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Neuropeptide K suppresses feeding in the rat
Regulatory Peptides, 23 (1988) 135 143 135
Elsevier RPT 00745
Neuropeptide K suppresses feeding in the rat Abhiram Sahu, Pushpa S. Kalra, Michael G. D u b e and Satya P. Kalra Department q[' Obstetrics and Gynecoh~gy, University q/Florida College q/" Medicine, Gainesville. FL 32610 (U.S.A.) (Received 22 April f988; revised version received and acccepted 15 June 1988)
Summary We studied the effects of neuropeptide K (NPK), a 36 amino acid residue peptide of the tachykinin family, on latency to onset of feeding and cumulative 1 and 2 h food intake in three experimetal paradigms. Intraperitoneal injection of NPK (1.25 and 3.14 nmol) to food-deprived rats delayed the onset of feeding and significantly decreased the cumulative food intake, lntraperitoneal injection of NPK (1.25 and 3.14 nmol) to water-deprived rats produced no effect on subsequent drinking behavior. Similarly, intraperitoneal injection of NPK (3.14 nmol) 15 min before onset of the dark phase (of the light-dark cycle) significantly delayed the occurrence of ingestive behavior and the cumulative food intake was markedly suppressed. Furthermore, administration of NPK intraperitoneally (0.5 3.14 nmol) 15 min before intraventricular (i.c.v.) injection of neuropeptide Y (NPY 0.47 nmol) to satiated rats significantly suppressed NPY-induced feeding and delayed the onset of ingestive behavior. However, when administered centrally prior to NPY injection, NPK delayed the onset of feeding response only. Collectively, these findings show that NPK can acutely and consistently suppress feeding behavior. Neuropeptide K; Feeding; Inhibition; Dark phase; Starvation; Neuropeptide Y
Introduction In recent years a number of neuropeptides have been shown to modulate feeding behavior in the rat [1 3]. Neuropeptide K (NPK), a 36 amino acid residue peptide Correspondence: S.P. Kalra, Department of Obstetrics and Gynecology, University of Florida College of Medicine, P.O. Box J-294 JHMHC, Gainesville, FL 326[0, U.S.A. 0167-0[ 15/87/$03,50 ,i!~ [988 Elsevier Science Publishers B.V. (Biomedical Division)
136 belonging to the tachykinin family, was originally isolated from porcine brain [4], and has been identified recently in selected neural sites in the rat brain [5,6]. Detailed immunocytochemical mapping of NPK immunoreactivity in the rat brain showed a large number of N P K immunoreactive groups of perikarya and fiber system in hypothalamic sites [6] previously implicated in the control of ingestive behavior in the rat [7]. In addition, NPK has been found in many neurons in the gastrointestinal tract [8]. During our investigation of the neuroendocrine effects of N P K , we did not observe any stimulatory effects of N P K on feeding in satiated female rats [9]. We now report that N P K suppresses feeding in 3 experimental paradigms in male rats, viz. feeding that occurs normally during the dark phase of the light dark cycle and that induced by either food deprivation [10,11] or neuropeptide Y (NPY) [12 15] administration in satiated rats.
Materials and Methods Adult male rats (CrI:CD)RR, Charles River Laboratories, Wilmington, MA) weighing 325 425 g were housed singly under controlled temperature (22°C) and lighting (lights on 05.00 19.00 h) with ad libitum access to Purina rat chow and water. The control and experimental groups in each of the 4 experiments were balanced for mean body weight. Water was available ad libitum during the experimental observation periods.
Experiment 1 The effects of i.p. injection of N P K (1.25 and 3.14 nmol in 0.1 ml saline, Peninsula Laboratories, Belmont, CA), or vehicle alone on food consumption by food-deprived (FD) rats were studied. The doses of N P K selected were based on our previous studies [9]. The food was removed from the cages for 24 h before the start of the experiments at 09.00 10.00 h. N P K or saline was administered 15 rain before the introduction of preweighed food pellets in each cage (0 min). The latency to onset of food intake (LOF) was recorded. At the end of the first hour, the remaining food pellets and spilled food were carefully collected and weighed to the nearest 0.001 g on a Mettler balance. Fresh, preweighed food pellets were replaced to determine food intake during the second hour by these rats. At the end of the second hour the remaining pellets and spilled food were collected and weighed. The cumulative 1 and 2 h food intake in response to N P K or saline were calculated. In the next experiment, the effect of N P K (l.25 and 3.14 nmol in 0.1 1331saline) or vehicle alone on drinking in water-deprived rats was studied. Water bottles were removed from the cage at 13.00 h and fk~od was available ad libitum. The next day at 09.00 09.30 h food cups were removed and 15 rain later N P K or vehicle alone was injected i.p. Access to ad libitum water was reinstituted 15 rain later. Latency to onset of drinking was recorded and the amount of water consumed at 15 and 45 min was calculated.
137
Experiment 2 The effect of i.p. NPK (3.14 nmol in 0.l ml saline) or vehicle alone on feeding that occurs after onset of the dark phase of the light-dark cycle was studied, The selection of the NPK dose was based on the results from Expt. 1. Food pellets were removed at 18.00 h and NPK or saline was injected at 18.45 h, 15 rain before lights-off (19.00 h). Preweighed food pellets were introduced into each cage at 19.00 h. On the first day of the experiment, control rats (n = 15) were injected with saline and the LOF and cumulative 1 and 2 h food intake were recorded as in Expt. 1. On the following day these rats received N P K and the LOF and food intake were similarly monitored. This experimental design allowed within-subject statistical comparisons of the LOF and food intake with each rat serving as its own control.
Experiment 3 Since we have previously reported that i.c.v, or i.p. injection of NPK did not affect food intake in satiated rats [9], we now explored the possibility whether NPK would influence feeding evoked by orexigenic peptides. The effects ofi.p. NPK (0.125, 0.5, 1.25 or 3.14 nmol/rat in 0.1 ml saline) or vehicle alone on NPY-induced feeding in satiated rats was studied in this experiment. The selection of 0.47 nmol dose of NPY was based on our previous observation that it invariably produced near-maximal feeding in satiated rats [12-15]. Permanent stainless-steel cannulae were placed stereotaxically into the third ventricle of the brain under pentobarbital anesthesia (40 mg/kg, i.p., [16]) and the rats allowed to recover for 2 weeks. On the day of the experiment (09.00 10.00 h), patency of the i.c.v, cannula was confirmed by efflux of cerebrospinal fluid (CSF) and only those rats showing good efftux of CSF were used. Food was removed from the cage 1 h before the start of the experiment. Rats were divided into two groups on the day of the experiment. One group received one of the 4 doses of NPK and the other received saline i.p. 15 min before the i.c.v, injection of NPY in 3/~1 saline to both groups. Immediately after NPY injection (0 min), preweighed food pellets were placed in each cage. The LOF and cumulative 1 and 2 h food intake were calculated as described above. Thus, for assessing the effectiveness of each dose of NPK to modulate NPY-induced feeding, we concurrently ran a control group receiving saline instead of NPK followed by i.c.v. NPY. In addition, a group of control rats received i.c.v, saline instead of NPY.
Experiment 4 In this experiment the effect of centrally administered N P K (0.5 or 1.25 nmol) on NPY-induced (0.47 nmol) feeding in satiated rats was studied. The experimental design was the same as in Expt. 3 except that NPK or vehicle (3/A saline) was injected i.c.v. 15 rain before the i.c.v, injection of NPY. The LOF and cumulative food intake were calculated as described above. The data for Expts. 1 and 4 were analyzed by one-way analysis of variance followed by Duncan multiple range test. Data for Expt. 3 were compared by Student's t-test. For Expt. 2, paired t-test was used to compare the saline (day 1) and NPK-induced effects (day 2) on LOF and food intake in the same rat.
138
Results The effect of NPK on LOF and food intake in FD rats (Fig. 1) I.p. a d m i n i s t r a t i o n o f 1.25 or 3.14 n m o l N P K 15 rain before the i n t r o d u c t i o n o f food pellets significantly d e l a y e d the onset o f feeding in F D rats. W h e r e a s salineinjected F D rats d i s p l a y e d eating b e h a v i o r within 2.7 + 0.9 min after i n t r o d u c t i o n o f f o o d pellets, p r e t r e a t m e n t with either o f the two doses o f N P K significantly delayed the onset o f feeding: the L O F was 8.9 + 2.1 a n d 9.6 + 2.2 min after 1.25 a n d 3.14 n m o l N P K , respectively (Fig. 1A). In a d d i t i o n , N P K p r e t r e a t m e n t significantly decreased c u m u l a t i v e f o o d i n t a k e in a d o s e - r e l a t e d fashion at the end o f the first h o u r (Fig. IB); the 2 h c u m u l a t i v e food intake was also significantly suppressed in N P K treated F D rats. In c o n t r a s t , the two doses o f N P K failed to exert any effect on d r i n k i n g in w a t e r - d e p r i v e d rats (Table I).
The effect of NPK on LOF andJbod consumption a/?er the onset of the dark phase (Fig.
2) As in the case o f F D rats (Fig. 1), injection o f N P K 15 rain before lights-off significantly d e l a y e d the onset o f ingestive b e h a v i o r d u r i n g the d a r k phase (Fig. 2A). W h e r e a s the saline-treated rats s t a r t e d to eat within 4.8 i 1.3 min, the L O F in N P K - t r e a t e d rats was 16.7 + 3.9 rain ( P < 0.01). N P K p r e t r e a t m e n t also significantly decreased c u m u l a t i v e 1 a n d 2 h f o o d i n t a k e (Fig. 2B).
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Fig. 1. The effects of i.p. injection of NPK on latency to onset of feeding (A) and food intake in fooddeprived rats (B). NPK was administered 15 min before the introduction of food pellets. Values are mean + S.E.M. for the number of rats inset within the bar. * P < 0.05: ** P < 0.01 vs saline and a - P < 0.05 vs 1.25 nmol NPK group.
i39
TABLE i Effect of NPK on water intake
Treatment
n
Latency to drinking (rain)
Water intake, ml 15 min
45 rain
Saline NPK, 1.25 nmol
7 7
1.7 ± 0.8 1.5 -t: 0.7
9.3 + 0.8 7.9 J- 0.5
I1.1 ~ 0.8 10.6 J: 0.8
NPK, 3.[4 nmol
7
7.2 ~- 3.2
6.6 ± 1.2
10.4 ± 0.6
Rats were water-deprived 18 h before i.p. injection of vehicle (saline) or NPK.
The effect of peripheral N P K on the NP Y-induced food intake in satiated rats (Fig. 3) The effects of various doses of N P K injected intraperitoneally 15 rain before central administration of NPY are shown in Fig. 3. Each dose of NPK was tested on a separate group of rats with a corresponding control group. Only 1/7 of the control group which received saline i.c.v, instead of NPY ate 1.9 g (data not shown in Fig. 3). On the other hand, as expected from earlier studies [12 15], 0.47 nmol NPY stimulated food intake in all 24 i.p. saline-injected control rats and the variability in food intake was not significantly different (Fig. 3). Whereas 0.125 nmol pretreatment did
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Fig. 2. The effects of i.p. injection of NPK on latency to onset of feeding (A) and food intake (B) at the onset of the dark phase. N P K was administered 15 rain prior to lights-off. Values are mean ± S.E.M. tbr the number of rats inset within the bar. * P < 0.05 and ** P < 0.01.
140
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Fig. 3. The effects of NPK on NPY-mduced feeding. Latency to onset of feeding (A), and I h (B) and 2 h (C) food intake were measured in satiated rats injected i.c.v, with NPY (0.47 nmol) 15 rain after i.p. injection of N PK (0.125, 0.5, 1.25 or 3.14 nmol represented in various shaded bars) or saline (white bars). Values are mean ± S.E.M. liar 6 rats/group. * P < 0.05 and ** P < 0.01.
not significantly influence either the L O F or the c u m u l a t i v e food intake, each of the 3 higher doses of N P K significantly increased the L O F and suppressed N P Y - i n d u c e d feeding. Also, N P K suppressed N P Y - i n d u c e d food intake response in a dose-related fashion because the decrease from their respective controls induced by 1.25 nmol and 3.14 nmol N P K was 33 a n d 78(¼,, respectively (P < 0.05).
The effect q/' centrally administered N P K on the N P Y-induced L O F and fi, eding in satiated rats ( F(~. 4) E n c o u r a g e d by the o b s e r v a t i o n that i.p. N P K effectively suppressed feeding (Fig. 3), we examined the effects of centrally administered N P K on N P Y - i n d u c e d feeding behavior. As was observed after i.p. injection, i.c.v. N P K markedly delayed the onset of the N P Y - i n d u c e d feeding response in a dose-related fashion (Fig. 4A). In contrast, i.c.v. N P K was not as effective as i.p. N P K in suppressing the N P Y - i n d u c e d food
141
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Fig. 4. The effects of i.c.v. N P K on latency to onset of feeding (A) and food i n t a k e (B) in response to i,c.v. injection of N P Y (0.47 nmol) in satiated male rats. Values are m e a n ± S.E.M. for the n u m b e r of rats inset within the bar. ** P < 0.01 v s s a l m e a n d a = P < 0.05vs0.5 nmolNPKgroup.
intake because each of the two NPK doses tested failed to significantly attenuate the 1 and 2 h cumulative food consumption as compared to their respective control groups (Fig. 4B).
Discussion The results of these studies show that NPK can suppress the two components of ingestive behavior. We observed that NPK decreased feeding that occurs normally during the dark phase of the light-dark cycle and that induced by starvation or by administration of NPY [12 15]. Of further significance is the revelation that the i.p. route of NPK exerted suppressive effects in all 3 paradigms of feeding tests employed. On the other hand, central NPK was only partially effective because the LOF but not the cumulative food intake was modifed to any significant manner by those two NPK doses that when injected i.p. clearly diminished not only the NPY- but also the dark phase-induced feeding. The dichotomy in the effectiveness of two routes of NPK administration is intriguing, but the observations that i.c.v, administered neuropeptides rapidly attain high concentrations in the peripheral blood [17,19] and NPK immunoreactive neurons innervate the hypothalamus [6], raise several possibilities. In view of the possible leakage ofi.c.v. NPK into the systemic circulation, the increase in LOF after i.c.v. NPK may be a consequence of peripheral NPK action. If this is true, then it is reasonable to conclude that NPK is primarily a peripheral signal capable of modulating feeding behavior in the rat. However, the alternate possibility, that NPK
I42 may also act centrally at receptive sites located within or outside the hypothalamus that are innervated by N P K immunoreactive neurons [6], cannot yet be excluded. Accordingly, one suspects that N P K may have dual sites of action, it may act centrally to delay the onset of feeding and act peripherally with a major effect to delay the onset and to decrease food consumption. Admittedly, further investigations are warranted to verify these possible modes of NPK action. Recently, a number of studies have reported high concentrations of tachykinins, including N P K , in the plasma and tumor tissue from patients with carcinoid tumors [20,21]. Our findings that peripheral N P K can reliably suppress feeding suggest that some of the clinical symptoms of gastrointestinal tract in carcinoid patients may be mediated by the reported high circulating N P K concentrations in these patients. Additionally, a large number of gastrointestinal and pancreatic peptides and brain peptides have been shown to suppress feeding in the rat {2,3,10,11,22,23]. A few of these peptides were reported to be effective only after central administration [1,2,11,22,23] while others such as cholecystokinin (CCK) and bombesin suppressed feeding after peripheral or central administration, but produced more potent effects when administered i.p. than when administered into the cerebral ventricles [2,3,10,11]. Our findings indicate that the suppressive effects of N P K on ingestive behavior may be similar to those of CCK and bombesin. However, whether N P K is an independent peripheral signal or whether it acts in concert with C C K and bombesin to regulate satiety in rats remains to be ascertained. We cannot completely rule out the possibility that the delay in feeding response and decrease in food consumption may be secondary to a general malaise or taste aversion in NPK-treated rats. However, this possibility is less likely in view of our observation that N P K failed to exert any affect on drinking. In the present investigation we did not notice any general untoward behavioral change after NPK administration, but occasionally the high dose of 3.14 nmol i.p. and 0.5 3.14 mnol i.c.v., like other tachykinins [24], produced salivation and lachrymal secretion. In summary, our studies show that N P K can acutely and consistently suppress feeding and not drinking behavior in rats. Further studies are underway to assess how N P K acts to suppress feeding in the rat.
Acknowledgements These sudies were supported by a grant from the N I H (DK 37273). Thanks are due to Ms. Sally McOonell for secretarial assistance.
References 1 Kalra, S.P., Allen, L.G., Clark, J.T., Crowley, W.R. and Kalra, P.S., Neuropeptide Y - - and integrator of reproductive and appetitive functions. In T. Moody (Ed.), Neural and Endocrine Peptides and Receptors, Plenum. New York, 1986, pp. 353 366. 2 Morley, J.E., Neuropeptide regulation of appetite and weight, Endocr. Rev., 8 (1987) 256 287. 3 Leibowitz, S.F. and Stanley, G., Brain peptides and the control of eating behavior. In T.W. Moody {Ed.), Neural and Endocrine Peptides and Receptors, Plenum, New York. 1986, pp. 333 352.
143 4 Tatemoto, K., Lundberg, J.M., Jornvall, H. and Mutt, V., Neuropeptide K: isolation, structure and biological activities of a novel brain tachykinin, Biochem. Biophys. Res. Commun.. 128 (1985) 947 953. 5 Arai, H. and Emson, P.C., Regional distribution of neuropeptide K and other tachykinins (neurokinin A, neurokinin B, and substance P) in rat central nervous system, Brain Res.. 399 (1986) 240 249. 6 Valentino, K.L., Tatemoto, K., Hunter, J. and Barchas, J.D., Distribution of neuropeptide K-immunoreactivity in the rat central nervous system, Peptides, 7 (1986) 1043-1059. 7 Leibowitz, S.F., Neurochemical systems of the hypothalamus. Control of feeding and drinking behavior and water-electrolyte excretion. In P. Morgane and J. Panksepp (Eds.), Handbook of the ttypothalamus, Vol. 3, Part A, Dekker, New York, 1980, pp. 299 437. 8 Deacon, C.F., Agoston, D.V., Nau, R. and Conlon, J.M., Conversion of neuropeptidc K to neurokmin A and vesicular localization of neurokinin A and substance P in neurons of the guinea pig small intestine, J. Neurochem.. 48 (1987) 141 146. 9 Sahu, A., Crowley, W.R., Tatemoto, K., Balasubramaniam, A. and Kalra, S.P., Effects of neuropeptide Y, NPY analog (norleucine4-NPY), galanin and neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen progesterone-treated rats, Peptides, 8 (1987) 921 926. 10 Gibbs, J. and Smith, G.P,, The neuroendocrinology of postprandial satiety. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. 8, Raven, New York, 1984, pp. 223-245. 11 Gibbs, J, Fauser, D.J., Rowe, E.A., Rolls, B.J., Rolls, E.T. and Maddison, S.P., Bombcsin suppresses feeding in rats, Nature (Lond.), 282 (1979) 208-210. 12 Clark, J.T., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancrcatic polypeptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427~429. 13 Clark, J.T., Kalra, P.S. and Kalra. S.P., Neuropeptide Y stimulates feeding but inhibits sexual behavior in male rats, Endocrinology, 117 (1985) 2435-2442. 14 Allen, L.G., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Comparison of the effect of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats, Life Sci., 37 (1985) 617 623. 15 Clark, J.T., Sahu, A., Kalra, P.S., Balasubramaniam, A. and Kalra, S.P., Neuropcptide Y (NPY)induced feeding behaviour in female rats: comparison with human NPY (Methionine ~7-NPY), NPY analog (Norleucine'*-NPY) and peptide YY. Reg. Pept,, 17 (1987) 31-39. 16 Kalra, S.P. and Gallo, R.V., Effects of inlraventricular catecholamines on luteinizing hormone release in morphine-treated rats, Endocrinology, 113 (1983) 23 28. 17 Ben-Jonathan, N., Mical, R.S. and Porter, J.C., Transport of LRF from CSF to hypophysial portal and systemic blood and release of LH, Endocrinology, 95 (1974) 18 25. 18 Oliver, C., Ben Jonathan, N., Mical, R.S. and Porter, J.C., Transport ofthyrotropin-relcasing hormone from cerebrospinal fluid to hypophysial portal blood and release of thyrotropin, Endocrinology, 97 (1975) 1138-1143. 19 Tannebaum, G. and Patel, Y.L., On the role of centrally administered somatostatin in the rat, Massive bypersomatostatinemia resulting from leakage into the peripheral circulation has effects on growth hormone secretion and glucoregulation, Endocrinology, 118 (1986) 2137--2141. 20 Theodorsson-Norheim, E., Norheim, 1., Oberg, K., Brodin, E., Lundberg, J.M., Tatemoto, K. and Lindgren, P.G., Neuropeptide K: a major tachykinin in plasma and tumor tissues from carcim)id patients. Biochem. Biophys. Res. Commun., 131 (1985) 77 83. 21 Conlon, J.M., Deacon, C.F., Richter, G., Stockmann, F. and Creutzfeldt, W., Circulating tachykinins (Substance P, Neurokinin A, Neuropeptide K) and carcinoid flush, Scand. J. Gastrocntcrol., 22 (1985) 97 105. 22 Arase, K., Sakaguchi, T., Takahashi, M., Bray, G.A. and Ling, N., Effects of feeding behavior on rats of a cryptic peptide from C-terminal end of prepro-growth hormone releasing factor, Endocrinology, 121 (1987) 1960 1965. 23 Jaworek, J., Schally, A.V., Coy, D.H. and Colaluca, J., Effects of analogs of(pyro)Glu-His-Gly-OH on food consumption and gastric acid secretion in rats, Life Sci., 34 (1984) 2597 2603. 24 Takano, Y., Takeda, Y., Doteuchi, M,, Inouye, K. and Kamiya, H., Effect of a novel tachykinin, substance K, on salivation in rats, Eur. J. PharmacoI., 111 (1985) 381-383.
Elsevier RPT 00745
Neuropeptide K suppresses feeding in the rat Abhiram Sahu, Pushpa S. Kalra, Michael G. D u b e and Satya P. Kalra Department q[' Obstetrics and Gynecoh~gy, University q/Florida College q/" Medicine, Gainesville. FL 32610 (U.S.A.) (Received 22 April f988; revised version received and acccepted 15 June 1988)
Summary We studied the effects of neuropeptide K (NPK), a 36 amino acid residue peptide of the tachykinin family, on latency to onset of feeding and cumulative 1 and 2 h food intake in three experimetal paradigms. Intraperitoneal injection of NPK (1.25 and 3.14 nmol) to food-deprived rats delayed the onset of feeding and significantly decreased the cumulative food intake, lntraperitoneal injection of NPK (1.25 and 3.14 nmol) to water-deprived rats produced no effect on subsequent drinking behavior. Similarly, intraperitoneal injection of NPK (3.14 nmol) 15 min before onset of the dark phase (of the light-dark cycle) significantly delayed the occurrence of ingestive behavior and the cumulative food intake was markedly suppressed. Furthermore, administration of NPK intraperitoneally (0.5 3.14 nmol) 15 min before intraventricular (i.c.v.) injection of neuropeptide Y (NPY 0.47 nmol) to satiated rats significantly suppressed NPY-induced feeding and delayed the onset of ingestive behavior. However, when administered centrally prior to NPY injection, NPK delayed the onset of feeding response only. Collectively, these findings show that NPK can acutely and consistently suppress feeding behavior. Neuropeptide K; Feeding; Inhibition; Dark phase; Starvation; Neuropeptide Y
Introduction In recent years a number of neuropeptides have been shown to modulate feeding behavior in the rat [1 3]. Neuropeptide K (NPK), a 36 amino acid residue peptide Correspondence: S.P. Kalra, Department of Obstetrics and Gynecology, University of Florida College of Medicine, P.O. Box J-294 JHMHC, Gainesville, FL 326[0, U.S.A. 0167-0[ 15/87/$03,50 ,i!~ [988 Elsevier Science Publishers B.V. (Biomedical Division)
136 belonging to the tachykinin family, was originally isolated from porcine brain [4], and has been identified recently in selected neural sites in the rat brain [5,6]. Detailed immunocytochemical mapping of NPK immunoreactivity in the rat brain showed a large number of N P K immunoreactive groups of perikarya and fiber system in hypothalamic sites [6] previously implicated in the control of ingestive behavior in the rat [7]. In addition, NPK has been found in many neurons in the gastrointestinal tract [8]. During our investigation of the neuroendocrine effects of N P K , we did not observe any stimulatory effects of N P K on feeding in satiated female rats [9]. We now report that N P K suppresses feeding in 3 experimental paradigms in male rats, viz. feeding that occurs normally during the dark phase of the light dark cycle and that induced by either food deprivation [10,11] or neuropeptide Y (NPY) [12 15] administration in satiated rats.
Materials and Methods Adult male rats (CrI:CD)RR, Charles River Laboratories, Wilmington, MA) weighing 325 425 g were housed singly under controlled temperature (22°C) and lighting (lights on 05.00 19.00 h) with ad libitum access to Purina rat chow and water. The control and experimental groups in each of the 4 experiments were balanced for mean body weight. Water was available ad libitum during the experimental observation periods.
Experiment 1 The effects of i.p. injection of N P K (1.25 and 3.14 nmol in 0.1 ml saline, Peninsula Laboratories, Belmont, CA), or vehicle alone on food consumption by food-deprived (FD) rats were studied. The doses of N P K selected were based on our previous studies [9]. The food was removed from the cages for 24 h before the start of the experiments at 09.00 10.00 h. N P K or saline was administered 15 rain before the introduction of preweighed food pellets in each cage (0 min). The latency to onset of food intake (LOF) was recorded. At the end of the first hour, the remaining food pellets and spilled food were carefully collected and weighed to the nearest 0.001 g on a Mettler balance. Fresh, preweighed food pellets were replaced to determine food intake during the second hour by these rats. At the end of the second hour the remaining pellets and spilled food were collected and weighed. The cumulative 1 and 2 h food intake in response to N P K or saline were calculated. In the next experiment, the effect of N P K (l.25 and 3.14 nmol in 0.1 1331saline) or vehicle alone on drinking in water-deprived rats was studied. Water bottles were removed from the cage at 13.00 h and fk~od was available ad libitum. The next day at 09.00 09.30 h food cups were removed and 15 rain later N P K or vehicle alone was injected i.p. Access to ad libitum water was reinstituted 15 rain later. Latency to onset of drinking was recorded and the amount of water consumed at 15 and 45 min was calculated.
137
Experiment 2 The effect of i.p. NPK (3.14 nmol in 0.l ml saline) or vehicle alone on feeding that occurs after onset of the dark phase of the light-dark cycle was studied, The selection of the NPK dose was based on the results from Expt. 1. Food pellets were removed at 18.00 h and NPK or saline was injected at 18.45 h, 15 rain before lights-off (19.00 h). Preweighed food pellets were introduced into each cage at 19.00 h. On the first day of the experiment, control rats (n = 15) were injected with saline and the LOF and cumulative 1 and 2 h food intake were recorded as in Expt. 1. On the following day these rats received N P K and the LOF and food intake were similarly monitored. This experimental design allowed within-subject statistical comparisons of the LOF and food intake with each rat serving as its own control.
Experiment 3 Since we have previously reported that i.c.v, or i.p. injection of NPK did not affect food intake in satiated rats [9], we now explored the possibility whether NPK would influence feeding evoked by orexigenic peptides. The effects ofi.p. NPK (0.125, 0.5, 1.25 or 3.14 nmol/rat in 0.1 ml saline) or vehicle alone on NPY-induced feeding in satiated rats was studied in this experiment. The selection of 0.47 nmol dose of NPY was based on our previous observation that it invariably produced near-maximal feeding in satiated rats [12-15]. Permanent stainless-steel cannulae were placed stereotaxically into the third ventricle of the brain under pentobarbital anesthesia (40 mg/kg, i.p., [16]) and the rats allowed to recover for 2 weeks. On the day of the experiment (09.00 10.00 h), patency of the i.c.v, cannula was confirmed by efflux of cerebrospinal fluid (CSF) and only those rats showing good efftux of CSF were used. Food was removed from the cage 1 h before the start of the experiment. Rats were divided into two groups on the day of the experiment. One group received one of the 4 doses of NPK and the other received saline i.p. 15 min before the i.c.v, injection of NPY in 3/~1 saline to both groups. Immediately after NPY injection (0 min), preweighed food pellets were placed in each cage. The LOF and cumulative 1 and 2 h food intake were calculated as described above. Thus, for assessing the effectiveness of each dose of NPK to modulate NPY-induced feeding, we concurrently ran a control group receiving saline instead of NPK followed by i.c.v. NPY. In addition, a group of control rats received i.c.v, saline instead of NPY.
Experiment 4 In this experiment the effect of centrally administered N P K (0.5 or 1.25 nmol) on NPY-induced (0.47 nmol) feeding in satiated rats was studied. The experimental design was the same as in Expt. 3 except that NPK or vehicle (3/A saline) was injected i.c.v. 15 rain before the i.c.v, injection of NPY. The LOF and cumulative food intake were calculated as described above. The data for Expts. 1 and 4 were analyzed by one-way analysis of variance followed by Duncan multiple range test. Data for Expt. 3 were compared by Student's t-test. For Expt. 2, paired t-test was used to compare the saline (day 1) and NPK-induced effects (day 2) on LOF and food intake in the same rat.
138
Results The effect of NPK on LOF and food intake in FD rats (Fig. 1) I.p. a d m i n i s t r a t i o n o f 1.25 or 3.14 n m o l N P K 15 rain before the i n t r o d u c t i o n o f food pellets significantly d e l a y e d the onset o f feeding in F D rats. W h e r e a s salineinjected F D rats d i s p l a y e d eating b e h a v i o r within 2.7 + 0.9 min after i n t r o d u c t i o n o f f o o d pellets, p r e t r e a t m e n t with either o f the two doses o f N P K significantly delayed the onset o f feeding: the L O F was 8.9 + 2.1 a n d 9.6 + 2.2 min after 1.25 a n d 3.14 n m o l N P K , respectively (Fig. 1A). In a d d i t i o n , N P K p r e t r e a t m e n t significantly decreased c u m u l a t i v e f o o d i n t a k e in a d o s e - r e l a t e d fashion at the end o f the first h o u r (Fig. IB); the 2 h c u m u l a t i v e food intake was also significantly suppressed in N P K treated F D rats. In c o n t r a s t , the two doses o f N P K failed to exert any effect on d r i n k i n g in w a t e r - d e p r i v e d rats (Table I).
The effect of NPK on LOF andJbod consumption a/?er the onset of the dark phase (Fig.
2) As in the case o f F D rats (Fig. 1), injection o f N P K 15 rain before lights-off significantly d e l a y e d the onset o f ingestive b e h a v i o r d u r i n g the d a r k phase (Fig. 2A). W h e r e a s the saline-treated rats s t a r t e d to eat within 4.8 i 1.3 min, the L O F in N P K - t r e a t e d rats was 16.7 + 3.9 rain ( P < 0.01). N P K p r e t r e a t m e n t also significantly decreased c u m u l a t i v e 1 a n d 2 h f o o d i n t a k e (Fig. 2B).
12 A
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Fig. 1. The effects of i.p. injection of NPK on latency to onset of feeding (A) and food intake in fooddeprived rats (B). NPK was administered 15 min before the introduction of food pellets. Values are mean + S.E.M. for the number of rats inset within the bar. * P < 0.05: ** P < 0.01 vs saline and a - P < 0.05 vs 1.25 nmol NPK group.
i39
TABLE i Effect of NPK on water intake
Treatment
n
Latency to drinking (rain)
Water intake, ml 15 min
45 rain
Saline NPK, 1.25 nmol
7 7
1.7 ± 0.8 1.5 -t: 0.7
9.3 + 0.8 7.9 J- 0.5
I1.1 ~ 0.8 10.6 J: 0.8
NPK, 3.[4 nmol
7
7.2 ~- 3.2
6.6 ± 1.2
10.4 ± 0.6
Rats were water-deprived 18 h before i.p. injection of vehicle (saline) or NPK.
The effect of peripheral N P K on the NP Y-induced food intake in satiated rats (Fig. 3) The effects of various doses of N P K injected intraperitoneally 15 rain before central administration of NPY are shown in Fig. 3. Each dose of NPK was tested on a separate group of rats with a corresponding control group. Only 1/7 of the control group which received saline i.c.v, instead of NPY ate 1.9 g (data not shown in Fig. 3). On the other hand, as expected from earlier studies [12 15], 0.47 nmol NPY stimulated food intake in all 24 i.p. saline-injected control rats and the variability in food intake was not significantly different (Fig. 3). Whereas 0.125 nmol pretreatment did
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Fig. 2. The effects of i.p. injection of NPK on latency to onset of feeding (A) and food intake (B) at the onset of the dark phase. N P K was administered 15 rain prior to lights-off. Values are mean ± S.E.M. tbr the number of rats inset within the bar. * P < 0.05 and ** P < 0.01.
140
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.125
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Fig. 3. The effects of NPK on NPY-mduced feeding. Latency to onset of feeding (A), and I h (B) and 2 h (C) food intake were measured in satiated rats injected i.c.v, with NPY (0.47 nmol) 15 rain after i.p. injection of N PK (0.125, 0.5, 1.25 or 3.14 nmol represented in various shaded bars) or saline (white bars). Values are mean ± S.E.M. liar 6 rats/group. * P < 0.05 and ** P < 0.01.
not significantly influence either the L O F or the c u m u l a t i v e food intake, each of the 3 higher doses of N P K significantly increased the L O F and suppressed N P Y - i n d u c e d feeding. Also, N P K suppressed N P Y - i n d u c e d food intake response in a dose-related fashion because the decrease from their respective controls induced by 1.25 nmol and 3.14 nmol N P K was 33 a n d 78(¼,, respectively (P < 0.05).
The effect q/' centrally administered N P K on the N P Y-induced L O F and fi, eding in satiated rats ( F(~. 4) E n c o u r a g e d by the o b s e r v a t i o n that i.p. N P K effectively suppressed feeding (Fig. 3), we examined the effects of centrally administered N P K on N P Y - i n d u c e d feeding behavior. As was observed after i.p. injection, i.c.v. N P K markedly delayed the onset of the N P Y - i n d u c e d feeding response in a dose-related fashion (Fig. 4A). In contrast, i.c.v. N P K was not as effective as i.p. N P K in suppressing the N P Y - i n d u c e d food
141
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Fig. 4. The effects of i.c.v. N P K on latency to onset of feeding (A) and food i n t a k e (B) in response to i,c.v. injection of N P Y (0.47 nmol) in satiated male rats. Values are m e a n ± S.E.M. for the n u m b e r of rats inset within the bar. ** P < 0.01 v s s a l m e a n d a = P < 0.05vs0.5 nmolNPKgroup.
intake because each of the two NPK doses tested failed to significantly attenuate the 1 and 2 h cumulative food consumption as compared to their respective control groups (Fig. 4B).
Discussion The results of these studies show that NPK can suppress the two components of ingestive behavior. We observed that NPK decreased feeding that occurs normally during the dark phase of the light-dark cycle and that induced by starvation or by administration of NPY [12 15]. Of further significance is the revelation that the i.p. route of NPK exerted suppressive effects in all 3 paradigms of feeding tests employed. On the other hand, central NPK was only partially effective because the LOF but not the cumulative food intake was modifed to any significant manner by those two NPK doses that when injected i.p. clearly diminished not only the NPY- but also the dark phase-induced feeding. The dichotomy in the effectiveness of two routes of NPK administration is intriguing, but the observations that i.c.v, administered neuropeptides rapidly attain high concentrations in the peripheral blood [17,19] and NPK immunoreactive neurons innervate the hypothalamus [6], raise several possibilities. In view of the possible leakage ofi.c.v. NPK into the systemic circulation, the increase in LOF after i.c.v. NPK may be a consequence of peripheral NPK action. If this is true, then it is reasonable to conclude that NPK is primarily a peripheral signal capable of modulating feeding behavior in the rat. However, the alternate possibility, that NPK
I42 may also act centrally at receptive sites located within or outside the hypothalamus that are innervated by N P K immunoreactive neurons [6], cannot yet be excluded. Accordingly, one suspects that N P K may have dual sites of action, it may act centrally to delay the onset of feeding and act peripherally with a major effect to delay the onset and to decrease food consumption. Admittedly, further investigations are warranted to verify these possible modes of NPK action. Recently, a number of studies have reported high concentrations of tachykinins, including N P K , in the plasma and tumor tissue from patients with carcinoid tumors [20,21]. Our findings that peripheral N P K can reliably suppress feeding suggest that some of the clinical symptoms of gastrointestinal tract in carcinoid patients may be mediated by the reported high circulating N P K concentrations in these patients. Additionally, a large number of gastrointestinal and pancreatic peptides and brain peptides have been shown to suppress feeding in the rat {2,3,10,11,22,23]. A few of these peptides were reported to be effective only after central administration [1,2,11,22,23] while others such as cholecystokinin (CCK) and bombesin suppressed feeding after peripheral or central administration, but produced more potent effects when administered i.p. than when administered into the cerebral ventricles [2,3,10,11]. Our findings indicate that the suppressive effects of N P K on ingestive behavior may be similar to those of CCK and bombesin. However, whether N P K is an independent peripheral signal or whether it acts in concert with C C K and bombesin to regulate satiety in rats remains to be ascertained. We cannot completely rule out the possibility that the delay in feeding response and decrease in food consumption may be secondary to a general malaise or taste aversion in NPK-treated rats. However, this possibility is less likely in view of our observation that N P K failed to exert any affect on drinking. In the present investigation we did not notice any general untoward behavioral change after NPK administration, but occasionally the high dose of 3.14 nmol i.p. and 0.5 3.14 mnol i.c.v., like other tachykinins [24], produced salivation and lachrymal secretion. In summary, our studies show that N P K can acutely and consistently suppress feeding and not drinking behavior in rats. Further studies are underway to assess how N P K acts to suppress feeding in the rat.
Acknowledgements These sudies were supported by a grant from the N I H (DK 37273). Thanks are due to Ms. Sally McOonell for secretarial assistance.
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143 4 Tatemoto, K., Lundberg, J.M., Jornvall, H. and Mutt, V., Neuropeptide K: isolation, structure and biological activities of a novel brain tachykinin, Biochem. Biophys. Res. Commun.. 128 (1985) 947 953. 5 Arai, H. and Emson, P.C., Regional distribution of neuropeptide K and other tachykinins (neurokinin A, neurokinin B, and substance P) in rat central nervous system, Brain Res.. 399 (1986) 240 249. 6 Valentino, K.L., Tatemoto, K., Hunter, J. and Barchas, J.D., Distribution of neuropeptide K-immunoreactivity in the rat central nervous system, Peptides, 7 (1986) 1043-1059. 7 Leibowitz, S.F., Neurochemical systems of the hypothalamus. Control of feeding and drinking behavior and water-electrolyte excretion. In P. Morgane and J. Panksepp (Eds.), Handbook of the ttypothalamus, Vol. 3, Part A, Dekker, New York, 1980, pp. 299 437. 8 Deacon, C.F., Agoston, D.V., Nau, R. and Conlon, J.M., Conversion of neuropeptidc K to neurokmin A and vesicular localization of neurokinin A and substance P in neurons of the guinea pig small intestine, J. Neurochem.. 48 (1987) 141 146. 9 Sahu, A., Crowley, W.R., Tatemoto, K., Balasubramaniam, A. and Kalra, S.P., Effects of neuropeptide Y, NPY analog (norleucine4-NPY), galanin and neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen progesterone-treated rats, Peptides, 8 (1987) 921 926. 10 Gibbs, J. and Smith, G.P,, The neuroendocrinology of postprandial satiety. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. 8, Raven, New York, 1984, pp. 223-245. 11 Gibbs, J, Fauser, D.J., Rowe, E.A., Rolls, B.J., Rolls, E.T. and Maddison, S.P., Bombcsin suppresses feeding in rats, Nature (Lond.), 282 (1979) 208-210. 12 Clark, J.T., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancrcatic polypeptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427~429. 13 Clark, J.T., Kalra, P.S. and Kalra. S.P., Neuropeptide Y stimulates feeding but inhibits sexual behavior in male rats, Endocrinology, 117 (1985) 2435-2442. 14 Allen, L.G., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Comparison of the effect of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats, Life Sci., 37 (1985) 617 623. 15 Clark, J.T., Sahu, A., Kalra, P.S., Balasubramaniam, A. and Kalra, S.P., Neuropcptide Y (NPY)induced feeding behaviour in female rats: comparison with human NPY (Methionine ~7-NPY), NPY analog (Norleucine'*-NPY) and peptide YY. Reg. Pept,, 17 (1987) 31-39. 16 Kalra, S.P. and Gallo, R.V., Effects of inlraventricular catecholamines on luteinizing hormone release in morphine-treated rats, Endocrinology, 113 (1983) 23 28. 17 Ben-Jonathan, N., Mical, R.S. and Porter, J.C., Transport of LRF from CSF to hypophysial portal and systemic blood and release of LH, Endocrinology, 95 (1974) 18 25. 18 Oliver, C., Ben Jonathan, N., Mical, R.S. and Porter, J.C., Transport ofthyrotropin-relcasing hormone from cerebrospinal fluid to hypophysial portal blood and release of thyrotropin, Endocrinology, 97 (1975) 1138-1143. 19 Tannebaum, G. and Patel, Y.L., On the role of centrally administered somatostatin in the rat, Massive bypersomatostatinemia resulting from leakage into the peripheral circulation has effects on growth hormone secretion and glucoregulation, Endocrinology, 118 (1986) 2137--2141. 20 Theodorsson-Norheim, E., Norheim, 1., Oberg, K., Brodin, E., Lundberg, J.M., Tatemoto, K. and Lindgren, P.G., Neuropeptide K: a major tachykinin in plasma and tumor tissues from carcim)id patients. Biochem. Biophys. Res. Commun., 131 (1985) 77 83. 21 Conlon, J.M., Deacon, C.F., Richter, G., Stockmann, F. and Creutzfeldt, W., Circulating tachykinins (Substance P, Neurokinin A, Neuropeptide K) and carcinoid flush, Scand. J. Gastrocntcrol., 22 (1985) 97 105. 22 Arase, K., Sakaguchi, T., Takahashi, M., Bray, G.A. and Ling, N., Effects of feeding behavior on rats of a cryptic peptide from C-terminal end of prepro-growth hormone releasing factor, Endocrinology, 121 (1987) 1960 1965. 23 Jaworek, J., Schally, A.V., Coy, D.H. and Colaluca, J., Effects of analogs of(pyro)Glu-His-Gly-OH on food consumption and gastric acid secretion in rats, Life Sci., 34 (1984) 2597 2603. 24 Takano, Y., Takeda, Y., Doteuchi, M,, Inouye, K. and Kamiya, H., Effect of a novel tachykinin, substance K, on salivation in rats, Eur. J. PharmacoI., 111 (1985) 381-383.