effects of intraventricular administration of neuropeptide y on feeding behavior in fasted female...

E L S E V I E R Regulatory Peptides 52 (1994)47-52

Effects of intraventricular administration of neuropeptide Y on feeding behavior in fasted female rats with ventromedial hypothalamic

lesions

Y a s u o T a n a k a , M a s a t o E g a w a , S h u j i I n o u e * , Y u t a r o T a k a m u r a

Third Department of Internal Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Japan

(Received 9 August 1993; revised version received 24 January 1994; accepted 9 March 1994)

Abstract

The role of ventromedial hypothalamic nucleus (VMN) in feeding behavior induced by central administration of porcine neuropeptide Y (NPY) in fasted female rats was investigated focusing on the effect of the intracerebro-ventricular (i.c.v.) injection of NPY on food intake in rats with VMN lesions after 24 h of food deprivation. Cumulative food intake was measured 1, 3 and 6 h after injection. In Exp. 1, the i.c.v, injection of NPY into intact rats stimulated food intake compared with the injection of saline 1 and 3 h after the injections in a dose dependent manner (1 h; NPY 1 #g, 3.29 + 0.43, 2/ag, 4.64 _+ 0.88, 5 #g, 5.15 + 0.61 vs. saline 2.48 + 0.42 g, P<0.05 in 2 and 5 #g). 5 pg of NPY significantly stimulated food intake 6 h later. In Exp. 2, i.c.v, injection of NPY (2/~g) in VMN-lesioned rats showed no significant effect on food intake compared with the injection of saline 3 weeks after VMN lesioning (i h; NPY 2 gg, 2.46 + 0.58 vs. saline 2.39 _+ 0.12 g). These results suggest that central administration of NPY enhances food intake in fasted female rats and that the VMN is one of the crucial sites of NPY induced feeding.

Key words." Ventromedial hypothalamic nucleus; Neuropeptide Y; Feeding behavior; Fasting; Intraventricular injection; Female

I. Introduction

Neuropeptide Y (NPY) is known to be a stimulator of food intake when administered intracerebro- ventricular (i.c.v.) in satiated and fasted rats. The effects of N P Y on feeding behavior in satiated rats

* Corresponding author. Fax: + 81 45 7843546.

0167-0115/94/57.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 2 3 - Q

were extensively studied [ 1-4]. In fasted rats, the effects ofi.c.v, injection of N P Y were examined only in male rats [5,6] and the effect in fasted female rats has not previously been reported.

Many brain-gut peptides are known as food intake regulators. /~-endorphin [7,8], dynorphin [9] are stimulators, and others are suppressants [10], and many of them have been well discussed with

48 Y, Tanaka et al. / Regulatoo' Peptides 52 (1994) 47-52

regard to their working sites. One of the potent peptides, corticotropin-releasing hormone (CRH) de- creased food intake only when injected into the intact paraventricular nucleus (PVN) [10]. I.c.v. injection of CRH also depressed food intake acutely and chronically in ventromedial hypothalamic nucleus (VMN) lesioned rats [11], suggesting that VMN is not the critical site that mediates the effect of CRH. On the other hand, neuropeptide Y stimulates food intake when injected i.c.v. [4,12] into the perifornical hypothalamus (PFH) [ 13], PVN, VMN, or the lateral hypothalamic area (LHA) [6,14,15]. However, the role of VMN, which is presumed to mediate the stimulatory effect of NPY, has never been examined on feeding behavior using VMN- lesioned rats. We therefore sought to determine whether NPY is similarly effective in fasted rats with VMN lesions.

2. Materials and methods

2.1. Animals

Female Sprague-Dawley rats weighing 200 to 250 g were used in this study. Rats were housed in in- dividual stainless steel cages under a controlled tem- perature (24 + 2°C) and a 12-h diurnal light cycle (lights on 08:00 h/lights off 20:00 h) with free access to powdered chow which was placed in a stainless steel food box with an acrylic holder to prevent spill- age. Each rat was subjected to only one administration test at 10:00 h.

2.2. Experiment 1: effect of i.c.v, administration of NPY on food intake in fasted female rats

porcine NPY (1, 2 and 5 gg) (Peptide Institute, Osaka, Japan) dissolved in 5 gl saline through a cannula using a 28 gauge stainless steel injector con- nected to a silastic tube and microinjector. To avoid back-flow through the tract, the injector was left in place for 10 min following this procedure. It was then pulled out and rats were returned to their cages with pre-weighed powdered chow. 1, 3 and 6 h after injection, the cumulative amount of food intake was measured.

2.3. Experiment 2: effect of i,c.v, administration of NPY on food intake in fasted female rats with VMN lesions

Under hexobarbital anesthesia, the rat's head was fixed using a stereotaxic instrument (David Kopf Instruments, Model 900, Tujunga, CA, USA). Bi- lateral electrolytic lesions were created by passing a 2.0 mA direct cathodat current for 20 s through stainless steel electrodes (insect pins size 00 insu- lated with epoxylite, except for a 0.5 mm area at the tip). The electrodes were placed 2.8 mm posterior to the bregma, 0.7 mm bilateral to the sagittal sinus, and 9.5 mm below the surface of the skull, with the incisor bar set 3.3 mm below the interaural line [ 16]. After 7 days, if VMN is appropriately destroyed, rats show hyperphagia and increase in body weight. Rats with an increase of more than 40 g a week were used as VMN-lesioned rats in this study [17]. A guide cannula was implanted into the third ventricle by a method similar to that used in Exp. 1. After 7 more days of recovery, saline or NPY was injected into the third ventricle and cumulative food intake was measured 1, 3 and 6 h thereafter.

Rats were anesthetized with 50 mg/kg i.p. hexobarbital, then stereotaxically implanted with a 23 gauge 15 mm stainless steel guide cannula aimed at the third cerebroventricle. Each rat was handled for a minimum of 5 min/day. After 7 days of recovery, rats were deprived of food for 24 h, and then were injected over a 5 min period with 5 /~1 saline or

2.4. Histology

After the experiment, all rats were injected with dye (5/~1 methylene blue) for i.c.v, through the same injectors. 100 ml of a 10~o formalin solution was then perfused through the animals' hearts under an overdose of anesthesia, and the brains were removed

Y. Tanaka et aL / Regulatory Peptides 52 (1994) 47-52 49

and placed in 10~o formalin solution. They were sectioned to confirm that the VMN had been appropriately lesioned [ 18].

2.5. Statistical analysis

Data were expressed as means + S.E.M. Statisti- cal analysis were made by one-way analysis of var- iance (ANOVA) using NCSS program (NCSS, Kaysville, UT, USA) for an IBM PC computer. In the presence of significance, within-treatment com- parisons were performed using the method of Duncan's multiple comparison test. Statistical significance was taken as P<0.05.

3. Results

cantly increased food intake after 1 h to more than that of the saline-injected group (n = 6) by post hoc test (P<0.05) following ANOVA (F(3,23)=4.45, P<0.05). 3 h after the injections, food intake of all three NPY injected groups (1/~g, n = 7; 2 #g, n = 5; 5/~g, n = 6) were significantly higher than that of the saline-injected group (P< 0.05 and P < 0.01 by post hoc, after ANOVA F(3,23)= 6.11, P<0.005). With the highest dose of NPY (5 #g), the cumulative food intake 6 h after the injections was significantly augmented compared to that of the saline-injected group (P < 0.05 by post hoc, after ANOVA F(3,23)= 3.44, P<0.05).

3.2. Experiment 2: effect of i.c.v, administration of NPY on food intake in fasted female rats with VMN lesions

3.1. Experiment 1: effect of i.c.v, administration of NPY on food intake in fasted female rats

As shown in Fig. 1, i.c.v, administration of two high doses of NPY (2/~g, n = 5; 5 Fg, n --- 6) signifi-

To examine whether or not the VMN is involved in the NPY induced feeding behavior, VMN-lesioned rats were used for this experiment. Fig. 2 indicates that food intake of VMN-lesioned rats injected with 2 Fg of NPY (n -- 6) was not significantly different from that of VMN-lesioned rats injected with saline (n = 5) at three time points. There were no significant

5 , "~

~ s d .

0 0 1 2 3 4 5 Tim6e(h)

Fig. 1. Cumulative food intake 1,3 and 6 h after 1, 2 and 5 Fg of NPY i.c.v, administration in fasted female rats. Each point represents mean _+ S.E. of 5-7 rats. O : saline, • : 1/tg of NPY, • : 2 #g of NPY, m: 5 pg of NPY. *P<0.05, **P<0.01, compared to value of saline-injected rats at the same time point.

differences in the extent or placement of VMN lesions between the groups with and without NPY.

Fig. 2. Cumulative food intake in fasted VMN-lesioned rats 1, 3 and 6 h after 2 #g of NPY i.c.v, administration. Each point represents mean _+ S.E. of 5-6 rats. O: saline, &: 2 pg of NPY.

50 Y. Tanaka et al. / Regulator), Peptides 52 (1994) 47 -52

pV, N, ,,-]r-.,

, / , ' , B R E G M A

', . . . . / i l J ' , 6 -1.8

VM'

1 . 0 m m

Fig. 3. Schematic representations of the HA lesions in Exp. 2. Areas of lesions in all rats and other areas affected in some rats are shown by black and shaded areas on the Paxinos atlas [ 16]. The AP level of each section is indicated by the numbers at right. There was no significant difference in the VMN lesions between the groups with and without NPY.

They did not extend to the PFH or the PVN dorsally but occasionally reached the base of the brain ven- trally (Fig. 3).

4. Discussion

In fasted female rats, two of the high doses (2 and 5 #g) of NPY increased food intake 1 h after i.c.v. injection, all three doses of NPY increased it after 3 h, and the highest dose (5 #g) increased it after 6 h.

The effects of NPY on food intake were thus dose- dependent. These observations are compatible with the study by Schick et al. [6] in male rats under fasted conditions, who showed that food intake was significantly augmented by i.c.v, injection of NPY after 24 h of fasting.

A recent study suggests that PFH is the most sensitive site of NPY in the hypothalamus [13]. VMN has also been established as a feeding regulatory area, and has responded to microinjection with/~-endorphin [ 19], norepinephrine [20], GABA [21] and neuropeptide Y [6,15], resulting in increased food intake in rats. To examine the influence of this nucleus on NPY mediated increase in food intake, VMN-lesioned rats were tested for their feeding response to this neuropeptide. The feeding response disturbed in this study, suggesting that VMN is involved in the mediation of NPY-induced feeding. This is consistent with the results that microinjections of NPY into VMN reportedly stimu- late food intake [6,15]. There are two subtypes of NPY agonists: [Leu31, Pro34]NPY, (Y1)NPY agonist, and NPY13-36, (Y2)NPY agonist. Central administration of Y1 but not Y2 stimulates feeding behavior [12,15,22] and this effect is mediated by hypothalamic sites. Consequently, we presume that the feeding response of NPY is mediated through the (Y1)NPY receptor.

Although further investigations are needed, there are three possible explanations for the role of VMN in the mechanism of NPY mediated increase in food intake. First, it is reasonable to consider that VMN is one of the neural pathways which connects to PFH or PVN in the NPY induced feeding behavior, since the potent effect of NPY on feeding behavior is reported to be within PFH and PVN [13,14]. Indeed, projections from PFH and PVN to VMN are reported [23]. Second, it may also be possible that NPY inhibits the satiety role of VMN through the NPY receptors, independent of other hypothalamic sites. This speculation is compatible with the stimulatory effect of microinjections of pentobarbital into VMN on feeding behavior [24]. A dose response

Y. Tanaka et al. / Regulatory Peptides 52 (1994) 47-52 51

curve might demonstrate differences in VMN re- sponsiveness to NPY. Another possibility is that the effect of NPY through VMN is mediated by changes of peripheral metabolites such as plasma glucose and insulin [25]. Cholecystokinin (CCK) is known to be a feeding suppressant and its action is assumed to be due to the hyperglycemia it produces [26]. VMN-lesioned rats show abnormal circadian rhythms in feeding behavior [27], hormones and metabolites [28], and these abnormalities might inhibit the stimulatory effect of NPY in these rats, even if NPY, as a potent stimulator of food intake, were to act within hypothalamus. This is unlikely, however, because i.c.v, injections of CRH, a potent suppressant of intake, did reduce food intake in VMN- lesioned rats [ 11 ], suggesting that peripheral abnormalities of VMN lesions do not inhibit the potent effects of centrally administered peptide hormones on feeding behavior.

Since the loss of circadian rhythm in VMN- lesioned rats might influence the effect of the NPY under ad libitum situation, we preferred the fasted condition which assures the same condition in both control and VMN-lesioned rats. It is possible that fasted VMN-lesioned rats might consume food at a maximal rate during the first hour after being offered food, and that the effects of NPY might be con- founded under these circumstances. However, there was no significant difference in food intake during the first hour between control rats and VMN- lesioned rats after saline injection (2.48 + 0.42 vs. 2.39 + 0.12 g), suggesting that the effect of fasting on feeding behavior was not different between the two groups. In the present study, we examined the effect in female rats, since the effect of VMN lesions on body weight gain following lesioning was more re- markable in females than in males [29]. However, the effect of gender difference on the results requires further investigation.

In summary, i.c.v, injection of NPY increased food intake in fasted female rats, and the results with VMN-lesioned rats suggested that VMN plays an important role in NPY mediated eating behavior in

hypothalamus, presumably through the NPY receptor in VMN or disturbance of the neural pathway to PFH or PVN.

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effects of intraventricular administration of neuropeptide y on feeding behavior in fasted female...

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