Opposite effects on feeding of suprachiasmatic nucleus neuropeptide Y administration in rats

Peptides 20 (1999) 601– 609

Opposite effects on feeding of suprachiasmatic nucleus neuropeptide Y administration in rats L. Thibaulta,*, N. Komenamib a

School of Dietetics and Human Nutrition, McGill University, Quebec H9X 3V9, Canada Domestic Science Department, Osaka International College for Women, Osaka 570, Japan

b

Received 16 September 1998; accepted 18 December 1998

Abstract The effects of injecting or infusing neuropeptide Y (NPY) into the suprachiasmatic nucleus of rats on patterns of individual macronutrient and water intake were examined during the following 2 h and also across 12 and 24 h light/dark cycles. Increased total energy intake (218 and 170%) and energy intake from the dextrin/sucrose diet (499 and 247%) were observed in the 2 h following injection of 100 pmol NPY at early light and early dark, respectively, and in the following 24 h (total energy: 67%, dextrin/sucrose: 73%). Nocturnal casein energy intake was also increased (258%) following NPY injection. Continuous infusion of 10 pmol/h of NPY suppressed nocturnal total energy (36%) and dextrin/sucrose intake (36%) as well as 24 h energy intake from casein (43%). These results demonstrate divergent effects of NPY subsequent to different mode of administration. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptide Y; Macronutrient intake; Injection; Infusion; Suprachiasmatic nucleus; Rats

1. Introduction The SCN is acknowledged to be an important brain site that controls a variety of circadian processes [17], and neuropeptide Y (NPY) is one of several neuroactive compounds previously identified as contributing to certain aspects of these regulatory controls. A body of evidence exists that strongly suggests that elevation of NPY within the brain enhances food intake. A bimodal rhythm of NPY levels in the SCN was reported, with a peak at dark onset and a peak at light onset, that corresponds to the activity-rest transition phases in the 24 h light/dark cycle [1,5,20,21]. In vitro NPY application in rat SCN slices produced a significant phase advance shown to be mediated by Y2 receptor [4]. We have reported a time-dependent effect of NPY in the SCN in rats fed a choice among dextrin/sucrose, casein and vegetable shortening/oil mixture diets, where NPY injected at a dose of 100 pmol at 4 different time points of the day was effective at stimulating energy intake from the dextrin/ sucrose diet when injected after lights-on [9]. However, a temporally dependent increased carbohydrate effect of NPY was not found across studies measuring intake of simulta* Corresponding author. Tel.: ⫹1-514-398-7848; fax: ⫹1-514-3987739. E-mail address: [email protected] (L. Thibault)

neously offered macronutrient rich diets and using other routes of administration. For example, PVN or LCV administration of NPY and PYY (5 ␮g, 78, 100 and 235 pmol) either every 8 h, at early light or at early dark consistently stimulated the intake of the carbohydrate-rich diet (corn starch, sucrose/corn starch/dextrin, dextrin/sucrose) [10,11, 13,25,26,28,29]. In addition, we failed to demonstrate an orexigenic action on NPY when 10 or 100 pmol/h were continuously infused for 7 days in the SCN of rats [8]. Specifically, the 24 h intake of a balanced semi-purified single diet was significantly decreased with NPY at a dose of 10 pmol/h, but unaltered at 100 pmol/h. Therefore, the rationale of the present study was to compare the effect of NPY chronically infused into the SCN or injected at early light and dark on feeding when rats were given a choice among macronutrient rich diets. 2. Methods 2.1. Experiment 1 2.1.1. Animals and diets Male Wistar rats (Charles River, Quebec) initially weighing 280 –300 g were individually housed in a room maintained at 24 ⫾ 1°C and 60% relative humidity, under a

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12–12 h light/dark schedule with lights on at 9:00 a.m. Rats were offered ad lib water and 3 macronutrient rich diets, separately providing carbohydrate, protein and fat (Table 1). Diet rations were presented individually in spill-proof stainless feeders and water in a drinking bottle held by a bottle holder, placed on weight scales that were connected to a Diet–Scan analyzer (AccuScan, Columbus, OH) connected to a Packard Bell computer. The system consisted of individual acrylic cages (size 41.75 ⫻ 41.75 ⫻ 31.5 cm) equipped with electronic balances for each diet and water. 2.1.2. Experimental procedures After 7 days of acclimatization to the experimental diets and environment, rats were stereotaxically implanted (Stoelting, Wood Dale, IL) under pentobarbital anesthesia (40 mg/kg) with an intracranial cannula (28 stainless gauge steel, Plastic One Inc., Roanoke, VA) connected to a piece of polyethylene tubing (PE-10, Clay Adams, NJ) toward SCN. The brain coordinates were AP: 1.3 mm posterior to the bregma; L, 0 mm; V, 8.5 mm below the skull surface, according to the brain atlas of Paxinos and Watson [14]. Animals were allowed to recover from the surgery for 4 days before the experimental protocol began. Rats were injected with 10 ␮l of 0.9% saline using a Hamilton syringe twice a day: 30 min after the beginning of the light period (early light) and 30 min after the beginning of the dark period (early dark) for 2 days to obtain food and water intake baseline. Then porcine NPY (Peninsula Laboratories Inc., Belmont, CA) was injected twice a day for the next 2 days. NPY was diluted with 0.9% saline to the concentration of 100 pmol/10 ␮l. Five ␮l of 0.9% saline were used to rinse tubing between injections. Food intake from the three macronutrient rich diets and water intake were measured during the 2 and 12 h subsequent to injections for the 4 experimental days using the DietScan system (AccuScan, Columbus, OH). 2.2. Experiment 2 2.2.1. Animals and diets Male Wistar rats (Charles River, Quebec) initially weighing 230 –260 g were individually housed and fed as described in Experiment 1. 2.2.2. Experimental procedures After 7 days of acclimatization to the experimental diets and environment, rats were stereotaxically implanted under pentobarbital anesthesia (40 mg/kg) with an intracranial cannula (28 stainless gauge steel, Plastic One Inc., Roanoke, VA) toward SCN. The coordinates were AP: 1.3 mm posterior to the bregma; L, 0 mm; V, 8.5 mm below the skull surface, according to the brain atlas of Paxinos and Watson [14]. Animals were allowed to be recovered from the surgery for 3 days. Then the animals were placed in the DietScan system (AccuScan, Columbus, OH) and food intake from the three macronutrient rich diets and water intake

Table 1 Composition of diets (g/100 g)* Ingredients

Carbohydrate

Protein

Fat

Casein, high nitrogen Sucrose Dextrin Oil mixture Vegetable shortening Cellulose, alphacel AIN salt mixture AIN vitamin mixture Chlorine chloride Energy density (kcal/g diet)

— 15.0 77.9 — — 2.0 4.0 1.0 0.1 3.81

92.9 — — — — 2.0 4.0 1.0 0.1 3.81

— — — 20.0 68.3 2.0 7.6 1.9 0.2 7.36

* Prepared in our laboratory. Diet ingredients were purchased from ICN Biochemical (Cleveland, OH), except for the vegetable shortening (Crisco) purchased at local supermarket.

were recorded every 12 h for 7 days (pre-infusion period). While animals were under ether anesthesia, an osmotic pump (model 2001, pump rate 1 ␮l/h, Alza Corporation, Palo Alto, CA) filled with 170 ␮l porcine NPY (Peninsula Laboratories Inc., Belmont, CA) or 0.9% saline was connected to the brain cannula with polyethylene tubing (PE-10 and PE-60, Clay Adams, NJ) and inserted in the subcutaneous (s.c.) pocket in the midscapular area of the back of the rats. NPY was diluted with 0.9% saline to the concentration of 10 pmol (0.042 ␮g)/␮l. This surgery was performed between 1000 and 1200 h. Food intake and feeding pattern were measured every 12 h during the 7-day infusion and 7-day post-infusion periods. 2.2.3. Histologic examination At the end of the experiment, rats were given an overdose of sodium pentobarbital (60 mg/kg) and the brains were perfused with 0.9% saline, followed by 10% phosphate buffered formalin for 15 min. Brains were excised and placed in 10% sucrose-buffered formalin solution. Frozen coronal sections of 30 ␮M thick were cut with a cryotome (Model 620 M, Shandon) and stained with cresyl violet (modified method of 32). The brain sections were then examined under a microscope equipped with a camera and the location of the cannula tip was determined according to the brain atlas of Koenig and Klippel [7]. Examination of correct positioning of cannulae brought the final number of animals to 6 in Experiment 1 and 12 in Experiment 2. 2.2.4. Statistical analysis Results are expressed as means ⫾SEM. Data from Experiment 1 were analyzed statistically by a three-way analysis of variance (ANOVA). The main effects of treatment (saline, NPY), exposure (first, second) and time (early light, early dark) were tested. The interaction between treatment, exposure and time were also tested. Experiment 2 data were analyzed with a two-way ANOVA, with the main effects of treatment (saline, NPY), period (pre-infusion, infusion, post-infusion) and their interaction tested. When the main

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Fig. 1. Water intake (g) and total energy intake (kcal) for 2 h after injection of saline (Days 1 and 2) or 10 ␮l NPY (100 pmol, Days 3 and 4) into the SCN at early light and early dark. Each bar represents the mean ⫾ SEM of six rats. See text for significance.

effects were significant, multiple comparisons were tested for significance by Tukey’s test. Comparisons were also made with contrast statements. A probability of less than 5% was considered significant. Statistical analysis was carried out using Super ANOVA computer program for Macintosh (version 1.11).

3. Results 3.1. Experiment 1 Water intake (g), total energy (kcal) intake and energy intake from the 3 macronutrient rich diets for 2 h after a first and a second exposure to saline or NPY injection into the SCN at two different time points of the day are shown in Figs. 1 and 2. Water intake was not significantly affected by treatment, time or exposure. Treatment significantly af-

fected total energy intake, F(1,40) ⫽ 15.8, P ⫽ 0.0003, and energy intake from the dextrin/sucrose mixture diet, F(1,40) ⫽ 16.7, P ⫽ 0.0002, with rats injected with NPY ingesting more total energy and more energy from the carbohydrate rich diet than when injected with saline. Moreover, the treatment by exposure interaction was significant for total energy intake, F(1,40) ⫽ 3.97, P ⫽ 0.05, and contrast revealed a significantly (P ⬍ 0.0001) higher total energy intake on the first exposure to NPY than on the first exposure to saline. A significant time effect was found for total energy intake, F(1,40) ⫽ 4.73, P ⫽ 0.036, and energy intake from the protein rich diet, F(1,40) ⫽ 4.63, P ⫽ 0.038, that were significantly higher at early dark than at early light. Water, total energy and energy intake from the three macronutrient rich diets over 24 h and at 12 h following a first and a second exposure to saline or NPY injection into the SCN at two different time points of the day are presented in Figs. 3 and 4. Treatment significantly affected 24 h

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Fig. 2. Macronutrient intake (kcal) for 2 h after injection of saline (Days 1 and 2) or 10 ␮l NPY (100 pmol, Days 3 and 4) into the SCN at early light and early dark. Each bar represents the mean ⫾ SEM of 6 rats. See text for significance.

total energy intake, F(1,20) ⫽ 7.6, P ⫽ 0.01, and energy intake from the dextrin/sucrose mixture diet, F(1,20) ⫽ 6.1, P ⫽ 0.02, and the casein diet, F(1,20) ⫽ 14.2, P ⫽ 0.001, with rats injected with NPY ingesting more total energy and more energy from the carbohydrate and the protein rich diets than when injected with saline. Time of the day significantly affected water intake, F(1,40) ⫽ 23.3, P ⫽ 0.0001, total energy intake, F(1,40) ⫽ 16.2, P ⫽ 0.0002, and energy intake from the dextrin/sucrose mixture diet, F(1,40) ⫽ 13.1, P ⫽ 0.0008, and the casein diet, F(1,40) ⫽ 17.3, P ⫽ 0.0002, with nocturnal intakes being higher than

diurnal intakes. Treatment also affected the 12 h light and dark total energy intake, F(1,40) ⫽ 11.3, P ⫽ 0.002, and energy intake from the dextrin/sucrose mixture diet, F(1,40) ⫽ 8.2, P ⫽ 0.007, and the casein diet, F(1,40) ⫽ 21.8, P ⫽ 0.0001, with higher intakes in animals injected with NPY. In addition, a significant time by treatment interaction was found for energy intake from the casein diet, F(1,40) ⫽ 10.4, P ⫽ 0.003, and contrast revealed a significantly higher (P ⬍ 0.0001) nocturnal protein intake in rats injected with NPY when compared to diurnal intake after NPY injection and to nocturnal intake after saline injection.

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Fig. 3. Water intake (g) and total energy intake (kcal) for 24 h, 12 h dark and 12 h light after injection of saline (Days 1 and 2) or 10 ␮l NPY (100 pmol, Days 3 and 4) into the SCN at early light and early dark. Each bar represents the mean ⫾ SEM of six rats. See text for significance.

3.2. Experiment 2 As shown in Figs. 5, 6 and 7, in all rats energy intakes decreased during the first day of the infusion period, immediately after surgical insertion of the osmotic pump. Total energy intake over 24 h and during the 12 h dark and light phases in rats infused with saline or NPY at 100 pmol into the SCN is shown in Fig. 5. Period significantly affected 24 h, F(2,246) ⫽ 23.7, P ⫽ 0.0001, and 12 h dark, F(2,246) ⫽ 29.2, P ⫽ 0.0001, total energy intake, with a lower energy intake during the infusion period than during the preand post-infusion periods. Nocturnal energy intake was also affected by treatment, F(1,246) ⫽ 14.8, P ⫽ 0.0002, with rats infused with NPY ingesting less total energy than rats infused with saline. Water intake was affected by period only during the 12 h dark phase, F(2,246) ⫽ 3.11, P ⬍ 0.05, where it was decreased during infusion (18.1 ⫾ 0.90 g)

Fig. 4. Macronutrient intake (kcal) for 24 h, 12 h dark and 12 h light after injection of saline (Days 1 and 2) or 10 ␮l NPY (100 pmol, Days 3 and 4) into the SCN at early light and early dark. Each bar represents the mean ⫾ SEM of 6 rats. See text for significance.

when compared to pre-infusion (20.7 ⫾ 0.56 g). As presented in Fig. 6, 24 h energy intake from the protein rich diet was affected by treatment, F(1,246) ⫽ 5.48, P ⫽ 0.02, with NPY diminishing casein energy intake. A period effect was also found for 24 h, F(2,246) ⫽ 21.2, P ⫽ 0.001, and 12 h dark, F(2,246) ⫽ 22.8, P ⫽ 0.0001, energy intake from the protein diet, and intakes were lower during infusion than during the pre- and post-infusion periods. Caloric intake from the dextrin/sucrose mixture (Fig. 7) was modified by

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Fig. 5. Total energy intake (kcal) for 24 h, 12 h dark and 12 h light in rats infused with 10 pmol/h of NPY into the SCN. Values are expressed as the mean ⫾ SEM of 6 rats. See text for significance.

Fig. 6. Energy intake from casein (kcal) for 24 h, 12 h dark and 12 h light in rats infused with 10 pmol/h of NPY into the SCN. Values are expressed as the mean ⫾ SEM of 6 rats. See text for significance.

treatment over 24 h, F(1,246) ⫽ 6.86, P ⫽ 0.009, and the 12 h dark phase, F(1,246) ⫽ 25.9, P ⫽ 0.0001. Specifically, animals infused with NPY ingested less energy from the carbohydrate rich diet than those infused with saline. Period also significantly affected 24 h, F(2,246) ⫽ 14.4, P ⫽ 0.0001, and 12 h dark, F(2,246) ⫽ 18.2, P ⫽ 0.0001, energy intake from the carbohydrate rich diet, that were decreased during infusion when compared to before infusion. Energy intake from the oil mixture/vegetable shortening diet was

affected by period during the 12 h dark phase, F(2,246) ⫽ 3.69, P ⫽ 0.03, and fat energy intake during infusion (14.0 ⫾ 1.0 g) was lower than that observed before infusion (18.3 ⫾ 1.2 g). During the experimental period, mean body weights in the two experimental groups remained stable during the pre-infusion (317 ⫾ 2.7 g) and infusion (326 ⫾ 3.9 g) periods but increased significantly at post-infusion (350 ⫾ 5.8 g), F(2,239) ⫽ 17.1, P ⫽ 0.0001.

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Fig. 7. Energy intake from dextrin/sucrose mixture (kcal) for 24 h, 12 h dark and 12 h light in rats infused in the 10 pmol/h of NPY into the SCN. Values are expressed as the mean ⫾ SEM of 6 rats. See text for significance.

4. Discussion The present study reports that 100 pmol of NPY injected in the SCN at light and dark onset caused an increase in total energy intake and energy intake from the dextrin/sucrose mixture during the following 2 and 24 h. The increased 2 h total energy intake was more prominent on the first exposure to NPY. Nocturnal 12 h protein energy intake was augmented in animals injected with NPY when compared to

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saline injection. Chronic and continuous infusion of 10 pmol/h of NPY into the SCN rather suppressed nocturnal total energy intake, 24 h and nocturnal energy intake from the carbohydrate rich diet, and 24 h energy intake from the protein rich diet. The increased energy intake from the dextrin/sucrose mixture effect of 100 pmol of NPY injected in the SCN at early light is consistent with results obtained in a previous study in which adult Wistar male rats provided with choice among casein, dextrin/sucrose and vegetable shortening/oil mixture diets received 100 pmol of NPY injected into the SCN or the LCV at 4 different time points of the day [9]. The latter study had revealed that only SCN NPY was effective at increasing energy intake from the dextrin/sucrose diet at early light, but not at late light, early dark or late dark, whereas this carbohydrate enhancing effect is observed at early light and dark in the present study. This different effect of time of day between our previous and the present work could be related to the fact that the former animals were fasted for 2 h before a daily NPY injection, whereas rats that were not fasted (freely fed) before being injected twice a day with NPY were used in the present study. The carbohydrate enhancing effect at early dark could be absent during restricted feeding because there is insufficient protein intake to induce a carbohydrate appetite and to motivate selection of the carbohydrate rich diet. Perifonical hypothalamic injection of NPY at a dose of 78 pmol administered at six different time points of the day consistently increased intake from a Purina rat chow/sucrose/Carnation evaporated milk mixture in freely fed adult Sprague–Dawley male rats [27]. Exogenous NPY stimulation of intake from carbohydrate-rich diets is a consistent effect across experiments using different dietary choices. Doses of 78 and 100 pmol of NPY injected in the PVN of adult Sprague–Dawley male rats at dark onset caused an increase in energy intake from a sucrose/corn starch/dextrin mixture during the following 1, 2 and 3 h in choice among casein, sucrose/corn starch/ dextrin and lard diets [10,11,28]. The carbohydrate facilitating effect of exogenous NPY was always tested with dextrin-containing preparation. To confirm that this represents a carbohydrate specific selection effect, NPY injection in the SCN should be tested with other carbohydrate preparations. Inconsistent effects on protein intake have been observed with NPY. Protein intake (kcal) was decreased during the following 3 and 12 h when NPY was administered at a dose of 100 pmol at dark onset in the PVN of adult Sprague– Dawley male rats freely feeding on a choice among casein, sucrose/dextrin/corn starch and lard diets [10]. However, in the same study, the selective NPY2 receptor agonist NPY 2–36 injected at 100 pmol in the PVN increased intake (kcal) from the casein diet during the following 3 and 12 h. In the present study, when NPY was infused in the SCN at a dose of 10 pmol/h for 7 days, it depressed 24 h energy intake from the casein and the dextrin/sucrose mixture diets

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while these intakes were augmented following NPY injection at 100 pmol. Thus, in the same experimental conditions, injection versus infusion mode of administration had opposite effects on 24 h energy intake from the casein and the dextrin/sucrose mixture diets. The present results confirm the 24 h food intake suppressive effect (g) of 10 pmol/h (0.042 ␮g) of NPY infused for 7 days in the SCN reported in a previous study using a balanced semi-purified single diet [8]. Indeed, when we expressed the present 24 h kcal total intake data into grams, the statistical analysis revealed a significant effect of treatment, F(1,246) ⫽ 5.66, P ⫽ 0.02, with NPY treated rats ingesting less food (17.7 ⫾ 0.53 g) than saline treated animals (19.3 ⫾ 0.53 g), and a period effect, F(1,246) ⫽ 26.2, P ⫽ 0.0001, with lower intake during infusion (15.7 ⫾ 0.69 g) than during pre-infusion (21.7 ⫾ 0.52 g). In agreement with our findings is the study from Chance et al. [2] reporting that chronic intrahypothalamic infusion of NPY at 0.125 ␮g/h did not alter food intake (g/100g body weight) whereas daily intrahypothalamic injections of NPY (250 and 500 ng, and 1, 2, and 4 ␮g) at early dark stimulated 24 h intake (g/100 g body weight) in male Fisher 344 rats. In the same study, chromatography of the pooled residual in the minipumps revealed intact NPY, ruling out the possibility that results are due to NPY degradation. Therefore, the authors suggested that the absence of feeding response to NPY infusion could have been due to down-regulation of NPY receptors in the presence of a constant supply of exogenous peptide. Recent findings also reported paradoxical effects of NPY on food intake such as the absence of effect of low doses of intraventricularly administered NPY to baboons, but a suppressive effect at higher doses [22] and the increase intraoral intake after food deprivation but not after NPY administered at 3, 9.5 and 30 ␮g in the third ventricle [19]. It is also possible that NPY can have both orexigenic and aversive properties due to the existence of different populations of central NPY receptors. The effect of NPY on feeding is indeed thought to be mediated by more than one class of NPY receptors. Although NPY binds to both Y1 and Y2 receptors, it was proposed that the NPY Y5 receptor subtype mediates the feeding response to exogenous and endogenous NPY [18]. However, the recent pharmacological demonstration of the expression and discrete localization of the Y5 receptor protein in the rat brain revealed only low densities of Y5 binding sites in the hypothalamus [3]. In addition, it was demonstrated that the NPY Y5 receptor was not a critical physiological feeding receptor in mice [12], in rats [6] and in humans [15]. Rather, a potential role for NPY Y5 receptor in motor activity was suggested from studies where NPY administered intracerebroventricularly reduced motor seizures induced by kainic acid [31] and motor score of the morphine withdrawal reaction [30] in rats. The injection of 1␮g of NPY into the juxtafornical hypothalamus promoted feeding and enhanced locomotor activity, and it was demonstrated that the NPY-induced rise in overall

metabolic rate is entirely accounted for by the enhancement in locomotor activity [16]. An increase in the locomotor and exploratory activity was also reported after injection of NPY into the frontal cortex of rats [23,24].

Acknowledgements This research was supported by a grant from Natural Sciences and Engineering Research Council of Canada and Japan Sciences and Technology Funds. The authors wish to thank and gratefully acknowledge the technical assistance of Luotja Yang and Elise Mok.

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