- Email: [email protected]
Similar feeding patterns are induced by perifornical neuropeptide Y injection and by food deprivation
Brain Research 782 Ž1998. 271–280
Research report
Similar feeding patterns are induced by perifornical neuropeptide Y injection and by food deprivation Carrie Lynn Marın ´ Bivens a
a,1
, William J. Thomas
b,2
, B. Glenn Stanley
b, )
Department of Psychology and Neuroscience Research Institute, UniÕersity of California, Santa Barbara, CA 93106, USA b The Departments of Neuroscience and Psychology, UniÕersity of California, RiÕerside, CA 92521, USA Accepted 14 October 1997
Abstract Although hypothalamic injections of neuropeptide Y ŽNPY. induce robust feeding, there is little information about the patterns of feeding elicited by this peptide. To reveal these patterns, NPY Ž0, 8, 24, 78, 235 pmolr10 nl. was injected into the perifornical hypothalamus ŽPFH. of satiated adult male rats and their subsequent food intake was monitored every minute for 24 h. For comparison, feeding patterns were similarly observed following fasts of 0, 3, 6, 9, 12, and 24 h. The results demonstrated that NPY and food deprivation both produced dose- or deprivation-dependent increases in food intake that were most evident in the first 6 h. The increased intakes induced by NPY were characterized by combinations of increased meal size and frequency, with the predominant effects being increases in the size of and decreased latency to eat the first meal. Similarly, fasting progressively increased food intake by combinations of increased meal size and frequency, with the predominant effects being increases in the size of and decreased latency to eat the first meal. These similarities between NPY-induced and food deprivation-induced feeding are consistent with a stimulatory role for endogenous NPY in deprivation-induced feeding. These findings also suggest that NPY may increase eating by acting on mechanisms of both meal initiation and of meal termination. q 1998 Elsevier Science B.V. Keywords: Feeding; Food deprivation; Meal patterns; Neuropeptide Y; Perifornical hypothalamus
1. Introduction Food is usually consumed in discrete meals that vary in size and frequency in patterns characteristic of particular physiological and environmental conditions. Manipulations that enhance food intake increase either meal size or meal frequency w6,17x and the regulation of these parameters may be partially independent w32x. The neural systems regulating meal size and frequency may be distinct neurochemically and anatomically because intracranial injection of some feeding-stimulatory neurotransmitters preferentially increase meal size, while others preferentially increase meal frequency w36x. Thus, analysis of the meal patterns produced by central injection of a neurotransmitter may provide insights about which aspects of eating are
)
Corresponding author. Fax: q 1-909-787-3985; [email protected] 1 Tel.: q1-805-893-7376; fax: q1-805-893-4303. 2 Tel.: q1-909-787-5625; fax: q1-909-787-3985.
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 2 8 9 - 4
regulated by the neurons which employ that neurotransmitter. Neuropeptide Y ŽNPY., a neurochemical abundant within many neurons originating or terminating in the hypothalamus w14x, has been implicated in the regulation of eating behavior and body weight. Intracranial injection of this phylogenetically conserved peptide induces eating across a wide range of vertebrates, in neonatal as well as adult rats, and throughout the circadian cycle w52x. Endogenous NPY may contribute to feeding because manipulations that suppress endogenous NPY suppress spontaneous feeding w1,37,50,60x, and manipulations that increase food intake, such as food deprivation, may elevate NPY release, tissue content and synthesis, and gene expression w4,28,47,67x. Although these studies suggest that endogenous NPY may regulate feeding behavior, there is limited data on the patterns of eating elicited by this peptide w27,35,38,39,58x. Previous meal pattern analyses have shown that low doses of NPY injected into the paraventricular hypothalamus ŽPVN. elicits the consumption of a single meal w35,58x and that continuous intracerebroventricular Ži.c.v.. injec-
272
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
tion produces consumption of discrete meals at low doses, but induces persistent nibbling at high doses w27x. Since i.c.v. injection of NPY produces a variety of physiological effects w25x that may alter feeding patterns, we chose to characterize the feeding patterns induced by NPY microinjected into the perifornical hypothalamus ŽPFH., the most sensitive known site for NPY-induced feeding w61x. The major questions were: Is the feeding elicited by PFH injection of NPY organized into discrete meals and, if so, are both meal size and meal frequency increased? Further, since similarities in the feeding patterns induced by NPY and natural conditions may suggest shared neural substrates, we also examined feeding patterns induced by food deprivation, to determine the extent to which they were comparable. Portions of these finding have previously been presented in preliminary form w62,63x.
measured included: latency to eat, meal size, meal duration, meal frequency, and intermeal interval. Fresh food was provided 24 h before testing. 2.3. Intracranial injection procedures Using a previously described nanoliter injection system w61x, porcine NPY ŽPeninsula Laboratories, Belmont, CA. was dissolved in sterile, artificial cerebrospinal fluid ŽaCSF; Naq 147 mM, Cly 154 mM, Kq 3.0 mM, Caqq 1.2 mM, Mgqq 0.9 mM, pH s 7.4. and injected into the PFH in a volume of 10 nl. The five doses of NPY were 0, 8, 24, 78, 235 pmol Ž0, 33, 100, 330, 1000 ng, respectively. injected in counterbalanced order, with each subject tested in every condition. The rats were injected during the first hour of the light-phase of the photoperiod, with 3–5 days between tests to allow for recovery of baseline food intake.
2. Materials and methods 2.1. Subjects and surgery Twenty adult, Sprague–Dawley, male rats ŽSimonsen Laboratories, Gilroy, CA., weighing 350–400 g at the start of the experiment, were used. Ten were stereotaxically implanted with an unilateral, 26-gauge, stainless-steel, guide cannula while under Metofane anesthesia. The guide cannula was positioned 1.0 mm dorsal to the PFH. With the incisor bar at y3.3 mm, the stereotaxic coordinates were 7.2 mm anterior to the interaural line, 1.0 mm lateral to the midsagittal sinus, and 7.7 mm ventral to the surface of the skull. The cannula was fixed to the skull surface with dental acrylic and screws just penetrating the skull. Surgery was not performed on the remaining 10 animals that were used in the food-deprivation experiment. 2.2. Housing and meal pattern data collection The rats were housed individually and tested in clear, rectangular, plastic cages Ž33 = 21 = 18 cm. that were equipped with an automatic watering system, in a room with a 12 h light:12 dark h photoperiod Žlights on 08:00.. A small cubicle was attached to the outside of the cage to allow access to food bowls, which were located through a hole in the bottom of the cubicle. Food bowls were filled with a mixture of Purina Rodent Chow Ž46%., sucrose Ž37%. and Carnation Evaporated Milk Ž17%. and placed directly beneath the hole so that any spilled food would fall back into the bowl. Food bowl weight was registered by an electronic balance ŽOHAUS port-a-gram., and was recorded every minute for 24 h after NPY injection or food deprivation using meal pattern software, with all balances connected to a single IBM-compatible computer. Meals were defined as a minimum weight decrement of 0.2 g separated from other feeding episodes by at least 10 min. The variables
Fig. 1. Neuropeptide Y injection ŽA: top panel. and food deprivation ŽB: bottom panel. both enhance food intake Žmean g"S.E.M.. during the 12 h light phase ŽLight Phase Intake. immediately after injection or refeeding, respectively. Light phase intakes increase as a function of the dose of neuropeptide Y Žpm s pmol of NPY. and as a function of the hrs of food deprivation ŽDeprivation Time.. ‘Dark Phase Intake’ represent the food intakes during the 12 h of darkness that follow this light phase and the ‘Total Intake’ represents the combined light and dark phase intakes. ) p- 0.05, ) ) p- 0.01 by Dunnet’s tests compared to intakes in the same phase of the light–dark cycle after vehicle injection Ž0 pmol of neuropeptide Y. or 0 h of food deprivation.
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
273
were perfused transcardially with 10% formalin. The perfused brains were removed, frozen, and sliced in 100 m m coronal sections using a microtome. The brain sections were mounted onto slides, stained with cresyl violet and examined microscopically. One- or two-way repeated measures ANOVA or ANCOVA and post-hoc Dunnet’s tests were used to analyze the effects of deprivation period, NPY dose, photoperiod and time on meal size, meal frequency, latency to eat, intermeal interval, and feeding rate, when appropriate. The criterion for statistical significance was p F 0.05 for all statistical tests.
Fig. 2. Mean meal size Žg"S.E.M.. of the first through fourth meals ŽMeal No. 1–4. occurring during the 12 h light phase after injection of neuropeptide Y ŽA: top panel. or the first through fifth meals occurring after the termination of food deprivation ŽB: bottom panel. as a function of dose of NPY Žpm s pmol. or ‘hours of deprivation,’ respectively. ) p- 0.05, ) ) p- 0.01 by Dunnet’s tests greater than intakes in the same meal after vehicle injection Ž0 pmol of NPY. or ‘0 h of food deprivation’. For cases when no meal occurred after ‘0 h of food deprivation’ Ži.e., meals 3, 4, and 5., the statistical comparison was to the averaged size of the first and second meals. For simplicity, the data from meal number 4 was included for the 8 and 24 pmol doses of NPY even though most of those meals occurred after the onset of the dark phase.
2.4. Food depriÕation procedures Food was removed 0, 3, 6, 9, 12, or 24 h before light onset, in counterbalanced order, and was returned at the onset of the light cycle to the otherwise undisturbed subjects. Their meal patterns were measured, as described above, during the subsequent 24 h. These tests were separated by at least four days, after all subjects returned to baseline feeding patterns for a minimum of two days. 2.5. Histology and statistics After testing was completed, the animals were euthanized by carbon dioxide inhalation and cannulated rats
Fig. 3. Mean time Žmin"S.E.M.. from NPY injection ŽA: top panel. or from postfasting return of food ŽB: bottom panel. to eat the first meal ŽLatency to Meal 1., as well as the times between the first and second meal ŽIMI 1s intermeal interval 1., second and third ŽIMI 2. and third and fourth meals ŽIMI 3.. ) p- 0.05, ) ) p- 0.01 by Dunnet’s tests different from latencies or IMI in the same meal after vehicle injection Ž0 pmol of NPY. or ‘0 h of food deprivation’. For cases when no meal occurred after ‘0 h of food deprivation’ Ži.e., IMI 2 and IMI 3. then the statistical comparison was to IMI 1.
274
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
3. Results
3.2. NPY-induced meal patterns
3.1. Histology
As shown in Fig. 1A, NPY injections produced dosedependent increases in food intake of up to 230% during the light-phase Ž F4,36 s 48.4, p - 0.001.. These increases were compensated by complementary dose-dependent decreases in food intake during the subsequent dark-phase Ž F4,36 s 48.4, p - 0.001., such that there was no significant increase in total 24 h food intake Ž F4,36 s 0.48, NS..
The injection sites Žnot shown. were centered in the PFH just ventromedial to the fornix at the coronal level of the posterior border of the PVN, an area shown to be maximally sensitive to the feeding-stimulatory effect of NPY w61x.
Fig. 4. Meal patterns in the 12 h following injection of neuropeptide Y Žleft side. or postfasting return of food Žright side. averaged across subjects. The height of each bar represents the mean size Žg " S.E.M.. of that meal and the width of each bar represent its duration Žh.. Additionally, the placement of each bar on the abscissa represents the average time during the 12 h light phase in which that meal occurred and the number of bars represents the average number of meals that occurred in each condition. The shaded horizontal line in each panel provides a comparison to the mean meal size of the four meals that occurred after vehicle injection Žleft side; 0 pmol. or of the two meals that occurred in rats that were not deprived Žright side; no deprivation.. ) p - 0.05, ) ) p - 0.01 by Dunnet’s tests different from the averaged meal sizes after vehicle injection Ž0 pmol of NPY. or ‘no deprivation’.
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
As shown in Fig. 2A, increases in the size of several meals contributed to the increased intakes Ž F16,144 s 8.9, p - 0.01., with the size of the first meal being most prominently elevated. The increase in the size of the first meal was statistically significant even at 8 pmol of NPY and was extremely large at the 235 pmol dose. Specifically, in comparison to the mean first meal size of 1.5 g after vehicle injection, this meal averaged 9.6 g after injection of 235 pmol of NPY. The size of the second meal increased significantly at the 78 and 235 pmol doses, while third meal did not increase at any dose, and the fourth meal was increased significantly at 8, 24, and 235 pmol NPY. The apparent increase in the size of the fourth meal at the 8 and 24 pmol doses of NPY was probably an artifact of the inclusion of several individual meals that actually occurred during the dark phase, when the size of spontaneously occurring meals is larger w17x. This is evident in Figs. 3 and 4 Žleft panel., which shows that an average of only three meals occurred in the 12 h light-phase after injection of 8 and 24 pmol of NPY. In addition to the augmentations of meal size, both decreases in the latency to eat the first meal and increases in frequency of subsequent meals contributed to the increased light-phase food intake ŽFig. 3A.. The latency to eat the first meal declined in a progressive dose-dependent manner, from 108 min after vehicle injection to 9.5 min after injection of the highest dose of NPY Ž F4,36 s 4.6 p - 0.01.. Also shown in Fig. 3A is that the intervals between the first and second meals Ži.e., IMI 1. and between the second and third meals Ži.e., IMI 2. declined at the 78 and 235 pmol doses. In contrast, the onset of the third meal was delayed at 24 pmol, possibly because of the satiating effects of the previously ingested food. That NPY affects both meal size and meal frequency is clearly shown in the left panel of Fig. 4. The increased food intake at the lowest NPY doses Ž8 and 24 pmol. was produced almost exclusively by increased meal size and decreased latency to eat the first meal. Higher doses further enhanced these effects, and additionally increased the size and frequency of subsequent meals, such that at 235 pmol, the size of the fifth postinjection meal, which occurred nearly 7 h postinjection, was increased significantly. There were no effects of dose of NPY on the eating rate Ž0.25–0.28 grmin across doses; F4,36 s 1.8, p ) 0.1, NS. and consequently meal duration increased in proportion to meal size Že.g., the 9.6 g first meal at 235 pmol of NPY lasted 39.6 min.. No statistically significant relationship existed between meal size and the subsequent intermeal interval Ž r s y0.2; F16,144 s 0.74, p ) 0.56 by ANCOVA.. 3.3. Effects of food depriÕation As shown in Fig. 1B, food deprivation increased subsequent total daily food intake Ž F5,56 s 62.8, p - 0.001.. This was mostly due to the light-phase food intakes in-
275
creasing as a function of increased length of food deprivation Ž F5,56 s 23.2, p - 0.01.. These increases were only partially offset by deprivation-related declines in darkphase food intake Ž F5,56 s 16.38, p - 0.05., yielding a significant interaction between the light- and dark-phase intakes Ž F5,56 s 83.5, p - 0.0001.. The increases in feeding during the daytime were produced by increases both in meal size and in meal frequency. As shown in Fig. 2B, with prolonged food deprivation Ž9, 12, or 24 h. there were prominent increases in the size of the first meal Ž F5,54 s 15.6, p - 0.01.. With deprivations of 12 or 24 h, the sizes of each of the subsequent four meals also increased but to a lesser extent Žranging from F5,54 s 5.9, p - 0.05 to F5,54 s 55.3, p 0.0001, for these meals.. As shown in Fig. 3B, increased meal frequency Ž F5,54 s 11.6, p - 0.01. and decreased latency to eat the first meal Ž F5,54 s 17.7, p - 0.001. also contributed to the augmented food intake. This was particularly evident with brief deprivations of 3 or 6 h, when the increased intakes were due almost exclusively to decreases in the latency to eat the first meal ŽFig. 3B. and to increases in the number of meals occurring in the light phase ŽFig. 4, right panel. with little change in meal size. In contrast, deprivations of 9 to 24 h produced no further increases in meal frequency but rather increased meal size. The overall pattern shown in Fig. 4 Žright panel., reveals that with brief food deprivation periods the increased food intake is due to an increase in the number of meals eaten, with little or no increase in the size of the meals. With more prolonged deprivations, meal frequency reached a plateau, with further increments in food intake due to marked increases in the size of the first meal and to smaller but significant increases in the size of the subsequent three to four meals.
4. Discussion 4.1. Feeding patterns induced by PFH injection of NPY These observations demonstrate that, across a wide range of doses, the feeding elicited in rats by acute PFH injection of NPY occurs in discrete meals that are clearly separated by periods without eating. This complements earlier findings that acute PVN injection of a low dose of NPY induces the consumption of a single large meal w58x, and that continuous i.c.v. injection of low NPY doses elicited consumption of several meals, whereas, higher doses induced persistent nibbling w27x. That NPY-induced feeding in a meal-like pattern is consistent with the hypothesis that NPY affects neural pathways that mediate natural eating behavior. Consistent with earlier reports w15,58x, NPY injected during the light-phase dose-dependently increases eating. These increases were compensated by reciprocal decreases in dark-phase feeding, resulting in the maintenance of 24 h
276
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
food intakes at baseline levels across doses. The accuracy of this compensation suggests that the decreases in darkphase food intake may have been due to the delayed satiating effects of the large amounts of previously ingested food after the injected NPY became ineffective, rather than to a potential latent feeding-suppressive effect of NPY. Further, tolerance to NPY is unlikely to have accounted for the decreased eating because repeated or continuous injections do not alter sensitivity to NPY w5,42,56x. This finding is consistent with NPY acting to elicit eating, in part, by suppressing the satiating effect of food w39x. Perhaps the most salient effect of NPY was the pronounced dose-dependent increase in the size of the first meal. This augmentation was apparent even at 8 pmol, the lowest dose of NPY yet shown to significantly increase food intake; at the 235 pmol dose the first meal was enormous, larger even than the first meal eaten after a 24 h fast. This suggests that the dramatic eating reportedly produced by hypothalamic NPY injections w59x is due in large part to the ingestion of a single enormous meal soon after injection. This elicited gorging is also consistent with the hypothesis that NPY-like peptides may contribute to binging patterns in bulimic humans w7x. In addition to increasing the size of and speed to initiate the first meal, NPY increased both the size and frequency of subsequent meals. This was most evident at the 235 pmol dose, which elicited a series of four abnormally large meals in the 6–7 h after injection, a time during which only one meal occurred after control injections. This feeding pattern suggests that NPY-induced feeding was produced both by stimulating meal initiation mechanisms, as indicated by the increased meal frequency and decreased latency to eat, and by inhibiting meal termination mechanisms, as indicated by the increased meal sizes and durations w32x. To the extent that distinct meal initiation and termination mechanisms actually exist in the brain and reflect the perceptual states of hunger and satiety, these findings suggest that NPY may elicit eating both by activating brain mechanisms of hunger and by inhibiting those producing satiety. Consistent with previous reports w59x, the eating-stimulatory effects of acute NPY injection was protracted, lasting at least 6–7 h after injection of the highest dose, as indicated by the increased meal sizes at that time. These increases are especially noteworthy, as they occurred despite the expected satiating effects of the enormous amount of food that had been eaten in the hours prior to that time. Although it is possible that levels of extracellular NPY might have been maintained at high levels and thus contributed to the duration of the effects, this is unlikely to have been the main factor because brain NPY seems to be degraded rapidly, as indicated by the previously observed rapid fluctuations in extracellular levels of endogenous NPY, and by the rapid reductions in the brain levels of radiolabeled NPY postinjection w28,61x. More likely is that
NPY’s long-lasting feeding-stimulatory effects were mediated by the activation of a G protein linked second messenger systemŽs., as has been suggested previously w13x and demonstrated for NPY in other systems w23x. Additionally, mediation by second messengers might contribute to the relatively longer observed latency to eat after NPY injection, as compared to some other neurochemical stimulants of eating w31,34,55x. Consistent with this is the demonstration that the latency to eat after NPY injection is reduced to seconds when the peptide is repeatedly injected w44x, possibly because the initial injections induced relevant changes in second messenger systems, which then allowed the later injections to act rapidly. Perhaps relevant to second messenger mediation of feeding is our recent demonstration that intense eating may be elicited by PFH injections of compounds that stimulate intracellular levels of cAMP w21x. 4.2. Feeding patterns produced by fasting A striking effect of fast duration was the markedly different impact of short- and long-term deprivations on meal patterns. Short-term deprivations of up to 6 h increased food intake by substantially increasing the number of meals eaten after food was returned, without increasing the size of those meals. Longer deprivations did not further increase meal frequency, rather food intake was enhanced by increases in meal size. The first meal was always the largest, although in most cases it was only fractionally larger than the subsequent light-phase meals. Further, this meal occurred almost immediately upon the return of food with any deprivation of 6 or more hours. These findings, in contrast to most of the published literature showing that fasting increases meal size w3,33x, show that short-term fasting induces eating by increasing meal frequency without increasing the size of the first or subsequent meals. This contrast may result primarily from differences in the time of the light–dark cycle in which the subjects were refed. Unlike most other studies, which usually refed their subjects in the middle of the light- or at some point in the dark-phase, we refed the animals in the first hour of the light, a time when rats do not normally eat w64x. Studies suggest that rats do not normally eat at this time primarily because they are metabolizing the stored food and metabolic fuels that were consumed during the latter portion of the night-phase, in anticipation of the following light-phase fast w2,29,51,64x. Specifically, it has been shown that there are daily cycles of hepatic and muscle glycogen, adipose tissue, and gastric food contents, with each peaking between the latter portion of the dark phase and the first portion of the light phase, times when the stomach is hard-packed with food w2,20,32,46x. Deprivation during the latter portion of the dark-phase induces eating during the following light-phase w29x, suggesting that freely feeding subjects do not eat at this time because they are utilizing the stored, gastric food, hepatic and
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
muscle glycogen, and adipose tissue. Thus, the progressive decrease in the latency to eat the first meal after short deprivation in our study may have resulted from depletion of the smaller food stores that had been accumulated prior to the fast. With 3 h of deprivation, these stores were presumably sufficient to last until about 3 to 4 h after light onset w45x, the time when 3 h deprived rat typically ate their first meal. At 6 h of deprivation, the first meal occurred almost immediately and was essentially of normal size, suggesting that the gastricrhepatic reserves were depleted Žor reduced to a critical level., provoking the consumption of a single meal. The subsequent meals are suggested to occur as the food from each preceding meal is metabolized to some critical level w32x. With longer deprivations Ž9 to 24 h., the size of the first immediately occurring meal and the size of subsequent meals increased progressively, presumably in response to signals relating to longer-term fuel sources. Other studies, examining eating patterns at other points in the light–dark cycle, observed increases in meal size, we suggest, because there were insufficient gastric or hepatic fuel reserves to blunt the effects of fasting on meal size. 4.3. Comparisons of feeding patterns produced by fasting and NPY injection Similar patterns of eating produced by fasting and NPY injection would support but not prove actions via shared neural substrates. However, even under ideal circumstances, the bolus injection of a single neurotransmitter into a single brain area can simulate only limited aspects of the patterned release of a multitude of interacting neurochemicals occurring within many different brain regions during normal eating. Therefore differences in feeding patterns should be expected. Given this, there is a surprisingly good correspondence between the feeding patterns produced by fasting and NPY injection. Among these are that: the animals ate food in meals; there were progressive increases in both meal size and meal frequency produced by increased levels of fasting and doses of NPY; the largest effects were the increases in the size of the first meals and; the sizes of subsequent meals were also increased but to a lesser extent. These similarities were most apparent between the highest dose of NPY and the longest fasts. In those circumstances, the increased intakes were due to the consumption of an initial enormous meal, followed for several hours by a series of smaller but still oversized meals. There were also distinct differences between the feeding patterns produced by progressive fasting and doses of NPY. With fasting, increased intakes were first expressed by increased numbers of meals, with no increase in meal size; with longer deprivation by increased meal size without any further increases in meal frequency. In contrast, with increased NPY doses meal size and meal frequency increased in parallel. Further, the rate of eating
277
increased with longer fasts but not with higher doses of NPY. These similarities are consistent with previous evidence suggesting that increase activity of endogenous NPY might participate in mediating the increased eating that occurs after fasting. This was first suggested by evidence that tissue concentrations of NPY increase within the PVN of fasted rats and normalize with refeeding w47x. This pattern also applies to NPY gene expression w10,43x and extracellular concentrations of NPY w28x. That this increase in hypothalamic NPY activity may be necessary for the normal expression of feeding has been suggested by studies showing that i.c.v. or PVN injection of antisera to NPY significantly reduces eating induced by food deprivation w50,60x. The similar patterns of feeding produced by fasting and NPY injection in the present study adds to this evidence, by showing that increasing the activity of NPY within the PFH is sufficient to reproduce some aspects of the feeding patterns produced by fasting. As to the observed differences in the rates and patterns of eating induced by NPY and fasting, it is unclear to what extent these were due to: patterns of endogenous NPY release that were not replicated by the bolus injection; actions of endogenous NPY within multiple brain sites in fasted rats; the modulation of NPY’s actions by other neurotransmitters in the intact animal and; actions of neurotransmitters other than or in addition to NPY in the mediation of eating in the fasted rat. It may also be noteworthy that in previous comparisons of NPY’s and deprivation’s effects on behavioral patterns that substantial differences were observed w38,39x. However, in those studies the NPY was administered i.c.v. Since NPY administered by this route has been shown to produce a variety of behavioral, autonomic and endocrine effects in addition to feeding stimulation w22,25x, it seems likely that these other effects would have contaminated those that might otherwise have been produced by a more specific stimulation of eating control mechanisms. 4.4. InÕolÕement of NPY in other conditions associated with enhanced feeding It has been suggested that NPY participates in increased eating in a wide range of physiological conditions w52x. Among these are circadian rhythms of eating; overeating in the obese Zucker rat; and overeating in diabetics. Each of these will be briefly discussed, with emphasis on comparing the feeding patterns associated with each of these conditions to those elicited by NPY injection. NPY has been implicated in the increased eating that occurs at the onset of the active phase of the light–dark cycle w27,54,65x. Supporting this is that: PVN and lateral hypothalamic NPY levels increase at the onset of the dark-phase in rats w24,40x; feeding during this time was suppressed by PVN injection of a putative NPY antagonist
278
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
w37x; rats eating at the onset of the dark phase and those injected with NPY prefer carbohydrate diets w35,42,54,66x; and the sensitivity of the PVN to NPY exhibits a circadian rhythm, with a peak a the onset of the dark phase w65x. Here we show that NPY can increase both meal size and frequency. This may support a role for endogenous NPY in nocturnal eating, since meal size and frequency both increase in spontaneously eating rats at the onset of the dark phase w17,30x. There are parallels between the NPY induced feeding patterns we observed and those of obese Zucker rats that support a role of NPY in this form of genetic obesity. Zucker rats overeat primarily by consuming extremely large meals with a marginal increase in meal frequency w6,12,19x. We found that acute injections of NPY produce a similar pattern, rats ate large meals and meal frequency increased slightly. Similarly, chronic injections of NPY reportedly produce Zucker-obese-like feeding patterns and obesity w5x. Further, fat and carbohydrate-rich foods are preferred both by the Zucker obese and NPY-treated rat w19,53x, and the eating effect is dependent on circulating corticosterone levels in each condition w11,57x. The Zucker obese rat has higher levels of NPY and NPY mRNA in the hypothalamus than does its lean counterpart w4,48x. Additionally, NPY-injected and Zucker obese rats have other behavioral, hormonal and metabolic commonalties, such as decreased metabolic rates and suppressed reproductive function w4,8,9,41x. Enhanced NPY activity has also been suggested to participate in the overeating exhibited by diabetic animals w68x. Streptozotocin ŽSTZ. diabetic rats reportedly eat moderately larger meals during the entire photoperiod and increase their light-phase meal frequency w18x. We found that acute injections of NPY stimulate feeding in a similar manner, consistent with a role in diabetic hyperphagia. Supportive of this idea, STZ diabetic animals have higher levels of NPY and NPY mRNA in the hypothalamus w26,67,68x. The lack of insulin may partially cause the rise in NPY levels, since central insulin decreases the production of NPY in the arcuate nucleus w49x. Conversely, under conditions of hypoglycemia induced by hyperinsulinemia, hypothalamic NPY levels do not increase w16x. Collectively, these finding support a role for NPY in the overeating exhibited by diabetic rats. However, several aspects of diabetic meal patterns are different from NPY-induced meal patterns w18x and thus, caution is warranted. In conclusion, the present results demonstrating that PFH injection of NPY produces overeating by increasing both meal size and meal frequency adds to the evidence suggesting a role for this peptide in feeding induced by fasting, that occurring in the active phase of the light–dark cycle, that associated with some forms of genetic obesity, and that exhibited by diabetics. Collectively, this suggests that NPY may mediate normal eating in a wide variety of circumstances, as well as the abnormal eating associated with a number of conditions.
Acknowledgements We thank Jack B. Calderone for statistical and graphical consultation. Supported by NIH grant NS 24268.
References w1x A. Akabayashi, C. Wahlestedt, J.T. Alexander, S.F. Leibowitz, Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding behavior and insulin secretion, Mol. Brain Res. 21 Ž1994. 55–61. w2x S. Armstrong, J. Clarke, G. Coleman, Light–dark variations in laboratory rat stomach and small intestine content, Physiol. Behav. 21 Ž1978. 785–788. w3x J.K. Bare, G. Cicala, Deprivation and time of testing as determinants of food intake, J. Comp. Physiol. Psychol. 53 Ž1960. 151–154. w4x B. Beck, A. Burlet, J.-P. Nicolas, C. Burlet, Hypothalamic neuropeptide Y ŽNPY. in obese Zucker rats: implications in feeding and sexual behavior, Physiol. Behav. 47 Ž1990. 449–453. w5x B. Beck, A. Stricker-Krongrad, J.-P. Nicolas, C. Burlet, Chronic and continuous intracerbroventricular infusion of neuropeptide Y in Long–Evans rats mimics the feeding behavior of obese Zucker rats, Int. J. Obesity 16 Ž1992. 295–302. w6x E.E. Becker, J.A. Grinker, Meal patterns in the genetically obese Zucker rat, Physiol. Behav. 18 Ž1977. 685–692. w7x W.H. Berrettini, W.H. Kaye, H. Gwirtsman, A. Allbright, Cerebrospinal fluid peptide YY immunoreactivity in eating disorders, Neuropsychobiology 19 Ž1988. 121–124. w8x C.J. Billington, J.E. Briggs, M. Grace, A.S. Levine, Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism, Am. J. Physiol. 260 Ž1991. R321–R327. w9x C.L. Bivens, H.J. Carlisle, Intrahypothalamic injection of neuropeptide Y reduces metabolic rate and increases behavioral thermoregualtion, Soc. Neurosci. Abstr. 18 Ž1992. 373–375. w10x L.S. Brady, M.A. Smith, P.W. Gold, M. Herkenham, Altered expression of hypothalamic neuropeptide Y mRNAs in food-restricted and food-deprived rats, Neuroendocrinology 52 Ž1990. 441–447. w11x T.W. Castonguay, M.F. Dallman, J.S. Stern, Some metabolic and behavioral effects of adrenalectomy on obese Zucker rat, Am. J. Physiol. 251 Ž1986. R923–R933. w12x T.W. Castonguay, D.E. Upton, P.M.B. Leung, J.S. Stern, Meal patterns in the genetically obese Zucker rat: a reexamination, Physiol. Behav. 28 Ž1982. 911–916. w13x W.T. Chance, S. Sheriff, T. Foley-Nelson, J.E. Fischer, A. Balasubramaniam, Pertussis toxin inhibits neuropeptide Y-induced feeding in rats, Peptides 10 Ž1989. 1283–1286. w14x B.M. Chronwall, D.A. DiMaggio, V.J. Massari, V.M. Pickel, D.A. Ruggiero, T.L. O’Donohue, The anatomy of neuropeptide Y-containing neurons in the rat brain, Neuroscience 15 Ž1985. 1159–1181. w15x J.T. Clark, P.S. Kalra, W.R. Crowley, S.P. Kalra, Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology 115 Ž1984. 427–429. w16x S.E. Corrin, H.D. McCarthy, P.E. McKibbin, G. Williams, Unchanged hypothalamic neuropeptide Y concentrations in hyperphagic, hypoglycemic rats: evidence for specific metabolic regulation of hypothalamic NPY, Peptides 12 Ž1991. 425–430. w17x R.F. Davies, Long- and short-term regulation of feeding patterns in the rat, J. Comp. Physiol. Psychol. 91 Ž1977. 574–585. w18x J.M. de Castro, S. Balagura, Meal patterning in the streptozotocindiabetic rat, Physiol. Behav. 15 Ž1975. 259–263. w19x M.P. Enns, J.A. Grinker, Dietary self-selection and meal patterns of obese and lean Zucker rats, Appetite 4 Ž1983. 281–293.
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280 w20x R.W. Fuller, E.R. Diller, Diurnal varietions of liver glycogen and plasma free fatty acids in rats fed ad libitum or single daily meal, Metabolism 19 Ž1970. 226–229. w21x E.R. Gillard, A.M. Khan, A.U. Haq, R.S. Grewal, B. Mouradi, B.G. Stanley, Stimulation of eating by the second messenger cAMP in the perifornical and lateral hypothalamus, Am. J. Physiol. 273 Ž1997. R107–R112. w22x A. Harfstrand, Brain neuropeptide Y mechanisms. Basic aspects and involvement in cardiovascular and neuroendocrine regulation, Acta Physiol. Scand. 565 ŽSuppl.. Ž1987. 1–83. w23x H. Herzog, Y.J. Hort, H.J. Ball, G. Hayes, J. Shine, L.A. Selbie, Cloned human neuropeptide Y receptor couples to two different second messenger systems, Proc. Natl. Acad. Sci. USA 89 Ž1992. 5794–5798. w24x M. Jhanwar-Uniyal, B. Beck, C. Burlet, S.F. Leibowitz, Diurnal rhythm of neuropeptide Y-like immunoreactivity in the suprachiasmatic, arcuate and paraventricular nuclei and other hypothalamic sites, Brain Res. 536 Ž1990. 331–334. w25x F.B. Jolicoeur, J.-N. Michaud, R. Rivest, D. Menard, D. Gaudin, A. Fournier, S. St-Pierre, Neurobehavioral profile of neuropeptide Y, Brain Res. Bull. 26 Ž1991. 265–268. w26x P.M. Jones, A.M. Pierson, G. Williams, M.A. Ghatei, S.R. Bloom, Increased hypothalmaic neuropeptide Y messenger RNA levels in two models of diabetes, Diabetic Med. 9 Ž1991. 76–80. w27x S.P. Kalra, M.G. Dube, P.S. Kalra, Continuous intraventricular infusion of neuropeptide Y evokes episodic food intake in satiated female rats: effects of adrenalectomy and cholecystokinin, Peptides 9 Ž1988. 723–728. w28x S.P. Kalra, M.G. Dube, A. Sahu, C.P. Phelps, P.S. Kalra, Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food, Proc. Natl. Acad. Sci. USA 88 Ž1991. 10931–10935. w29x A. Kersten, J.H. Strubbe, N.J. Spiteri, Meal patterning of rats with changes in day length and food availability, Physiol. Behav. 25 Ž1980. 953–958. w30x H.R. Kissileff, Free feeding in normal and ‘recovered lateral’ rats monitored by a pellet-detecting eatometer, Physiol. Behav. 5 Ž1970. 163–173. w31x S.E. Kyrkouli, B.G. Stanley, S.F. Leibowitz, Galanin: stimulation of feeding induced by medial hypothalamic injection of this novel peptide, Eur. J. Pharmacol. 122 Ž1986. 159–160. w32x J. Le Magnen, The metabolic basis of dual periodicity of feeding in rats, Behav. Brain Sci. 4 Ž1981. 561–607. w33x J. Le Magnen, M. Devos, C. Larue-Achagiotis, Food deprivation induced parallel changes in blood glucose, plasma free fatty acids and feeding during two parts of the diurnal cycle in rats, Neurosci. Biobehav. Rev. 4 ŽSuppl. 1. Ž1980. 17–23. w34x S.F. Leibowitz, Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking, Pharmacol. Biochem. Behav. 8 Ž1978. 163–175. w35x S.F. Leibowitz, J.T. Alexander, Analysis of neuropeptide Y-induced feeding: dissociation of Y1 and Y2 receptor effects on natural meal patterns, Peptides 12 Ž1991. 1251–1260. w36x S.F. Leibowitz, G. Shor-Posner, Hypothalamic monoamine systems for control of food intake: analysis of meal patterns and macronutrient selection, in: M.O. Carruba, J.E. Blundell ŽEds.., Pharmacology of Eating Disorders: Theoretical and Clinical Developments, Raven Press, New York, 1986, pp. 29–49. w37x S.F. Leibowitz, M. Xuereb, T. Kim, Blockade of natural and neuropeptide Y-induced carbohydrate feeding by a receptor antagonist PYX-2, NeuroReport 3 Ž1992. 1023–1026. w38x A.S. Levine, M.A. Kuskowski, M. Grace, C.J. Billington, Food deprivation-induced feeding: a behavioral evaluation, Am. J. Physiol. 260 Ž1991. R546–R552. w39x W.C. Lynch, P. Hart, A.M. Babcock, Neuropeptide Y attenuates satiety: evidence from a detailed analysis of patterns of ingestion, Brain Res. 636 Ž1994. 28–34.
279
w40x P.E. McKibbin, P. Rogers, G. Williams, Increased neuropeptide-Y concentrations in the lateral hypothalamic area of the rat after the onset of darkness: possible relevance to the circadian periodicity of feeding behavior, Life Sci. 48 Ž1991. 2527–2533. w41x J.A. Menendez, I.S. McGregor, P.A. Healey, D.M. Atrens, S.F. Leibowitz, Metabolic effects of neuropeptide Y injections into the paraventricular nucleus of the hypothalamus, Brain Res. 516 Ž1990. 8–14. w42x J.E. Morley, A.S. Levine, B.A. Gosnell, J. Kneip, M. Grace, Effect of neuropeptide Y on ingestive behaviors in the rat, Am. J. Physiol. 252 Ž1987. R599–R609. w43x R.D. O’Shea, A.L. Gundlach, Preproneuropeptide Y messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat is increased in food deprivation or dehydration, J. Neuroendocrinol. 3 Ž1991. 11–14. w44x X. Paez, R.D. Myers, Insatiable feeding evoked in rats by recurrent perfusion of neuropeptide Y in the hypothalamus, Peptides 12 Ž1991. 609–616. w45x A. Palou, X. Remesar, L. Arola, E. Herrera, M. Alemany, Metabolic effects of short term food deprivation in the rat, Horm. Metab. Res. 13 Ž1981. 326–330. w46x J. Peret, I. MaCaire, M. Chanez, Schedule of protein ingestion, nitrogen and energy utilization and circadian rhythm of hepatic glycogen, plasma corticosterone and insulin in rats, J. Nutr. 103 Ž1973. 866–874. w47x A. Sahu, P.S. Kalra, S.P. Kalra, Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus, Peptides 9 Ž1988. 83–86. w48x G. Sanacora, M. Kershaw, J.A. Finkelstein, J.D. White, Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation, Endocrinology 127 Ž1990. 730–737. w49x M.W. Schwartz, J.L. Marks, A.J. Sipols, D.G. Baskin, S.C. Woods, S.E. Kahn, D. Porte, Central insulin administration reduces neuropeptide Y mRNA expression in the arcuate nucleus of food-deprived lean ŽFarFa. but not obese Žfarfa. Zucker rats, Endocrinology 128 Ž1991. 2645–2647. w50x T. Shibasaki, T. Oda, T. Imaki, N. Ling, H. Demura, Injection of anti-neuropeptide Y gamma-globulin into the hypothalamic paraventricular nucleus decreases food intake in rats, Brain Res. 601 Ž1993. 313–316. w51x N.J. Spiteri, A.B. Alingh Prins, J. Keyser, J.H. Strubbe, Circadian pacemaker control of feeding in the rat, at dawn, Physiol. Behav. 29 Ž1982. 1141–1145. w52x B.G. Stanley, Neuropeptide Y in multiple hypothalamic sites controls eating behavior, endocrine, and autonomic systems for body energy balance, in: W.F. Colmers, C. Wahlestedt ŽEds.., The Biology of Neuropeptide Y and Related Peptides, Humana Press, NJ, 1993, pp. 457–509. w53x B.G. Stanley, K.C. Anderson, M.H. Grayson, S.F. Leibowitz, Repeated hypothalamic stimulation with neuropeptide Y increases daily carbohydrate and fat intake and body weight gain in female rats, Physiol. Behav. 46 Ž1989. 173–177. w54x B.G. Stanley, D.R. Daniel, A.S. Chin, S.F. Leibowitz, Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion, Peptides 6 Ž1985. 1205–1211. w55x B.G. Stanley, L.H. Ha, L.C. Spears, M.G. Dee II, Lateral hypothalamic injections of glutamate, kainic acid, D,L-a-amino-3-hydroxy-5methyl-isoxazole propionic acid or N-methyl-D-aspartic acid rapidly elicit intense transient eating in rats, Brain Res. 613 Ž1993. 88–95. w56x B.G. Stanley, S.E. Kyrkouli, S. Lampert, S.F. Leibowitz, Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity, Peptides 7 Ž1986. 1189–1192. w57x B.G. Stanley, D. Lanthier, A.S. Chin, S.F. Leibowitz, Suppression of neuropeptide Y-elicited eating by adrenalectomy or hypophysectomy: reversal with corticosterone, Brain Res. 501 Ž1989. 32–36.
280
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
w58x B.G. Stanley, S.F. Leibowitz, Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci. 35 Ž1984. 2635–2642. w59x B.G. Stanley, S.F. Leibowitz, Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior, Proc. Natl. Acad. Sci. USA 82 Ž1985. 3940–3943. w60x B.G. Stanley, W. Magdalin, A. Seirafi, M.M. Nguyen, S.F. Leibowitz, Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a variant of the Y1 receptor mediating this peptide’s effect, Peptides 13 Ž1992. 581–587. w61x B.G. Stanley, W. Magdalin, A. Seirafi, W.J. Thomas, S.F. Leibowitz, The perifornical area: the major focus of a patchily distributed hypothalamic neuropeptide Y sensitive feeding systemŽs., Brain Res. 604 Ž1993. 304–317. w62x B.G. Stanley, W.J. Thomas, Patterns of eating behavior elicited by neuropeptide Y injected into the medial perifornical hypothalamus, Soc. Neurosci. Abstr. 16 Ž1990. 773. w63x B.G. Stanley, W.J. Thomas, C.L. Bivens, Gorging of food induced by intrahypothalamic injection of neuropeptide Y, Tenth Annual
w64x
w65x
w66x
w67x
w68x
Bristol–Myers SquibbrMead Johnson Symposium on Nutrition Research, Toronto, Canada, 1990. J.H. Strubbe, J. Keyser, T. Dijkstra, J.A. Prins, Interaction between circadian and caloric control of feeding behavior in the rat, Physiol. Behav. 36 Ž1986. 489–493. D.L. Tempel, S.F. Leibowitz, Diurnal variations in the feeding responses to norepinephrine, neuropeptide Y and galanin in the PVN, Brain Res. Bull. 25 Ž1990. 821–825. D.L. Tempel, G. Shor-Posner, D. Dwyer, S.F. Leibowitz, Nocturnal patterns of macronutrient intake in freely feeding and food-deprived rats, Am. J. Physiol. 256 Ž1989. R541–R548. G. Williams, J.S. Gill, Y.C. Lee, H.M. Cardoso, B.E. Okpere, S.R. Bloom, Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats, Diabetes 38 Ž1989. 321–327. G. Williams, J.H. Steel, H. Cardoso, M.A. Ghatei, Y.C. Lee, J.S. Gill, J.M. Burrin, J.M. Polak, S.R. Bloom, Increased hypothalamic neuropeptide Y concentrations in diabetic rat, Diabetes 37 Ž1988. 763–772.
Research report
Similar feeding patterns are induced by perifornical neuropeptide Y injection and by food deprivation Carrie Lynn Marın ´ Bivens a
a,1
, William J. Thomas
b,2
, B. Glenn Stanley
b, )
Department of Psychology and Neuroscience Research Institute, UniÕersity of California, Santa Barbara, CA 93106, USA b The Departments of Neuroscience and Psychology, UniÕersity of California, RiÕerside, CA 92521, USA Accepted 14 October 1997
Abstract Although hypothalamic injections of neuropeptide Y ŽNPY. induce robust feeding, there is little information about the patterns of feeding elicited by this peptide. To reveal these patterns, NPY Ž0, 8, 24, 78, 235 pmolr10 nl. was injected into the perifornical hypothalamus ŽPFH. of satiated adult male rats and their subsequent food intake was monitored every minute for 24 h. For comparison, feeding patterns were similarly observed following fasts of 0, 3, 6, 9, 12, and 24 h. The results demonstrated that NPY and food deprivation both produced dose- or deprivation-dependent increases in food intake that were most evident in the first 6 h. The increased intakes induced by NPY were characterized by combinations of increased meal size and frequency, with the predominant effects being increases in the size of and decreased latency to eat the first meal. Similarly, fasting progressively increased food intake by combinations of increased meal size and frequency, with the predominant effects being increases in the size of and decreased latency to eat the first meal. These similarities between NPY-induced and food deprivation-induced feeding are consistent with a stimulatory role for endogenous NPY in deprivation-induced feeding. These findings also suggest that NPY may increase eating by acting on mechanisms of both meal initiation and of meal termination. q 1998 Elsevier Science B.V. Keywords: Feeding; Food deprivation; Meal patterns; Neuropeptide Y; Perifornical hypothalamus
1. Introduction Food is usually consumed in discrete meals that vary in size and frequency in patterns characteristic of particular physiological and environmental conditions. Manipulations that enhance food intake increase either meal size or meal frequency w6,17x and the regulation of these parameters may be partially independent w32x. The neural systems regulating meal size and frequency may be distinct neurochemically and anatomically because intracranial injection of some feeding-stimulatory neurotransmitters preferentially increase meal size, while others preferentially increase meal frequency w36x. Thus, analysis of the meal patterns produced by central injection of a neurotransmitter may provide insights about which aspects of eating are
)
Corresponding author. Fax: q 1-909-787-3985; [email protected] 1 Tel.: q1-805-893-7376; fax: q1-805-893-4303. 2 Tel.: q1-909-787-5625; fax: q1-909-787-3985.
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 2 8 9 - 4
regulated by the neurons which employ that neurotransmitter. Neuropeptide Y ŽNPY., a neurochemical abundant within many neurons originating or terminating in the hypothalamus w14x, has been implicated in the regulation of eating behavior and body weight. Intracranial injection of this phylogenetically conserved peptide induces eating across a wide range of vertebrates, in neonatal as well as adult rats, and throughout the circadian cycle w52x. Endogenous NPY may contribute to feeding because manipulations that suppress endogenous NPY suppress spontaneous feeding w1,37,50,60x, and manipulations that increase food intake, such as food deprivation, may elevate NPY release, tissue content and synthesis, and gene expression w4,28,47,67x. Although these studies suggest that endogenous NPY may regulate feeding behavior, there is limited data on the patterns of eating elicited by this peptide w27,35,38,39,58x. Previous meal pattern analyses have shown that low doses of NPY injected into the paraventricular hypothalamus ŽPVN. elicits the consumption of a single meal w35,58x and that continuous intracerebroventricular Ži.c.v.. injec-
272
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
tion produces consumption of discrete meals at low doses, but induces persistent nibbling at high doses w27x. Since i.c.v. injection of NPY produces a variety of physiological effects w25x that may alter feeding patterns, we chose to characterize the feeding patterns induced by NPY microinjected into the perifornical hypothalamus ŽPFH., the most sensitive known site for NPY-induced feeding w61x. The major questions were: Is the feeding elicited by PFH injection of NPY organized into discrete meals and, if so, are both meal size and meal frequency increased? Further, since similarities in the feeding patterns induced by NPY and natural conditions may suggest shared neural substrates, we also examined feeding patterns induced by food deprivation, to determine the extent to which they were comparable. Portions of these finding have previously been presented in preliminary form w62,63x.
measured included: latency to eat, meal size, meal duration, meal frequency, and intermeal interval. Fresh food was provided 24 h before testing. 2.3. Intracranial injection procedures Using a previously described nanoliter injection system w61x, porcine NPY ŽPeninsula Laboratories, Belmont, CA. was dissolved in sterile, artificial cerebrospinal fluid ŽaCSF; Naq 147 mM, Cly 154 mM, Kq 3.0 mM, Caqq 1.2 mM, Mgqq 0.9 mM, pH s 7.4. and injected into the PFH in a volume of 10 nl. The five doses of NPY were 0, 8, 24, 78, 235 pmol Ž0, 33, 100, 330, 1000 ng, respectively. injected in counterbalanced order, with each subject tested in every condition. The rats were injected during the first hour of the light-phase of the photoperiod, with 3–5 days between tests to allow for recovery of baseline food intake.
2. Materials and methods 2.1. Subjects and surgery Twenty adult, Sprague–Dawley, male rats ŽSimonsen Laboratories, Gilroy, CA., weighing 350–400 g at the start of the experiment, were used. Ten were stereotaxically implanted with an unilateral, 26-gauge, stainless-steel, guide cannula while under Metofane anesthesia. The guide cannula was positioned 1.0 mm dorsal to the PFH. With the incisor bar at y3.3 mm, the stereotaxic coordinates were 7.2 mm anterior to the interaural line, 1.0 mm lateral to the midsagittal sinus, and 7.7 mm ventral to the surface of the skull. The cannula was fixed to the skull surface with dental acrylic and screws just penetrating the skull. Surgery was not performed on the remaining 10 animals that were used in the food-deprivation experiment. 2.2. Housing and meal pattern data collection The rats were housed individually and tested in clear, rectangular, plastic cages Ž33 = 21 = 18 cm. that were equipped with an automatic watering system, in a room with a 12 h light:12 dark h photoperiod Žlights on 08:00.. A small cubicle was attached to the outside of the cage to allow access to food bowls, which were located through a hole in the bottom of the cubicle. Food bowls were filled with a mixture of Purina Rodent Chow Ž46%., sucrose Ž37%. and Carnation Evaporated Milk Ž17%. and placed directly beneath the hole so that any spilled food would fall back into the bowl. Food bowl weight was registered by an electronic balance ŽOHAUS port-a-gram., and was recorded every minute for 24 h after NPY injection or food deprivation using meal pattern software, with all balances connected to a single IBM-compatible computer. Meals were defined as a minimum weight decrement of 0.2 g separated from other feeding episodes by at least 10 min. The variables
Fig. 1. Neuropeptide Y injection ŽA: top panel. and food deprivation ŽB: bottom panel. both enhance food intake Žmean g"S.E.M.. during the 12 h light phase ŽLight Phase Intake. immediately after injection or refeeding, respectively. Light phase intakes increase as a function of the dose of neuropeptide Y Žpm s pmol of NPY. and as a function of the hrs of food deprivation ŽDeprivation Time.. ‘Dark Phase Intake’ represent the food intakes during the 12 h of darkness that follow this light phase and the ‘Total Intake’ represents the combined light and dark phase intakes. ) p- 0.05, ) ) p- 0.01 by Dunnet’s tests compared to intakes in the same phase of the light–dark cycle after vehicle injection Ž0 pmol of neuropeptide Y. or 0 h of food deprivation.
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
273
were perfused transcardially with 10% formalin. The perfused brains were removed, frozen, and sliced in 100 m m coronal sections using a microtome. The brain sections were mounted onto slides, stained with cresyl violet and examined microscopically. One- or two-way repeated measures ANOVA or ANCOVA and post-hoc Dunnet’s tests were used to analyze the effects of deprivation period, NPY dose, photoperiod and time on meal size, meal frequency, latency to eat, intermeal interval, and feeding rate, when appropriate. The criterion for statistical significance was p F 0.05 for all statistical tests.
Fig. 2. Mean meal size Žg"S.E.M.. of the first through fourth meals ŽMeal No. 1–4. occurring during the 12 h light phase after injection of neuropeptide Y ŽA: top panel. or the first through fifth meals occurring after the termination of food deprivation ŽB: bottom panel. as a function of dose of NPY Žpm s pmol. or ‘hours of deprivation,’ respectively. ) p- 0.05, ) ) p- 0.01 by Dunnet’s tests greater than intakes in the same meal after vehicle injection Ž0 pmol of NPY. or ‘0 h of food deprivation’. For cases when no meal occurred after ‘0 h of food deprivation’ Ži.e., meals 3, 4, and 5., the statistical comparison was to the averaged size of the first and second meals. For simplicity, the data from meal number 4 was included for the 8 and 24 pmol doses of NPY even though most of those meals occurred after the onset of the dark phase.
2.4. Food depriÕation procedures Food was removed 0, 3, 6, 9, 12, or 24 h before light onset, in counterbalanced order, and was returned at the onset of the light cycle to the otherwise undisturbed subjects. Their meal patterns were measured, as described above, during the subsequent 24 h. These tests were separated by at least four days, after all subjects returned to baseline feeding patterns for a minimum of two days. 2.5. Histology and statistics After testing was completed, the animals were euthanized by carbon dioxide inhalation and cannulated rats
Fig. 3. Mean time Žmin"S.E.M.. from NPY injection ŽA: top panel. or from postfasting return of food ŽB: bottom panel. to eat the first meal ŽLatency to Meal 1., as well as the times between the first and second meal ŽIMI 1s intermeal interval 1., second and third ŽIMI 2. and third and fourth meals ŽIMI 3.. ) p- 0.05, ) ) p- 0.01 by Dunnet’s tests different from latencies or IMI in the same meal after vehicle injection Ž0 pmol of NPY. or ‘0 h of food deprivation’. For cases when no meal occurred after ‘0 h of food deprivation’ Ži.e., IMI 2 and IMI 3. then the statistical comparison was to IMI 1.
274
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
3. Results
3.2. NPY-induced meal patterns
3.1. Histology
As shown in Fig. 1A, NPY injections produced dosedependent increases in food intake of up to 230% during the light-phase Ž F4,36 s 48.4, p - 0.001.. These increases were compensated by complementary dose-dependent decreases in food intake during the subsequent dark-phase Ž F4,36 s 48.4, p - 0.001., such that there was no significant increase in total 24 h food intake Ž F4,36 s 0.48, NS..
The injection sites Žnot shown. were centered in the PFH just ventromedial to the fornix at the coronal level of the posterior border of the PVN, an area shown to be maximally sensitive to the feeding-stimulatory effect of NPY w61x.
Fig. 4. Meal patterns in the 12 h following injection of neuropeptide Y Žleft side. or postfasting return of food Žright side. averaged across subjects. The height of each bar represents the mean size Žg " S.E.M.. of that meal and the width of each bar represent its duration Žh.. Additionally, the placement of each bar on the abscissa represents the average time during the 12 h light phase in which that meal occurred and the number of bars represents the average number of meals that occurred in each condition. The shaded horizontal line in each panel provides a comparison to the mean meal size of the four meals that occurred after vehicle injection Žleft side; 0 pmol. or of the two meals that occurred in rats that were not deprived Žright side; no deprivation.. ) p - 0.05, ) ) p - 0.01 by Dunnet’s tests different from the averaged meal sizes after vehicle injection Ž0 pmol of NPY. or ‘no deprivation’.
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
As shown in Fig. 2A, increases in the size of several meals contributed to the increased intakes Ž F16,144 s 8.9, p - 0.01., with the size of the first meal being most prominently elevated. The increase in the size of the first meal was statistically significant even at 8 pmol of NPY and was extremely large at the 235 pmol dose. Specifically, in comparison to the mean first meal size of 1.5 g after vehicle injection, this meal averaged 9.6 g after injection of 235 pmol of NPY. The size of the second meal increased significantly at the 78 and 235 pmol doses, while third meal did not increase at any dose, and the fourth meal was increased significantly at 8, 24, and 235 pmol NPY. The apparent increase in the size of the fourth meal at the 8 and 24 pmol doses of NPY was probably an artifact of the inclusion of several individual meals that actually occurred during the dark phase, when the size of spontaneously occurring meals is larger w17x. This is evident in Figs. 3 and 4 Žleft panel., which shows that an average of only three meals occurred in the 12 h light-phase after injection of 8 and 24 pmol of NPY. In addition to the augmentations of meal size, both decreases in the latency to eat the first meal and increases in frequency of subsequent meals contributed to the increased light-phase food intake ŽFig. 3A.. The latency to eat the first meal declined in a progressive dose-dependent manner, from 108 min after vehicle injection to 9.5 min after injection of the highest dose of NPY Ž F4,36 s 4.6 p - 0.01.. Also shown in Fig. 3A is that the intervals between the first and second meals Ži.e., IMI 1. and between the second and third meals Ži.e., IMI 2. declined at the 78 and 235 pmol doses. In contrast, the onset of the third meal was delayed at 24 pmol, possibly because of the satiating effects of the previously ingested food. That NPY affects both meal size and meal frequency is clearly shown in the left panel of Fig. 4. The increased food intake at the lowest NPY doses Ž8 and 24 pmol. was produced almost exclusively by increased meal size and decreased latency to eat the first meal. Higher doses further enhanced these effects, and additionally increased the size and frequency of subsequent meals, such that at 235 pmol, the size of the fifth postinjection meal, which occurred nearly 7 h postinjection, was increased significantly. There were no effects of dose of NPY on the eating rate Ž0.25–0.28 grmin across doses; F4,36 s 1.8, p ) 0.1, NS. and consequently meal duration increased in proportion to meal size Že.g., the 9.6 g first meal at 235 pmol of NPY lasted 39.6 min.. No statistically significant relationship existed between meal size and the subsequent intermeal interval Ž r s y0.2; F16,144 s 0.74, p ) 0.56 by ANCOVA.. 3.3. Effects of food depriÕation As shown in Fig. 1B, food deprivation increased subsequent total daily food intake Ž F5,56 s 62.8, p - 0.001.. This was mostly due to the light-phase food intakes in-
275
creasing as a function of increased length of food deprivation Ž F5,56 s 23.2, p - 0.01.. These increases were only partially offset by deprivation-related declines in darkphase food intake Ž F5,56 s 16.38, p - 0.05., yielding a significant interaction between the light- and dark-phase intakes Ž F5,56 s 83.5, p - 0.0001.. The increases in feeding during the daytime were produced by increases both in meal size and in meal frequency. As shown in Fig. 2B, with prolonged food deprivation Ž9, 12, or 24 h. there were prominent increases in the size of the first meal Ž F5,54 s 15.6, p - 0.01.. With deprivations of 12 or 24 h, the sizes of each of the subsequent four meals also increased but to a lesser extent Žranging from F5,54 s 5.9, p - 0.05 to F5,54 s 55.3, p 0.0001, for these meals.. As shown in Fig. 3B, increased meal frequency Ž F5,54 s 11.6, p - 0.01. and decreased latency to eat the first meal Ž F5,54 s 17.7, p - 0.001. also contributed to the augmented food intake. This was particularly evident with brief deprivations of 3 or 6 h, when the increased intakes were due almost exclusively to decreases in the latency to eat the first meal ŽFig. 3B. and to increases in the number of meals occurring in the light phase ŽFig. 4, right panel. with little change in meal size. In contrast, deprivations of 9 to 24 h produced no further increases in meal frequency but rather increased meal size. The overall pattern shown in Fig. 4 Žright panel., reveals that with brief food deprivation periods the increased food intake is due to an increase in the number of meals eaten, with little or no increase in the size of the meals. With more prolonged deprivations, meal frequency reached a plateau, with further increments in food intake due to marked increases in the size of the first meal and to smaller but significant increases in the size of the subsequent three to four meals.
4. Discussion 4.1. Feeding patterns induced by PFH injection of NPY These observations demonstrate that, across a wide range of doses, the feeding elicited in rats by acute PFH injection of NPY occurs in discrete meals that are clearly separated by periods without eating. This complements earlier findings that acute PVN injection of a low dose of NPY induces the consumption of a single large meal w58x, and that continuous i.c.v. injection of low NPY doses elicited consumption of several meals, whereas, higher doses induced persistent nibbling w27x. That NPY-induced feeding in a meal-like pattern is consistent with the hypothesis that NPY affects neural pathways that mediate natural eating behavior. Consistent with earlier reports w15,58x, NPY injected during the light-phase dose-dependently increases eating. These increases were compensated by reciprocal decreases in dark-phase feeding, resulting in the maintenance of 24 h
276
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
food intakes at baseline levels across doses. The accuracy of this compensation suggests that the decreases in darkphase food intake may have been due to the delayed satiating effects of the large amounts of previously ingested food after the injected NPY became ineffective, rather than to a potential latent feeding-suppressive effect of NPY. Further, tolerance to NPY is unlikely to have accounted for the decreased eating because repeated or continuous injections do not alter sensitivity to NPY w5,42,56x. This finding is consistent with NPY acting to elicit eating, in part, by suppressing the satiating effect of food w39x. Perhaps the most salient effect of NPY was the pronounced dose-dependent increase in the size of the first meal. This augmentation was apparent even at 8 pmol, the lowest dose of NPY yet shown to significantly increase food intake; at the 235 pmol dose the first meal was enormous, larger even than the first meal eaten after a 24 h fast. This suggests that the dramatic eating reportedly produced by hypothalamic NPY injections w59x is due in large part to the ingestion of a single enormous meal soon after injection. This elicited gorging is also consistent with the hypothesis that NPY-like peptides may contribute to binging patterns in bulimic humans w7x. In addition to increasing the size of and speed to initiate the first meal, NPY increased both the size and frequency of subsequent meals. This was most evident at the 235 pmol dose, which elicited a series of four abnormally large meals in the 6–7 h after injection, a time during which only one meal occurred after control injections. This feeding pattern suggests that NPY-induced feeding was produced both by stimulating meal initiation mechanisms, as indicated by the increased meal frequency and decreased latency to eat, and by inhibiting meal termination mechanisms, as indicated by the increased meal sizes and durations w32x. To the extent that distinct meal initiation and termination mechanisms actually exist in the brain and reflect the perceptual states of hunger and satiety, these findings suggest that NPY may elicit eating both by activating brain mechanisms of hunger and by inhibiting those producing satiety. Consistent with previous reports w59x, the eating-stimulatory effects of acute NPY injection was protracted, lasting at least 6–7 h after injection of the highest dose, as indicated by the increased meal sizes at that time. These increases are especially noteworthy, as they occurred despite the expected satiating effects of the enormous amount of food that had been eaten in the hours prior to that time. Although it is possible that levels of extracellular NPY might have been maintained at high levels and thus contributed to the duration of the effects, this is unlikely to have been the main factor because brain NPY seems to be degraded rapidly, as indicated by the previously observed rapid fluctuations in extracellular levels of endogenous NPY, and by the rapid reductions in the brain levels of radiolabeled NPY postinjection w28,61x. More likely is that
NPY’s long-lasting feeding-stimulatory effects were mediated by the activation of a G protein linked second messenger systemŽs., as has been suggested previously w13x and demonstrated for NPY in other systems w23x. Additionally, mediation by second messengers might contribute to the relatively longer observed latency to eat after NPY injection, as compared to some other neurochemical stimulants of eating w31,34,55x. Consistent with this is the demonstration that the latency to eat after NPY injection is reduced to seconds when the peptide is repeatedly injected w44x, possibly because the initial injections induced relevant changes in second messenger systems, which then allowed the later injections to act rapidly. Perhaps relevant to second messenger mediation of feeding is our recent demonstration that intense eating may be elicited by PFH injections of compounds that stimulate intracellular levels of cAMP w21x. 4.2. Feeding patterns produced by fasting A striking effect of fast duration was the markedly different impact of short- and long-term deprivations on meal patterns. Short-term deprivations of up to 6 h increased food intake by substantially increasing the number of meals eaten after food was returned, without increasing the size of those meals. Longer deprivations did not further increase meal frequency, rather food intake was enhanced by increases in meal size. The first meal was always the largest, although in most cases it was only fractionally larger than the subsequent light-phase meals. Further, this meal occurred almost immediately upon the return of food with any deprivation of 6 or more hours. These findings, in contrast to most of the published literature showing that fasting increases meal size w3,33x, show that short-term fasting induces eating by increasing meal frequency without increasing the size of the first or subsequent meals. This contrast may result primarily from differences in the time of the light–dark cycle in which the subjects were refed. Unlike most other studies, which usually refed their subjects in the middle of the light- or at some point in the dark-phase, we refed the animals in the first hour of the light, a time when rats do not normally eat w64x. Studies suggest that rats do not normally eat at this time primarily because they are metabolizing the stored food and metabolic fuels that were consumed during the latter portion of the night-phase, in anticipation of the following light-phase fast w2,29,51,64x. Specifically, it has been shown that there are daily cycles of hepatic and muscle glycogen, adipose tissue, and gastric food contents, with each peaking between the latter portion of the dark phase and the first portion of the light phase, times when the stomach is hard-packed with food w2,20,32,46x. Deprivation during the latter portion of the dark-phase induces eating during the following light-phase w29x, suggesting that freely feeding subjects do not eat at this time because they are utilizing the stored, gastric food, hepatic and
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
muscle glycogen, and adipose tissue. Thus, the progressive decrease in the latency to eat the first meal after short deprivation in our study may have resulted from depletion of the smaller food stores that had been accumulated prior to the fast. With 3 h of deprivation, these stores were presumably sufficient to last until about 3 to 4 h after light onset w45x, the time when 3 h deprived rat typically ate their first meal. At 6 h of deprivation, the first meal occurred almost immediately and was essentially of normal size, suggesting that the gastricrhepatic reserves were depleted Žor reduced to a critical level., provoking the consumption of a single meal. The subsequent meals are suggested to occur as the food from each preceding meal is metabolized to some critical level w32x. With longer deprivations Ž9 to 24 h., the size of the first immediately occurring meal and the size of subsequent meals increased progressively, presumably in response to signals relating to longer-term fuel sources. Other studies, examining eating patterns at other points in the light–dark cycle, observed increases in meal size, we suggest, because there were insufficient gastric or hepatic fuel reserves to blunt the effects of fasting on meal size. 4.3. Comparisons of feeding patterns produced by fasting and NPY injection Similar patterns of eating produced by fasting and NPY injection would support but not prove actions via shared neural substrates. However, even under ideal circumstances, the bolus injection of a single neurotransmitter into a single brain area can simulate only limited aspects of the patterned release of a multitude of interacting neurochemicals occurring within many different brain regions during normal eating. Therefore differences in feeding patterns should be expected. Given this, there is a surprisingly good correspondence between the feeding patterns produced by fasting and NPY injection. Among these are that: the animals ate food in meals; there were progressive increases in both meal size and meal frequency produced by increased levels of fasting and doses of NPY; the largest effects were the increases in the size of the first meals and; the sizes of subsequent meals were also increased but to a lesser extent. These similarities were most apparent between the highest dose of NPY and the longest fasts. In those circumstances, the increased intakes were due to the consumption of an initial enormous meal, followed for several hours by a series of smaller but still oversized meals. There were also distinct differences between the feeding patterns produced by progressive fasting and doses of NPY. With fasting, increased intakes were first expressed by increased numbers of meals, with no increase in meal size; with longer deprivation by increased meal size without any further increases in meal frequency. In contrast, with increased NPY doses meal size and meal frequency increased in parallel. Further, the rate of eating
277
increased with longer fasts but not with higher doses of NPY. These similarities are consistent with previous evidence suggesting that increase activity of endogenous NPY might participate in mediating the increased eating that occurs after fasting. This was first suggested by evidence that tissue concentrations of NPY increase within the PVN of fasted rats and normalize with refeeding w47x. This pattern also applies to NPY gene expression w10,43x and extracellular concentrations of NPY w28x. That this increase in hypothalamic NPY activity may be necessary for the normal expression of feeding has been suggested by studies showing that i.c.v. or PVN injection of antisera to NPY significantly reduces eating induced by food deprivation w50,60x. The similar patterns of feeding produced by fasting and NPY injection in the present study adds to this evidence, by showing that increasing the activity of NPY within the PFH is sufficient to reproduce some aspects of the feeding patterns produced by fasting. As to the observed differences in the rates and patterns of eating induced by NPY and fasting, it is unclear to what extent these were due to: patterns of endogenous NPY release that were not replicated by the bolus injection; actions of endogenous NPY within multiple brain sites in fasted rats; the modulation of NPY’s actions by other neurotransmitters in the intact animal and; actions of neurotransmitters other than or in addition to NPY in the mediation of eating in the fasted rat. It may also be noteworthy that in previous comparisons of NPY’s and deprivation’s effects on behavioral patterns that substantial differences were observed w38,39x. However, in those studies the NPY was administered i.c.v. Since NPY administered by this route has been shown to produce a variety of behavioral, autonomic and endocrine effects in addition to feeding stimulation w22,25x, it seems likely that these other effects would have contaminated those that might otherwise have been produced by a more specific stimulation of eating control mechanisms. 4.4. InÕolÕement of NPY in other conditions associated with enhanced feeding It has been suggested that NPY participates in increased eating in a wide range of physiological conditions w52x. Among these are circadian rhythms of eating; overeating in the obese Zucker rat; and overeating in diabetics. Each of these will be briefly discussed, with emphasis on comparing the feeding patterns associated with each of these conditions to those elicited by NPY injection. NPY has been implicated in the increased eating that occurs at the onset of the active phase of the light–dark cycle w27,54,65x. Supporting this is that: PVN and lateral hypothalamic NPY levels increase at the onset of the dark-phase in rats w24,40x; feeding during this time was suppressed by PVN injection of a putative NPY antagonist
278
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
w37x; rats eating at the onset of the dark phase and those injected with NPY prefer carbohydrate diets w35,42,54,66x; and the sensitivity of the PVN to NPY exhibits a circadian rhythm, with a peak a the onset of the dark phase w65x. Here we show that NPY can increase both meal size and frequency. This may support a role for endogenous NPY in nocturnal eating, since meal size and frequency both increase in spontaneously eating rats at the onset of the dark phase w17,30x. There are parallels between the NPY induced feeding patterns we observed and those of obese Zucker rats that support a role of NPY in this form of genetic obesity. Zucker rats overeat primarily by consuming extremely large meals with a marginal increase in meal frequency w6,12,19x. We found that acute injections of NPY produce a similar pattern, rats ate large meals and meal frequency increased slightly. Similarly, chronic injections of NPY reportedly produce Zucker-obese-like feeding patterns and obesity w5x. Further, fat and carbohydrate-rich foods are preferred both by the Zucker obese and NPY-treated rat w19,53x, and the eating effect is dependent on circulating corticosterone levels in each condition w11,57x. The Zucker obese rat has higher levels of NPY and NPY mRNA in the hypothalamus than does its lean counterpart w4,48x. Additionally, NPY-injected and Zucker obese rats have other behavioral, hormonal and metabolic commonalties, such as decreased metabolic rates and suppressed reproductive function w4,8,9,41x. Enhanced NPY activity has also been suggested to participate in the overeating exhibited by diabetic animals w68x. Streptozotocin ŽSTZ. diabetic rats reportedly eat moderately larger meals during the entire photoperiod and increase their light-phase meal frequency w18x. We found that acute injections of NPY stimulate feeding in a similar manner, consistent with a role in diabetic hyperphagia. Supportive of this idea, STZ diabetic animals have higher levels of NPY and NPY mRNA in the hypothalamus w26,67,68x. The lack of insulin may partially cause the rise in NPY levels, since central insulin decreases the production of NPY in the arcuate nucleus w49x. Conversely, under conditions of hypoglycemia induced by hyperinsulinemia, hypothalamic NPY levels do not increase w16x. Collectively, these finding support a role for NPY in the overeating exhibited by diabetic rats. However, several aspects of diabetic meal patterns are different from NPY-induced meal patterns w18x and thus, caution is warranted. In conclusion, the present results demonstrating that PFH injection of NPY produces overeating by increasing both meal size and meal frequency adds to the evidence suggesting a role for this peptide in feeding induced by fasting, that occurring in the active phase of the light–dark cycle, that associated with some forms of genetic obesity, and that exhibited by diabetics. Collectively, this suggests that NPY may mediate normal eating in a wide variety of circumstances, as well as the abnormal eating associated with a number of conditions.
Acknowledgements We thank Jack B. Calderone for statistical and graphical consultation. Supported by NIH grant NS 24268.
References w1x A. Akabayashi, C. Wahlestedt, J.T. Alexander, S.F. Leibowitz, Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding behavior and insulin secretion, Mol. Brain Res. 21 Ž1994. 55–61. w2x S. Armstrong, J. Clarke, G. Coleman, Light–dark variations in laboratory rat stomach and small intestine content, Physiol. Behav. 21 Ž1978. 785–788. w3x J.K. Bare, G. Cicala, Deprivation and time of testing as determinants of food intake, J. Comp. Physiol. Psychol. 53 Ž1960. 151–154. w4x B. Beck, A. Burlet, J.-P. Nicolas, C. Burlet, Hypothalamic neuropeptide Y ŽNPY. in obese Zucker rats: implications in feeding and sexual behavior, Physiol. Behav. 47 Ž1990. 449–453. w5x B. Beck, A. Stricker-Krongrad, J.-P. Nicolas, C. Burlet, Chronic and continuous intracerbroventricular infusion of neuropeptide Y in Long–Evans rats mimics the feeding behavior of obese Zucker rats, Int. J. Obesity 16 Ž1992. 295–302. w6x E.E. Becker, J.A. Grinker, Meal patterns in the genetically obese Zucker rat, Physiol. Behav. 18 Ž1977. 685–692. w7x W.H. Berrettini, W.H. Kaye, H. Gwirtsman, A. Allbright, Cerebrospinal fluid peptide YY immunoreactivity in eating disorders, Neuropsychobiology 19 Ž1988. 121–124. w8x C.J. Billington, J.E. Briggs, M. Grace, A.S. Levine, Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism, Am. J. Physiol. 260 Ž1991. R321–R327. w9x C.L. Bivens, H.J. Carlisle, Intrahypothalamic injection of neuropeptide Y reduces metabolic rate and increases behavioral thermoregualtion, Soc. Neurosci. Abstr. 18 Ž1992. 373–375. w10x L.S. Brady, M.A. Smith, P.W. Gold, M. Herkenham, Altered expression of hypothalamic neuropeptide Y mRNAs in food-restricted and food-deprived rats, Neuroendocrinology 52 Ž1990. 441–447. w11x T.W. Castonguay, M.F. Dallman, J.S. Stern, Some metabolic and behavioral effects of adrenalectomy on obese Zucker rat, Am. J. Physiol. 251 Ž1986. R923–R933. w12x T.W. Castonguay, D.E. Upton, P.M.B. Leung, J.S. Stern, Meal patterns in the genetically obese Zucker rat: a reexamination, Physiol. Behav. 28 Ž1982. 911–916. w13x W.T. Chance, S. Sheriff, T. Foley-Nelson, J.E. Fischer, A. Balasubramaniam, Pertussis toxin inhibits neuropeptide Y-induced feeding in rats, Peptides 10 Ž1989. 1283–1286. w14x B.M. Chronwall, D.A. DiMaggio, V.J. Massari, V.M. Pickel, D.A. Ruggiero, T.L. O’Donohue, The anatomy of neuropeptide Y-containing neurons in the rat brain, Neuroscience 15 Ž1985. 1159–1181. w15x J.T. Clark, P.S. Kalra, W.R. Crowley, S.P. Kalra, Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology 115 Ž1984. 427–429. w16x S.E. Corrin, H.D. McCarthy, P.E. McKibbin, G. Williams, Unchanged hypothalamic neuropeptide Y concentrations in hyperphagic, hypoglycemic rats: evidence for specific metabolic regulation of hypothalamic NPY, Peptides 12 Ž1991. 425–430. w17x R.F. Davies, Long- and short-term regulation of feeding patterns in the rat, J. Comp. Physiol. Psychol. 91 Ž1977. 574–585. w18x J.M. de Castro, S. Balagura, Meal patterning in the streptozotocindiabetic rat, Physiol. Behav. 15 Ž1975. 259–263. w19x M.P. Enns, J.A. Grinker, Dietary self-selection and meal patterns of obese and lean Zucker rats, Appetite 4 Ž1983. 281–293.
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280 w20x R.W. Fuller, E.R. Diller, Diurnal varietions of liver glycogen and plasma free fatty acids in rats fed ad libitum or single daily meal, Metabolism 19 Ž1970. 226–229. w21x E.R. Gillard, A.M. Khan, A.U. Haq, R.S. Grewal, B. Mouradi, B.G. Stanley, Stimulation of eating by the second messenger cAMP in the perifornical and lateral hypothalamus, Am. J. Physiol. 273 Ž1997. R107–R112. w22x A. Harfstrand, Brain neuropeptide Y mechanisms. Basic aspects and involvement in cardiovascular and neuroendocrine regulation, Acta Physiol. Scand. 565 ŽSuppl.. Ž1987. 1–83. w23x H. Herzog, Y.J. Hort, H.J. Ball, G. Hayes, J. Shine, L.A. Selbie, Cloned human neuropeptide Y receptor couples to two different second messenger systems, Proc. Natl. Acad. Sci. USA 89 Ž1992. 5794–5798. w24x M. Jhanwar-Uniyal, B. Beck, C. Burlet, S.F. Leibowitz, Diurnal rhythm of neuropeptide Y-like immunoreactivity in the suprachiasmatic, arcuate and paraventricular nuclei and other hypothalamic sites, Brain Res. 536 Ž1990. 331–334. w25x F.B. Jolicoeur, J.-N. Michaud, R. Rivest, D. Menard, D. Gaudin, A. Fournier, S. St-Pierre, Neurobehavioral profile of neuropeptide Y, Brain Res. Bull. 26 Ž1991. 265–268. w26x P.M. Jones, A.M. Pierson, G. Williams, M.A. Ghatei, S.R. Bloom, Increased hypothalmaic neuropeptide Y messenger RNA levels in two models of diabetes, Diabetic Med. 9 Ž1991. 76–80. w27x S.P. Kalra, M.G. Dube, P.S. Kalra, Continuous intraventricular infusion of neuropeptide Y evokes episodic food intake in satiated female rats: effects of adrenalectomy and cholecystokinin, Peptides 9 Ž1988. 723–728. w28x S.P. Kalra, M.G. Dube, A. Sahu, C.P. Phelps, P.S. Kalra, Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food, Proc. Natl. Acad. Sci. USA 88 Ž1991. 10931–10935. w29x A. Kersten, J.H. Strubbe, N.J. Spiteri, Meal patterning of rats with changes in day length and food availability, Physiol. Behav. 25 Ž1980. 953–958. w30x H.R. Kissileff, Free feeding in normal and ‘recovered lateral’ rats monitored by a pellet-detecting eatometer, Physiol. Behav. 5 Ž1970. 163–173. w31x S.E. Kyrkouli, B.G. Stanley, S.F. Leibowitz, Galanin: stimulation of feeding induced by medial hypothalamic injection of this novel peptide, Eur. J. Pharmacol. 122 Ž1986. 159–160. w32x J. Le Magnen, The metabolic basis of dual periodicity of feeding in rats, Behav. Brain Sci. 4 Ž1981. 561–607. w33x J. Le Magnen, M. Devos, C. Larue-Achagiotis, Food deprivation induced parallel changes in blood glucose, plasma free fatty acids and feeding during two parts of the diurnal cycle in rats, Neurosci. Biobehav. Rev. 4 ŽSuppl. 1. Ž1980. 17–23. w34x S.F. Leibowitz, Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking, Pharmacol. Biochem. Behav. 8 Ž1978. 163–175. w35x S.F. Leibowitz, J.T. Alexander, Analysis of neuropeptide Y-induced feeding: dissociation of Y1 and Y2 receptor effects on natural meal patterns, Peptides 12 Ž1991. 1251–1260. w36x S.F. Leibowitz, G. Shor-Posner, Hypothalamic monoamine systems for control of food intake: analysis of meal patterns and macronutrient selection, in: M.O. Carruba, J.E. Blundell ŽEds.., Pharmacology of Eating Disorders: Theoretical and Clinical Developments, Raven Press, New York, 1986, pp. 29–49. w37x S.F. Leibowitz, M. Xuereb, T. Kim, Blockade of natural and neuropeptide Y-induced carbohydrate feeding by a receptor antagonist PYX-2, NeuroReport 3 Ž1992. 1023–1026. w38x A.S. Levine, M.A. Kuskowski, M. Grace, C.J. Billington, Food deprivation-induced feeding: a behavioral evaluation, Am. J. Physiol. 260 Ž1991. R546–R552. w39x W.C. Lynch, P. Hart, A.M. Babcock, Neuropeptide Y attenuates satiety: evidence from a detailed analysis of patterns of ingestion, Brain Res. 636 Ž1994. 28–34.
279
w40x P.E. McKibbin, P. Rogers, G. Williams, Increased neuropeptide-Y concentrations in the lateral hypothalamic area of the rat after the onset of darkness: possible relevance to the circadian periodicity of feeding behavior, Life Sci. 48 Ž1991. 2527–2533. w41x J.A. Menendez, I.S. McGregor, P.A. Healey, D.M. Atrens, S.F. Leibowitz, Metabolic effects of neuropeptide Y injections into the paraventricular nucleus of the hypothalamus, Brain Res. 516 Ž1990. 8–14. w42x J.E. Morley, A.S. Levine, B.A. Gosnell, J. Kneip, M. Grace, Effect of neuropeptide Y on ingestive behaviors in the rat, Am. J. Physiol. 252 Ž1987. R599–R609. w43x R.D. O’Shea, A.L. Gundlach, Preproneuropeptide Y messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat is increased in food deprivation or dehydration, J. Neuroendocrinol. 3 Ž1991. 11–14. w44x X. Paez, R.D. Myers, Insatiable feeding evoked in rats by recurrent perfusion of neuropeptide Y in the hypothalamus, Peptides 12 Ž1991. 609–616. w45x A. Palou, X. Remesar, L. Arola, E. Herrera, M. Alemany, Metabolic effects of short term food deprivation in the rat, Horm. Metab. Res. 13 Ž1981. 326–330. w46x J. Peret, I. MaCaire, M. Chanez, Schedule of protein ingestion, nitrogen and energy utilization and circadian rhythm of hepatic glycogen, plasma corticosterone and insulin in rats, J. Nutr. 103 Ž1973. 866–874. w47x A. Sahu, P.S. Kalra, S.P. Kalra, Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus, Peptides 9 Ž1988. 83–86. w48x G. Sanacora, M. Kershaw, J.A. Finkelstein, J.D. White, Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation, Endocrinology 127 Ž1990. 730–737. w49x M.W. Schwartz, J.L. Marks, A.J. Sipols, D.G. Baskin, S.C. Woods, S.E. Kahn, D. Porte, Central insulin administration reduces neuropeptide Y mRNA expression in the arcuate nucleus of food-deprived lean ŽFarFa. but not obese Žfarfa. Zucker rats, Endocrinology 128 Ž1991. 2645–2647. w50x T. Shibasaki, T. Oda, T. Imaki, N. Ling, H. Demura, Injection of anti-neuropeptide Y gamma-globulin into the hypothalamic paraventricular nucleus decreases food intake in rats, Brain Res. 601 Ž1993. 313–316. w51x N.J. Spiteri, A.B. Alingh Prins, J. Keyser, J.H. Strubbe, Circadian pacemaker control of feeding in the rat, at dawn, Physiol. Behav. 29 Ž1982. 1141–1145. w52x B.G. Stanley, Neuropeptide Y in multiple hypothalamic sites controls eating behavior, endocrine, and autonomic systems for body energy balance, in: W.F. Colmers, C. Wahlestedt ŽEds.., The Biology of Neuropeptide Y and Related Peptides, Humana Press, NJ, 1993, pp. 457–509. w53x B.G. Stanley, K.C. Anderson, M.H. Grayson, S.F. Leibowitz, Repeated hypothalamic stimulation with neuropeptide Y increases daily carbohydrate and fat intake and body weight gain in female rats, Physiol. Behav. 46 Ž1989. 173–177. w54x B.G. Stanley, D.R. Daniel, A.S. Chin, S.F. Leibowitz, Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion, Peptides 6 Ž1985. 1205–1211. w55x B.G. Stanley, L.H. Ha, L.C. Spears, M.G. Dee II, Lateral hypothalamic injections of glutamate, kainic acid, D,L-a-amino-3-hydroxy-5methyl-isoxazole propionic acid or N-methyl-D-aspartic acid rapidly elicit intense transient eating in rats, Brain Res. 613 Ž1993. 88–95. w56x B.G. Stanley, S.E. Kyrkouli, S. Lampert, S.F. Leibowitz, Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity, Peptides 7 Ž1986. 1189–1192. w57x B.G. Stanley, D. Lanthier, A.S. Chin, S.F. Leibowitz, Suppression of neuropeptide Y-elicited eating by adrenalectomy or hypophysectomy: reversal with corticosterone, Brain Res. 501 Ž1989. 32–36.
280
C.L. Marın ´ BiÕens et al.r Brain Research 782 (1998) 271–280
w58x B.G. Stanley, S.F. Leibowitz, Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci. 35 Ž1984. 2635–2642. w59x B.G. Stanley, S.F. Leibowitz, Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior, Proc. Natl. Acad. Sci. USA 82 Ž1985. 3940–3943. w60x B.G. Stanley, W. Magdalin, A. Seirafi, M.M. Nguyen, S.F. Leibowitz, Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a variant of the Y1 receptor mediating this peptide’s effect, Peptides 13 Ž1992. 581–587. w61x B.G. Stanley, W. Magdalin, A. Seirafi, W.J. Thomas, S.F. Leibowitz, The perifornical area: the major focus of a patchily distributed hypothalamic neuropeptide Y sensitive feeding systemŽs., Brain Res. 604 Ž1993. 304–317. w62x B.G. Stanley, W.J. Thomas, Patterns of eating behavior elicited by neuropeptide Y injected into the medial perifornical hypothalamus, Soc. Neurosci. Abstr. 16 Ž1990. 773. w63x B.G. Stanley, W.J. Thomas, C.L. Bivens, Gorging of food induced by intrahypothalamic injection of neuropeptide Y, Tenth Annual
w64x
w65x
w66x
w67x
w68x
Bristol–Myers SquibbrMead Johnson Symposium on Nutrition Research, Toronto, Canada, 1990. J.H. Strubbe, J. Keyser, T. Dijkstra, J.A. Prins, Interaction between circadian and caloric control of feeding behavior in the rat, Physiol. Behav. 36 Ž1986. 489–493. D.L. Tempel, S.F. Leibowitz, Diurnal variations in the feeding responses to norepinephrine, neuropeptide Y and galanin in the PVN, Brain Res. Bull. 25 Ž1990. 821–825. D.L. Tempel, G. Shor-Posner, D. Dwyer, S.F. Leibowitz, Nocturnal patterns of macronutrient intake in freely feeding and food-deprived rats, Am. J. Physiol. 256 Ž1989. R541–R548. G. Williams, J.S. Gill, Y.C. Lee, H.M. Cardoso, B.E. Okpere, S.R. Bloom, Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats, Diabetes 38 Ž1989. 321–327. G. Williams, J.H. Steel, H. Cardoso, M.A. Ghatei, Y.C. Lee, J.S. Gill, J.M. Burrin, J.M. Polak, S.R. Bloom, Increased hypothalamic neuropeptide Y concentrations in diabetic rat, Diabetes 37 Ž1988. 763–772.