 41. [32] Tarttelin MF, Gorski RA. The effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinol 1973;72:551– 68. [33] ter Haar MB. Circadian and estrual rhythms in food intake in the rat. Horm Behav 1972;3:213–9. [34] Wade GN. Gonadal hormones and behavioral regulation of body weight. Physiol Behav 1972;8:523–34. [35] Wade GN. Some effects of ovarian hormones on food intake and body weight in female rats. J Comp Physiol Psychol 1975;88:183– 93. [36] Wager–Srdar SA, Gannon M, Levine AS. The effect of cholecystokinin on food intake in gonadectomized and intacts rats: the influence of sex hormones. Physiol Behav 1987;40:25– 8. [37] Wank SA. G protein-coupled receptors in gastrointestinal physiology. I. CCK receptors: an exemplary family. Am J Physiol 1998;274: G607–13.
Endogenous cholecystokinin’s satiating action increases during estrus in female rats Lisa A. Eckel*, Nori Geary Joan and Sanford I. Weill Medical College of Cornell University, and the Edward W. Bourne Laboratory, New York Presbyterian Hospital-Westchester Division, White Plains, NY 10605, USA Received 24 August 1998; accepted 5 November 1998
Abstract Food intake and meal size are reduced in female Long–Evans rats during estrus. To investigate the contribution of the satiating action of endogenous cholecystokinin (CCK) to this, rats were injected with 1 mg/kg of the potent, selective CCKA receptor antagonist, devazepide, during diestrus, when meal size is maximal, and during estrus, when it is minimal. Devazepide increased spontaneous food intake and meal size during estrus, but not during diestrus. Meal frequency was not affected by devazepide treatment. These results indicate that the potency of the CCK satiety-signaling system increases during estrus. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Devazepide; Estradiol; Meal size; Meal patterns; Satiety; Ovarian cycle
1. Introduction Ovarian cycles are associated with numerous physiological and behavioral changes. For example, the increased sexual receptivity and locomotor activity that occur during estrus are accompanied by decreased food intake under many conditions in rats [2,4,9,28,33]. The decreased feeding occurs because rats eat smaller spontaneous meals during estrus, not because they change meal frequency [3,10, 15]. This suggests that the decreased feeding during estrus results from a selective change in the neurobiological controls of meal size rather than being secondary to an increase in competing behaviors, such as increased sexual receptivity and locomotor activity, that could divert the rats from initiating meals. The orosensory and postingestive consequences of feeding stimulate physiological feedback mechanisms that are integrated by central neural networks to control meal size [29]. Positive feedback signals arise primarily from the mouth and tend to sustain the meal. Negative feedback signals arise primarily from the stomach and small intestine and promote meal termination. Extensive research in male rats indicates that cholecystokinin (CCK) is one of the
* Corresponding author. Tel.: ⫹1-914-997-5935; fax: ⫹1-914-682-3793. E-mail address: [email protected] (L.A. Eckel)
negative feedback controls of meal size [23,27,30]. During a meal, CCK is released from the intestine in response to nutrient stimulation, acts on low affinity CCKA receptors in the gut, and initiates a vagal afferent satiety signal to the brain. Exogenous CCK produces a dose-dependent reduction in meal size, and antagonism of endogenous CCK increases meal size. The clearest antagonist effects come from work with devazepide, the most potent and selective CCKA receptor antagonist available [6,18]. In contrast to the extensive work in male rats, much less is known about the role of endogenous CCK in the control of meal size in female rats. There have been two studies of devazepide’s effect on feeding in intact adult female rats. Salorio et al. [26] reported that devazepide increased the size of scheduled test meals of a solution of 12.5% glucose that was presented to female rats after 5-h chow deprivation, but they did not monitor phase of the ovarian cycle. Huang et al. [17] detected no effects of devazepide on the size of chow meals induced by 18-h deprivation in female rats at any phase of the ovarian cycle. Therefore, we investigated the effects of devazepide-induced CCKA receptor blockade on spontaneous feeding at different phases of the ovarian cycle: diestrus, when meal size and food intake are maximal, and estrus, when they are minimal. Devazepide increased meal size and food intake during estrus, but not during diestrus. Thus, an enhancement in the potency of the peripheral CCK satiety-signaling system appears to contrib-
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ute to the decrease in meal size and food intake during estrus in female rats.
2. Method 2.1. Animals and housing Fourteen adult female Long–Evans hooded rats (Charles River Breeding Laboratories, Wilmington, MA) were housed individually in Plexiglas cages (floor area 475 cm2; height 50 cm) connected to stainless steel running wheels (35 cm in diameter) by a 5-cm Plexiglas tube (7 cm in diameter). A feeding niche (8 ⫻ 9 ⫻ 13 cm) protruded from each cage about 4 cm above the wire mesh floor. A circular opening (4.5 cm in diameter) in the floor of each niche allowed access to a spill-resistant food bowl that was mounted on an electronic balance (EW 300, A&D, Tokyo, Japan; ⫾ 0.1 g). Rats were given continual access to ground rat chow (#5001, Ralston Purina, St. Louis, MO) and tap water. The room was maintained at 20 ⫾ 2°C with a 12 light:12 dark cycle (lights off, 1:00 p.m. to 1:00 a.m.). A moderate-intensity white noise generator (Lafayette Instruments, Lafayette, IN) masked extraneous noise except from 8:30 to 9:30 a.m., when daily maintenance was done. The rats were adapted to the housing conditions and daily maintenance for at least 8 days before tests began. 2.2. Meal patterns The electronic balances were connected via an interface (Plus 8, Stargate Technologies, Solon, OH) into a computer. Custom-designed software recorded the weight of each balance at 30 s intervals (VZM Software, Entwicklung Kru¨gel, Munich, Germany), and converted these data into spontaneous feeding patterns (Cafe´ Mahlzeit, T.A. Houpt, Florida State University, Tallahassee, FL). A meal was defined as any feeding bout of at least 0.2 g that was separated from other bouts by at least 15 min as described previously [12]. With these criteria, rats consumed an average of 7–12 meals per day. Recorded meals accounted for 98% of total daily food intake. 2.3. Ovarian cycles Phase of the ovarian cycle was monitored by examination of vaginal smears taken daily 4 h prior to dark onset. A cotton swab moistened with warm physiological saline was inserted about 1.5 cm into the vagina, making gentle contact with the vaginal walls. The sample was transferred to a glass slide, fixed with alcohol (Surgipath Cytology Spray, Richmond, IL), and stained with hematoxylin and eosin (HHS-32 and HT40 –2-32, Sigma Diagnostics, St. Louis, MO). Microscopic examination of the sample was used to identify the phases of the ovarian cycle using standard criteria [20]. The first day of diestrus is characterized by
leukocytes interspersed with small clusters of non-nucleated cornified cells or nucleated epithelial cells. The second day of diestrus is characterized by leukocytes and nucleated epithelial cells. Proestrus is characterized by large clumps of round nucleated epithelial cells, the absence of leukocytes, and, occasionally, a few small clusters of cornified cells. Estrus is characterized by large clumps of non-nucleated squamous cornified cells. Because the luteinizing hormone surge, ovulation, increased sexual receptivity, and increased locomotor activity occur during the nocturnal period that precedes a fully cornified (estrus) smear, cycle phase labels were assigned to the 24-h period ending at the time of sampling. Using this scheme, metestrus, which lasts 8 –10 h in rats, was included in the first day of diestrus. Throughout the experiment, twelve rats had regular 4-day cycles and two rats had regular 5-day cycles, in which they spent an additional day in diestrus. Body weight, food intake, water intake, meal patterns, and activity levels did not differ between rats with 4- and 5-day cycles. 2.4. Procedure Each day at 8:30 a.m. the VZM system monitoring spontaneous meal patterns was halted for 0.5 h. During this time, activity (number of revolutions in the running wheels) and food intake (⫾ 0.1 g) for the previous 23.5 h, body weight (⫾ 1 g), and phase of the ovarian cycle were recorded. Beginning on the first day of diestrus, rats were adapted to a daily intraperitoneal (IP) injection of vehicle (0.5% carboxymethylcellulose dissolved in distilled water) in a volume of 1 ml/kg for one ovarian cycle. Because rats feed primarily during the nocturnal period (about 90% during adaptation here), injections were given at 12:00 p.m., 1 h prior to dark onset. The operation of the CCK satiety-signaling system was probed with devazepide (Merck, Sharpe, & Dohme Research Laboratories, West Point, PA). Devazepide is the most potent antagonist yet developed for CCKA receptors and is highly selective, with about 1000 fold less affinity for CCKB receptors than CCKA receptors and even less affinity for other receptors [6,18]. The use of devazepide to antagonize the actions of endogenous or exogenous CCK has produced crucial evidence for CCK’s physiological roles in feeding and other functions including gallbladder contraction, pancreatic exocrine secretion, gastric emptying, and gastric acid secretion [18,22,23,27,30,37]. We used a dose of 1 mg/kg devazepide because it has consistently attenuated or completely blocked the inhibitory effects of exogenous CCK on feeding and has dramatically increased feeding under several conditions when administered to male rats before meals [21,25]. Furthermore, 1 mg/kg devazepide produced a sustained increase in food intake for 21 h [25], the approximate duration of our test periods. During 2 consecutive ovarian cycles, rats received single IP injections of devazepide (1 mg/kg) suspended in 0.5% carboxymethylcellulose and vehicle alone (1 ml/kg) during the
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second day of diestrus, when meal size is maximal, and during estrus, when meal size is minimal. Cycle phases were verified by the appearance of the vaginal mucosa preceding and subsequent to the injections. A within-subjects design was used, and the order of devazepide and vehicle injections was randomized for each rat. On the intervening days (the first day of diestrus and proestrus of 4-day cycles; the first and third days of diestrus and proestrus of 5-day cycles), rats received vehicle injections. 2.5. Data analysis Changes in food intake, meal patterns, body weight and activity during the second day of diestrus and estrus were analyzed by the two-factor (cycle phase by drug treatment) repeated measures ANOVA. Food intake and meal patterns during the diurnal and nocturnal periods were analyzed separately because about 90% of total daily food intake occurred during the nocturnal period. Mean meal size during 3-h quartiles through the nocturnal period was analyzed by two-factor (nocturnal quartile by drug treatment) repeated measures ANOVA during diestrus and estrus. When significant effects were detected, differences between individual means were tested with Tukey’s honestly significant difference test. Differences were considered significant when P ⬍ 0.05. The standard error of the difference (SED) is presented as a measure of residual experiment-wide variability. Data were analyzed with SAS (Cary, NC) and BMDP (SOLO V6.0, SPSS, Chicago, IL) statistical packages.
3. Results 3.1. Food intake Devazepide increased nocturnal food intake during estrus, but not during diestrus, F(1,12) ⫽ 10.81, P ⬍ 0.005, SED ⫽ 0.5 g (Fig. 1A). The difference in nocturnal food intake between devazepide and vehicle treatment was significantly larger during estrus (5.3 ⫾ 1.5 g) than during diestrus (0.8 ⫾ 0.6 g), P ⬍ 0.01. Both devazepide- and vehicle-injected rats consumed less food during estrus than they did during diestrus (Ps ⬍ 0.05). Diurnal food intake accounted for only 5–15% of total daily food intake and was not affected by devazepide at either cycle phase, F(1,12) ⫽ 0.10, n.s., SED ⫽ 0.4 g (Fig. 1B).
Fig. 1. Nocturnal and diurnal food intake in female rats after injection of devazepide during diestrus and estrus. Devazepide (DEV) and vehicle (VEH) injections were administered 1 h prior to dark onset. Data are mean ⫾ SE. A. Devazepide increased nocturnal food intake during estrus, but not during diestrus. B. Devazepide did not alter diurnal food intake. *Nocturnal food intake after DEV larger than after VEH during estrus (P ⬍ 0.01), and DEV-VEH difference larger during estrus than diestrus (P ⬍ 0.01).
larger during estrus (0.7 ⫾ 0.2 g) than during diestrus (0.2 ⫾ 0.2 g), P ⬍ 0.05. It should be noted, however, that the typical reduction in meal size during estrus was observed in both devazepide- and vehicle-injected rats. That is, although devazepide-injected rats ate larger meals than vehicle-injected rats during estrus, these meals were still smaller than those during diestrus (see Fig. 2A). Because spontaneous meal size increases during the nocturnal period when rats have access to running wheels [15], we examined the effect of devazepide treatment on meal size during each 3 h quartile of the nocturnal period. During estrus, the effect of devazepide treatment varied by nocturnal quartile, F(3,34) ⫽ 42.00, P ⬍ 0.0001, SED ⫽ 0.2 g (Fig. 3, bottom). That is, devazepide significantly increased meal size during estrus both early in the dark (quartiles 1 and 2), when meals were very small, and late in the dark
3.2. Meal patterns Devazepide increased nocturnal feeding by increasing meal size, F(1,11) ⫽ 5.06, P ⬍ 0.05, SED ⫽ 0.1 g (Fig. 2A), not by changing meal frequency, F(1,11) ⫽ 0.44, NS, SED ⫽ 0.4 (Fig. 2B). The difference in nocturnal meal size between devazepide and vehicle treatment was significantly
Fig. 2. Devazepide increased nocturnal food intake by selectively increasing meal size. Data are mean ⫾ SE. A. Devazepide increased nocturnal meal size during estrus, but not during diestrus. B. Devazepide did not alter nocturnal meal number at either cycle phase. *Nocturnal meal size after DEV larger than after VEH during estrus (P ⬍ 0.05), and DEV-VEH difference larger during estrus than diestrus (P ⬍ 0.05).
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Fig. 4. Effects of devazepide on body weight and daily running wheel activity. Data are mean ⫾ SE. A. Devazepide increased body weight during estrus, but not during diestrus. B. Devazepide did not affect running wheel activity during either cycle phase. *Body weight after DEV larger than after VEH during estrus (P ⬍ 0.05). ‡Larger than same condition during diestrus (Ps ⬍ 0.01). Fig. 3. Devazepide increased meal size throughout the nocturnal period during estrus, but not during diestrus. Data are mean meal size per 3-h nocturnal quartile ⫾ SE. During diestrus (upper panel), devazepide failed to alter meal size during each nocturnal period. During estrus (lower panel), devazepide increased nocturnal meal size during three of the four nocturnal quartiles. Note that control meal size during the fourth nocturnal quartile of estrus, when devazepide increased meal size, was as large as control meal size during nocturnal quartiles 1 and 2 of diestrus, when devazepide was ineffective. *Nocturnal meal size after DEV larger than after VEH during estrus (Ps ⬍ 0.05).
(quartile 4), when meals approached the size of diestrus meals (Ps ⬍ 0.05). During diestrus (Fig. 3, top), devazepide did not significantly affect meal size during any 3-h nocturnal quartile. 3.3. Body weight Devazepide increased body weight during estrus, but not during diestrus, F(1,13) ⫽ 10.45, P ⬍ 0.01, SED ⫽ 0.6 g (Fig. 4A). The difference in body weight between devazepide and vehicle treatment was significantly larger during estrus (5 ⫾ 1 g) than during diestrus (1 ⫾ 1 g), P ⬍ 0.01. Body weight often decreases during estrus [3,4,15,32,34]. This occurred here in vehicle-treated rats (P ⬍ 0.05), but not in devazepide-treated rats. 3.4. Running wheel activity Devazepide did not alter activity levels during diestrus or estrus, F(1,10) ⫽ 1.48, NS, SED ⫽ 570 revolutions (Fig. 4B). Activity was increased during estrus as compared to diestrus in both groups (Ps ⬍ 0.001).
4. Discussion During estrus, when spontaneous meal size and food intake are typically smallest in Long–Evans and other
strains of rats [3,10,15], devazepide significantly increased food intake by increasing meal size, not by changing meal frequency. During diestrus, when meal size and food intake are largest [3,10,15], devazepide did not significantly increase nocturnal food intake, meal size, or meal frequency. Because devazepide is the most selective and potent antagonist of physiological functions mediated by CCKA receptors [6,12,18,27,30], these results indicate that the potency of the endogenous CCK satiety-signaling system varies across the ovarian cycle. That is, endogenous CCK contributes more to the control of meal size during estrus than during diestrus. We believe this to be the first direct indication that the satiating potency of CCK, or any other endogenous satiety mechanism, varies across the ovarian cycle. Extensive evidence indicates that CCK released from the small intestine during meals acts on low affinity CCKA receptors on vagal afferents in the pylorus or proximal duodenum to initiate a satiety signal [27,30]. This evidence includes demonstrations that infusions of CCK and devazepide into the superior pancreatic-duodenal artery are sufficient to affect feeding in doses that are inactive when infused into the general circulation [7,8] and that abdominal vagotomy blocks the effects of CCK and devazepide on feeding [13,24,31]. In view of these data, we conclude that devazepide increased meal size in female rats during estrus by acting on pyloric or proximal duodenal vagal CCKA receptors rather than on CCKA receptors at other peripheral or central sites. This change in the potency of the CCK satiety-signaling system may be due to changes in CCK secretion, CCK receptor function, transduction of receptor activation into a neural signal, or central processing of the peripheral CCK satiety signal. Devazepide’s stimulatory effect on meal size appeared physiologically and behaviorally specific. The appearance of the vaginal smears was normal during each phase of the ovarian cycle and each rat’s cycle length remained constant.
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Devazepide did not alter the ovarian rhythm of running wheel activity. Finally, the typical decreases in food intake and meal size during estrus compared to diestrus were still evident, although smaller in magnitude, after devazepide treatment. That devazepide-treated rats consumed smaller meals during estrus than diestrus indicates that CCK is not the only factor involved in mediating the reduction of meal size during estrus in female rats. For example, pancreatic glucagon [14] also appears to be involved. Because average nocturnal meal size here was almost twice as large during diestrus than estrus (3.3 ⫾ 0.2 g vs. 1.9 ⫾ 0.2 g, P ⬍ 0.05), it is possible that a ceiling masked devazepide’s effect during diestrus. That is, the rats may not have been able to increase meal size further during diestrus. Inspection of devazepide’s effects during the 3-h quartiles of the nocturnal period, however, discounts this possibility. This is because devazepide increased meal size during the fourth nocturnal quartile of estrus, but not during the first and second nocturnal quartiles of diestrus, despite that meal sizes were similar in each of these periods. Thus, the selective ability of devazepide to increase nocturnal meal size during estrus does not appear to be an artifact of the normal differences in meal size across the phases of the ovarian cycle. Rather, our findings suggest that endogenous CCK plays a greater role in the control of meal size during estrus than diestrus, presumably due to some neuroendocrine change across the ovarian cycle. We hypothesize that rising plasma estradiol levels early in the ovarian cycle enhance the potency of the CCK satiety-signaling system during estrus, and that this effect wanes by the subsequent diestrus. The results of ovariectomy indicate that the inhibitory influence of the hypothalamic-pituitary-gonadal (HPG) axis on feeding has two components: a phasic inhibition, revealed by the disappearance of the cyclic decrease in spontaneous meal size and food intake associated with estrus, and a tonic inhibition, revealed by an increase in the basal level of meal size and food intake over that occurring during any phase of the ovarian cycle in intact rats [10]. The effects of devazepide that we report here are consistent with the idea that endogenous CCK contributes to the phasic inhibitory effect of the HPG axis, but not to its tonic inhibitory effect. Devazepide increased meal size more during estrus than diestrus, indicating an involvement of endogenous CCK in the phasic inhibition of feeding during estrus. Because devazepide failed to increase food intake or meal size during diestrus, endogenous CCK does not appear to play an important role in the tonic inhibition of feeding. This is the first evidence that the phasic and tonic components of the inhibitory action of the HPG axis on feeding may be mediated by separate physiological mechanisms. Several lines of evidence link the influence of the HPG axis on meal size to variations in plasma estradiol concentration [34,35], and estradiol’s effects on meal size have been linked to CCK. In three studies, the satiating potency of exogenous CCK was increased by chronic estradiol treatment in ovariectomized rats [5,16,19]. The enhancement of
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the CCK satiety-signaling system during estrus that we report here extends these pharmacological studies by demonstrating that the normally functioning HPG axis can modulate the physiological action of endogenous CCK in the control of meal size in intact, spontaneously feeding rats. This effect of the HPG axis may be mediated by estradiol because devazepide increased meal size more in estradioltreated ovariectomized rats than in oil-treated ovariectomized rats [1,5]. An important aspect of the putative effect of estradiol on feeding is the phase lag between the changes in plasma estradiol concentration and in food intake during the ovarian cycle. This, like the similar phase lags of ovulation, sexual receptivity and increased locomotor activity, is presumed to be due to the time required by the activation of the physiological cascades initiated by the effects of estrogen receptor binding on gene expression. In three studies of exogenous CCK’s satiating effect across the ovarian cycle [11,17,36], CCK tended to decrease feeding more during diestrus than other phases of the ovarian cycle. That is, the modulation of exogenous CCK’s satiating potency was opposite to that of endogenous CCK reported here. However, it is possible that the larger amounts of baseline food intake during diestrus may permit the expression of larger inhibitory effects of exogenous CCK. Alternatively, the enhancement of the endogenous CCK satiety-signaling system during estrus that we observed here may reduce its accessibility to exogenous CCK. For example, if the enhanced satiating potency of the endogenous CCK-signaling system during estrus is due to increased CCK secretion during meals, exogenous CCK may compete less effectively for the CCKA receptors mediating satiety. In summary, devazepide produced a significantly larger increase in spontaneous meal size and food intake during estrus than diestrus in naturally cycling female Long–Evans rats. We believe this is the first report of a variation in the potency of an endogenous satiety signal across the ovarian cycle. The selective increase in meal size by devazepide during estrus, and the lack of effect of devazepide during diestrus, indicate that endogenous CCK is involved in the phasic, but not the tonic, inhibitory influence of the HPG axis on meal size. Because estradiol appears to link the HPG axis with the neural mechanisms controlling feeding, these results suggest that estradiol may act directly on interneuronal networks to produce a phasic increase in the potency of the CCK satiety-signaling system during the estrus phase of the ovarian cycle.
Acknowledgments This research was supported by a National Institute of Health Grant MH51135 (N.G.) and a Natural Sciences and Engineering Research Council of Canada Fellowship
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(L.A.E.). We thank L. Asarian, B.S., for helpful comments on a previous version of the manuscript.
References [1] Asarian L, Geary N. Cyclic estradiol treatment phasically potentiates endogenous cholecystokinin’s satiating action in ovariectomized rats. Peptides 1999;20:445–50. [2] Bartness TJ, Waldbillig RJ. Dietary self-selection in intact, ovariectomized, and estradiol-treated female rats. Behav Neurosci 1984;98: 125–37. [3] Blaustein JD, Wade GN. Ovarian influences on the meal patterns of female rats. Physiol Behav 1976;17:201– 8. [4] Brobeck JR, Wheatland M, Strominger JL. Variations in regulation of energy exchange associated with estrus, diestrus and pseudopregnancy in rats. Endocrinology 1947;40(2):65–72. [5] Butera PC. CNS steroids and the control of feeding. In: Stone TW, ed. CNS neurotransmitters and neuromodulators: neuroactive steroids. New York: CRC Press; 1996:211–35. [6] Chang RSL, Lotti VJ. L-364,718: a review of the biochemical and pharmacological characterization as a highly potent peripherally selective CCK antagonist. In: Wang RY, Schoenfeld R, eds. Cholecystokinin Antagonists. New York: Alan R. Liss, Inc.; 1988:13–28. [7] Cox JE. Cholecystokinin satiety involves CCKA receptors perfused by the superior pancreaticoduodenal artery. Am J Physiol 1998;274: R1390 – 6. [8] Cox JE, McCown SM, Bridges JM, Tyler WJ. Inhibition of sucrose intake by continuous celiac, superior mesenteric and intravenous CCK-8 infusions. Am J Physiol 1996;270:R319 –25. [9] Drewett RF. Oestrous and dioestrous components of the ovarian inhibition on hunger in the rat. Anim Behav 1973;21:772– 80. [10] Drewett RF. The meal patterns of the oestrous cycle and their motivational significance. Quart J Exp Psychol 1974;26:489 –94. [11] Dulawa SC, VanderWeele DA. Cholecystokinin and estradiol synergistically potentiate satiety in rats. Peptides 1994;15(5):913– 8. [12] Eckel LA, Langhans W, Kahler A, Campfield LA, Smith FJ, Geary N. Chronic administration of OB protein decreases food intake by selectively reducing meal size in female rats. Am J Physiol 1998;275: R186 –93. [13] Garlicki J, Konturek PK, Majka J, Kwiecien N, Konturek SJ. Cholecystokinin receptors and vagal nerves in control of food intake in rats. Am J Physiol 1990;258:E40 –5. [14] Geary N, Asarian L. Estradiol increases the satiating potency of endogenous glucagon in ovariectomized rats (Abstract). Appetite 31:273. [15] Geary N, Eckel LA. Decreased meal size and increased activity during estrus in rats (abstract). Int Behavioral Neurosci Soc 1997;6: 23. [16] Geary N, Trace D, McEwen B, Smith GP. Cyclic estradiol replacement increases the satiety effect of CCK-8 in ovariectomized rats. Physiol Behav 1994;56:281–9. [17] Huang YS, Doi R, Chowdhury P, Pasley JN, Nishikawa M, Huang TJ, Rayford PL. Effect of cholecystokinin on food intake at different stages of the estrous cycle in female rats. J Assoc Acad Minor Phys 1993;4:56 – 8. [18] Liddle RA. Cholecystokinin. In: Walsh JH, Dockray GJ, eds. Gut Peptides: Biochemistry and Physiology. New York: Raven Press, Ltd.; 1994:175–216.
[19] Linde´n A, Uvna¨s–Moberg K, Forsberg G, Bednar I, So¨dersten P. Involvement of cholecystokinin in food intake: III. Oestradiol potentiates the inhibitory effect of cholecystokinin octapeptide on food intake in ovariectomized rats. J Neuroendocrinol 1990;2:797– 801. [20] Long JA, Evans HM. The oestrous cycle in the rat and its associated phenomena. In: Leuschner AO, ed. Memoirs of the University of California, vol. 6. Berkely, CA: University of California Press; 1922: 1–148. [21] Moran TH, Ameglio PJ, Schwartz GJ, McHugh PR. Blockade of type A, not type B, CCK receptors attenuates satiety actions of exogenous and endogenous CCK. Am J Physiol 1992;262:R46 –50. [22] Moran TH, Field DG, Knipp S, Carrigan TS, Schwartz GJ. Endogenous CCK in the control of gastric emptying of glucose and maltose. Peptides 1997;18:547–50. [23] Moran TH, Katz LF, Plata–Salaman CR, Schwartz GJ. Disordered food intake and obesity in rats lacking cholecystokinin A receptors. Am J Physiol 1998;274:R618 –25. [24] Reidelberger RD. Abdominal vagal mediation of the satiety effects of exogenous and endogenous cholecystokinin in rats. Am J Physiol 1992;263:R1354 – 8. [25] Reidelberger RD, O’Rourke MF. Potent cholecystokinin antagonist L-364,718 stimulates food intake in rats. Am J Physiol 1989;257: R1512– 8. [26] Salorio CF, Hammond PB, Schwartz GJ, McHugh PR, Moran TH. Age-dependent effects of CCK and devazepide in male and female rats. Physiol Behav 1994;56:645– 8. [27] Schwartz GJ, Moran TH. Progress in psychobiology, and physiological psychology. In: Fluharty S, Morrison A, eds. Integrative gastrointestinal actions of the brain-gut peptide cholecystokinin in satiety. New York: Academic Press; 1998:1–34. [28] Slonaker JR. The effect of copulation, pregnancy, pseudopregnancy and lactation on the voluntary activity and food consumption of the albino rat. Am J Physiol 1925;71:362–94. [29] Smith GP. The direct and indirect controls of meal size. Neurosci Biobehav Rev 1996;20:41– 6. [30] Smith GP, Gibbs J. The development and proof of the CCK hypothesis of satiety. In: Dourish CT, Cooper SJ, Iversen SD, Iversen LL, eds. Multiple cholecystokinin receptors in the CNS. Oxford: Oxford University Press; 1992:166 – 82. [31] Smith GP, Jerome C, Norgren R. Afferent axons in abdominal vagus mediate satiety effect of cholecystokinin in rats. Am J Physiol 1985; 249:R638 – 41. [32] Tarttelin MF, Gorski RA. The effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinol 1973;72:551– 68. [33] ter Haar MB. Circadian and estrual rhythms in food intake in the rat. Horm Behav 1972;3:213–9. [34] Wade GN. Gonadal hormones and behavioral regulation of body weight. Physiol Behav 1972;8:523–34. [35] Wade GN. Some effects of ovarian hormones on food intake and body weight in female rats. J Comp Physiol Psychol 1975;88:183– 93. [36] Wager–Srdar SA, Gannon M, Levine AS. The effect of cholecystokinin on food intake in gonadectomized and intacts rats: the influence of sex hormones. Physiol Behav 1987;40:25– 8. [37] Wank SA. G protein-coupled receptors in gastrointestinal physiology. I. CCK receptors: an exemplary family. Am J Physiol 1998;274: G607–13.