- Email: [email protected]
Neuropeptide Y (NPY)-induced feeding behavior in female rats: comparison with human NPY ([Met17]NPY), NPY analog ([norLeu4]NPY) and peptide YY
Regulatory Peptides, 17 (1987) 31-39 Elsevier
31
RPT 00561
Neuropeptide Y (NPY)-induced feeding behavior in female rats: comparison with human NPY ([Met 17]NPY), NPY analog ([norLeu4]NpY) and peptide YY* John T. Clark T M , A b h i r a m Sahu 1, Pushpa S. Kalra 1, A m b i k a i p a k a n Balasubramaniam 2 and Satya P. Kalra 1 1Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. and 2Department of Surgery, University of Cincinnati Medical Center, Cincinnati, OH 45267, U.S.A. (Received 22 August 1986; revised version received 24 November 1986; accepted 24 November 1986)
Summary Porcine neuropeptide Y (pNPY) administered into the third ventricle of the brain is known to elicit a powerful feeding response in steroid-treated ovariectomized and intact male rats. The present study compared the effects of pNPY and 3 structurally related peptides, human NPY (hNPY), an analog of NPY (NPY-A, [norLeu4]NPY) and peptide YY (PYY) on feeding behavior in intact female rats. Intraventricular administration of pNPY, hNPY, NPY-A and PYY over a dose range of 0.5 to 10 #g evoked feeding behavior to a varying extent. Cumulative food intake during 60 and 120 min was increased in a dose-related fashion at 0.5 and 2.0 /~g for the 4 peptides. Whereas the 10-#g dose of pNPY evoked a feeding response smaller than that seen after 2/ag, the responses to either 10 #g hNPY or 10/~g PYY were similar to that seen after 2 #g. The effects of these peptides on the time spent eating were quite different: while pNPY increased the time spent eating, this effect was not dose-related, whereas hNPY, NPY-A and PYY produced dose-related increments in the time spent eating. The most dramatic increment in local eating rate was observed
*Preliminary data were presented at the Society for Neuroscience annual meeting in Dallas, TX, 1985. **Present address: Department of Physiology, Meharry Medical College, 1-5 D.B. Todd Blvd., Nashville, TN 37208, U.S.A. Correspondence: Satya P. Kalra, Ph.D., Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. 0167-0115/87/$03.50 O 1987 Elsevier Science Publishers B.V. (Biomedical Division)
32
after 2.0/~g pNPY, with lesser increments seen after 2.0/~g hNPY and NPY-A. This increased local eating was apparently responsible for the highest cumulative food intake observed. These results demonstrate that (a) 2 #g pNPY is equally effective in stimulating feeding behavior in intact female rats as it is in steroid-primed ovariectomized female and intact male rats; (b) like the other members of the pancreatic polypeptide family, the PYY-stimulated feeding response was smaller than seen after pNPY; (c) in general, the cumulative food intake response, when compared with that evoked by pNPY, is reduced after substitution of either single, as in hNPY and NPY-A, or multiple amino acids as in the case of the members of the pancreatic polypeptide family, including PYY and (d) surprisingly, unlike pNPY, the other peptides tested facilitate the time spent eating response in a dose-related fashion. Further studies are needed to identify those active moieties in pNPY and other polypeptides of the pancreatic polypeptide family that impart differential effects on components of the feeding behavior in rats. Food intake; Neuropeptide; Intraventricular; Grooming; Behavior; Latency
Introduction
For over a decade attention has been focused on the role of neuropeptides in the stimulation of feeding behavior in the rat. Endogenous opioid peptides, opiates and their analogs increase food intake under a variety of experimental conditions [1,2]. Recently the members of the pancreatic polypeptide family were shown to stimulate various aspects of ingestive behavior in the rat (Table I). Neuropeptide Y, isolated and chemically characterized from porcine brain by Tatemoto [3], is structurally related to the pancreatic polypeptide family of hormones [4]. Administration of porcine NPY (pNPY) intracerebroventricularly (i.v.t.) stimulated food intake in satiated TABLE I Amino acid sequences of pNPY. hNPY and other members of the pancreatic polypeptide family of horlYIOl'leS
(Homology 4
8
12
16_
20
24
28
pNPY:
Y P S K P D N P G E D A P A E D L A R Y Y S A L R H Y I
hNPY:
.
rPP:
A
P L E -
M Y -
-
D Y -
T H -
Q R -
Q -
E T Q -
-
hPP-
A P L E -
V Y -
-
D N -
T P -
Q M -
Q - A A D -
-
PYY:
-
E A . . . . .
S P -
E -
.
.
-
.
A
.
~
.
-
.
.
.
.
.
.
.
.
.
.
M .
.
.
S -
.
-
.
-
.
A
.
.
.
.
.
N L
I
T R Q R Y .
.
.
to NH 2
NPYL
100%
.
.
R -
-
-
T
L
-
-
P -
-
46%
R -
-
- M L
-
-
P -
-
46%
L
-
-
.
36
.
S . . . .
.
32
97%
V . . . . .
69%
[Norleu4]NPY is a synthetic analog differing from NPY only by substitution of a norleucine for lysine at the 4 position, rPP, rat pancreatic polypeptide; hPP, human pancreatic polypeptide. One-letter codes for amino acids are used. Dashes indicate identity with NPY (sources: refs. I, 2, I 1, 13-15).
33 steroid-pretreated ovariectomized rats, and we observed a dose-related stimulation of feeding in intact male rats in both diurnal and nocturnal tests [5-7]. Interestingly, the NPY-induced feeding responses seen in our studies [5-7] and subsequently corroborated by others [8,9] were reliably more robust as compared to those reported for opiates and endogenous opioid peptides and growth hormone-releasing factor [1,2,10]. Similarly, members of the pancreatic polypeptide (PP) family, which show considerable structural homologies with pNPY, stimulate feeding in satiated rats (Table I [4,5]). We found that human pancreatic peptide and rat pancreatic peptide stimulated feeding response in a manner quite different from that induced by pNPY [6,7]. In an attempt to further understand the structure-function relationships among the members of PP, we have now compared the effects of pNPY with those of a gut peptide (PYY [11]) and two related peptides, human NPY (hNPY) and a NPY analog (NPY-A) on key parameters of ingestive behavior in the female rat. The two peptides, hNPY and NPY-A, are of interest because they differ from pNPY in only one amino acid, methionine replaced leucine at the 17 position in hNPY [12] and norleucine replaced lysine at the 4 position in NPY-A (Table I [12]).
Materials and Methods
Adult female rats (CRL:CDtR~(SD)BR, Charles River Breeding Laboratories, Wilmington, MA) were maintained in individual wire-bottom cages under controlled temperature (22-23°C) and light (lights on 05.00-19.00 h) with ad libitum access to food and water. Rats were implanted with permanent cannulae in the third cerebral ventricle under pentobarbital anesthesia (40 mg/kg [6]). Ten to 12 days after surgery, food and water were removed (12.00-14.00 h) and patency of the i.v.t, cannula was checked by observing the efflux of cerebrospinal fluid. Only those females exhibiting efflux of fluid were used in this study. One to two hours later, the effects on food intake of i.v.t, injection of the saline vehicle (3/~1, n = 17), pNPY (0.5, n = 6; 2, n = 6; 10 #g, n = 9), hNPY (0.5, n = 6; 2, n = 6; 10 #g, n = 7) or PYY (0.5, n = 8; 2, n = 6; 10 #g, n = 5) or NPY-A (0.5, n = 6; 2/~g, n = 6) were assessed. NPY-A was not tested at the 10-#g dose due to a supply problem as well as the observation of a decreased response after 10/~g pNPY and hNPY. Water was not available during the test. Each rat was used only once. The peptides (NPY, hNPY, PYY) were obtained commercially from Peninsula Laboratories (Belmont, CA) and NPY-A was synthesized as described [13]. Immediately after the i.v.t, injection, rats were returned to their home cages which contained a known amount of standard rat chow and continuously observed for 60 min [14]. The behavior was recorded in 3 categories: eating, grooming and yawning. At the end of 1 h the remaining food was carefully collected and weighed on a Mettler balance to the nearest 0.01 g. From the record, together with the food weighing, the following measurements were derived: total food intake (g); latency to first feeding response (min); duration of time spent eating (min); latency to first grooming response (min); time spent grooming (min) and local eating rate (g/min). Local eating rate is an index of the amount of food consumed per unit
34 time spent eating [14]. Following the food weighing, the remaining food was replaced in the cage with the animal and food intake was recorded for the period of 60-120 min after treatment. Thus, the number of rats displaying feeding behavior during the initial 60 min after injection and in the subsequent 60-120-min period and cumulative 2-h food intake was recorded. Data are presented as mean + S.E.M. The number of rats initiating behavior was evaluated using the Fisher exact probability test (comparing to saline-treated rats) and the parameters of feeding behavior were evaluated by Dunnett's test (comparison with a common control) followed by Mann-Whitney U-tests, where appropriate. Comparisons of food intake were made for the two discrete intervals after i.v.t, administration as well as for cumulative 2-h food intake.
Results
Effects of pNPY, hNPY, NPY-A and P Y Y on cumulative food intake I.v.t. pNPY at doses of 0.5 and 2.0 #g stimulated a dose-related increase in cumulative food intake for 60 min, with the most dramatic increase seen after the 2#g (0.47-nmol) dose. However, a higher dose of 10 #g pNPY was only slightly effective; in fact, the response was drastically lower than that seen after the 2-#g dose. NPY-A enhanced food intake in a manner similar to pNPY at 0.5- and 2-#g doses. All doses of hNPY and PYY also increased food intake. However, the responses were different from that seen after pNPY. As in the case of pNPY, the maximal response was seen after a 2-#g dose, but higher 10 pg doses of hNPY or PYY, unlike pNPY, produced an equivalent feeding response. A comparative analysis of the results indicates that whereas 2 #g of these peptides produced maximal responses, the quantitative responses were different among the peptides; pNPY and NPY-A were the most effective followed by hNPY and PYY, in that order. The cumulative 2-h food intake was also increased by these peptides (Fig. 1). However, there were additional quantitative differences that were not apparent when 60-min cumulative food intake was analyzed. After 0.5 #g, the cumulative food intake was greater after hNPY or NPY-A than after pNPY or PYY (P < 0.05). However, the response was reversed somewhat with the 2-pg dose: pNPY now elicited food intake equivalent to NPY-A but significantly greater than hNPY and PYY (P < 0.05). Further, 10 #g hNPY produced the greatest food intake which was higher than pNPY or PYY. Effects of pNP Y, hNP Y, NP Y-A and P Y Y on parameters of ingestive behavior The latency to the initial feeding response was variable, but was usually less than 20 min after 2 #g i.v.t, administration of the 4 peptides (Table II). The lowest latency of 7.4 min to first feeding response was seen after 2 #g pNPY, which increased dramatically to 38.8 4- 7.6 after 10.0 pg injection. Thus, all peptides tested are similar with respect to their effect on the latency to initiate feeding. The time spent eating increased in a dose-related manner after administration of hNPY, NPY-A and PYY (Fig. 2). In the case of pNPY, the time spent eating re-
5/6 a 5/6" 4/6 a 5/8 b
2.0 2.0 2.0 2.0 10.0 10.0 10.0
pNPY hNPY NPY-A PYY
pNPY hNPY PYY
1.6d 3.0 a,, 8.4 a 3.5 a
11.4a 7.1
7.7 a
4.4 1.8a
7.6 13.1 4- 2.2 a,, 10.4 4- 2.6 d,c
44+ 4-
+ 44+ 4-
38.8 4-
7.4 14.9 18.4 13.1
53.2 18.9 24.0 24.6 40.0
Latency to initial feeding response (minI
" P < 0.01 compared to saline-treated females using the Fisher Exact Probability Test. b p < 0.02 compared to saline-treated females using the Fisher Exact Probability Test. c M a x i m u m latency of 60 rain used for those females not feeding. d p < 0.01 compared to saline-treated females using the Man-Whitney U-test. P < 0.05 compared to NPY-treated females using the M a n n - W h i t n e y U-test.
5/9 5/5" 5/5"
6/6 a 5/5" 5/6" 7/7"
2/17
0.5 0.5 0.5 0.5
Proportion of females exhibiting feeding within 60 min o f treatment
-
Dose (#g)
Saline pNPY hNPY NPY-A PYY
Treatment
4+ 4+
+ + + ± 42.6 4.0 6.3 0.4
1.4 3.1 1.0 2.2 1.5
8.7 4- 2.1 5.2 + 1.6 16.1 4- 11.0
9.5 10.5 10.1 3.7
6.8 9.3 3.3 6.7 6.6
Latency to initial grooming response (min)
44+ 4-
+ + 44+
0.2 a 2.0 0.6 d 2.3
0.8 0.8 1.5 1.6 1.7
5.3 4- 0.8 2.9 4- 1.9 1.0 4- 0.4 a
0.6 3.5 1.9 6.9
5.0 3.0 5.1 3.5 8.0
Time spent grooming in the 60 min after treatment (min)
Effects o f pNPY, hNPY, N P Y - A (synthetic) and P Y Y (porcine) on aspects o f feeding and grooming behavior after intraventricular administration
T A B L E II
36 o) Cumulohve 6 0
If
minutefoodintake:
•e5
1"25
2hfoodintake:
' 3"0 b) Cumulohve
3"0
b
L
b
ose:
t
~
0
i
1.5
Treatment:
[]
~ hNPY
• NPY-A
[] P Y Y
~ to.o,,.,q q
Fig. I. Sixty-min and 2-h food intake after i.v.t, administration of pNPY, hNPY, N P Y - A and PYY. a, P < 0.05 compared to saline-treated female rats; b, P < 0.01 compared to saline-treated female rats; c, P < 0.05 compared to pNPY-treated female rats. For saline, n = 17 and for the other treatments, 5-9.
mained unaltered at doses of 0.5-10 #g. In fact, NPY-A, PYY at 2 #g and hNPY and PYY at 10 #g dosage significantly stimulated the time spent eating over that seen after pNPY administration. Local eating rate (g/min) was increased over the controls by 2 #g of pNPY, NPY-A and hNPY. pNPY also dramatically increased the local eating rate at this dose over that produced by either of the 3 peptides or 0.5 #g pNPY ( x 10 compared to 0.5 #g). Grooming activity was significantly reduced only after 2 #g NPY, 2 #g NPY-A and 10 #g PYY (Table II) in association with the largest increments in food intake (2 #g pNPY and NPY-A) or time spent eating (PYY and NPY-A). 30
a) T~me spent eatmq..
ob
l
3O
3. 15
o
2"2
o
;I
FE'jhNPY
b) Local eotmq r a t e
~ 0.4[
Dose."
--
O
k
~-O'SflJq ~
Tree tmen t.
~PYY 0.2
F2.0pQ-~
Fig. 2. Effects of i.v.t, administration of pNPY, hNPY, N P Y - A and P Y Y on parameters of feeding behavior in the 60 min after treatment, a, P < 0.01 compared to saline-treated female rats; b, P < 0.01 compared to pNPY-treated rats.
37 Discussion We have previously shown that pNPY was more effective than human pancreatic peptide or rat pancreatic peptide in stimulating food intake in ovariectomized steroid-primed female, and intact male rats, respectively [7,8]. We have now extended these comparative studies to show that pNPY is equally effective in intact female rats, and in terms of maximal cumulative food intake, it was more effective than either hNPY or PYY. These observations are of considerable interest because they reveal, for the first time, that substitution occurring naturally either of a single amino acid at the 17 position as in hNPY [11] or of multiple amino acids, as in the case of PYY, hPP or rPP [5,14] (Table I), attenuated the cumulative food intake response compared to that produced by pNPY. In the case of NPY-A [12], an experimentally produced analog which has norleucine substituted for lysine at the 4 position, the food intake response was comparable to that seen after pNPY. We noted that the gut peptide PYY, which has identical amino acid residues in 25 positions [14] (Table I), was only about half as effective as pNPY in increasing food intake in satiated female rats. This observation is interesting because other studies have found PYY more potent than pNPY in male rats [15,16]. While it is possible that PYY-induced response may be different in the two sexes; there are other important variables between our studies and those of others that may explain these differences in feeding responses. These include: route of administration [11,12], the composition of food [10,16], prescreening of rats for feeding response tests [10,16] and the availability of water during the test period [9,10,16]. In our study water was not available during the experiment. This may be an important procedural difference because we have seen that the dose response for the elicitation of feeding behavior in satiated males is shifted to the left if the water is unavailable [7] compared to when water is available [8]. Despite these differences, our findings together with previous reports on hPP [6] and rPP [7] suggest that structural modifications of the pNPY molecule, as those occurring in nature in PP and hNPY may result in diminution of cumulative food intake as compared to pNPY. Interestingly, the 3 naturally occurring peptides also differ considerably when other parameters related to ingestive behavior were monitored. Whereas pNPY evoked similar increments in time spent eating after 0.5 or 2 pg, the 2-/~g dose evinced a dramatic increase in the local eating rate suggesting that the dramatic increment in food intake seen after 2 pg pNPY was probably due to the increased local rate. On the other hand, the two naturally occurring peptides, hNPY and PYY, and the experimentally produced analog NPY-A, enhanced the time spent eating in a doserelated fashion. Further, it is interesting to note that the higher 10-#g dose of pNPY was less effective than 2 pg, an observation suggestive of a bell-shaped response typically observed after central administration of peptides. On the other hand, hNPY and PYY produced similar food intake and the time spent eating responses after the 2 or 10 #g dose. Thus, we suggest that the mode of action of these peptides in enhancing food intake may be different from that produced by pNPY. In addition, our data suggest that the stimulation of feeding is behaviorally specific and not due to a general activation of arousal since opposite effects are seen on grooming and sexual behavior [7].
38
Although these findings consistently reveal that structural modifications of the pNPY molecule have profound effects on feeding behavior in rats, the precise physiological role of pNPY and other related members of the pancreatic polypeptide family remains to be clarified [17]. A number of studies suggest that pNPY or a related peptide in the rat may be the endogenous neurochemical signal in the hypothalamic circuitry involved in the regulation of feeding behavior [18-24]. PYY, a gut hormone of non-neural origin, has been localized in small amounts in the rat brain [20,24] and therefore its role in the neural control of food intake remains to be defined. On the other hand, it is tempting to infer from our studies that hNPY, isolated from human adrenal medullary pheochromocytoma tissue [12], may play an important role in the neural regulation of feeding in man. In summary, our comparative studies have consistently shown that pNPY evokes a robust feeding response, primarily by increasing the local eating rate and that other members of the pancreatic polypeptide family stimulate feeding to variable, and usually lesser, degrees, primarily by increasing the time spent eating.
Acknowledgements This research was supported by NIH DK 37273 and J.T.C. was supported by Postdoctoral Fellowship, HD 06660. The technical assistance of D.L. Heaton-Jones and secretarial assistance of Sally McDonell are gratefully acknowledged.
References 1 Grandison, L. and Guidotti, A., Stimulation of food intake by muscimol and beta endorphin, Neuropharmacology, 16 (1977) 533-536. 2 Morley, J., Levine, A., Yim, L. and Lowy, M., Opioid modulation of appetite, Neurosci. Behav. Rev., 7 (1983) 281-305. 3 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5485-5489. 4 Lin, T.M., Pancreatic polypeptides: isolation, chemistry and biological functions. In: G.B.J. Glass (Ed.), Gastrointestinal Hormones, Raven, New York, 1980, pp. 275-306. 5 Clark, J.T., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology, I 15 (1984) 427--429. 6 Clark, J.T., Kalra, P.S. and Kalra, S.P., Neuropeptide Y stimulates feeding but inhibits sexual behavior in rats, Endocrinology, 117 (1985) 2435-2442. 7 Allen, L.G., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Comparison of the effects of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats. Life Sci,, 37 (1985) 617623. 8 Levine, A.S. and Morley, J.E., Neuropeptide Y: a potent inducer of consummatory behavior in rats, Peptides, 5 (1984) 1025-1029. 9 Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci., 35 (1984) 2635-2642. 10 Vaccarino, F.J., Bloom, F.E., Rivier, J., Vale, W. and Koob, G.F., Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor, Nature (London), 314 (1985) 167-168.
39 11 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London), 296 (1982) 659~560. 12 Cordor, R., Emson, P. and Lowry, P., Purification and characterization of human neuropeptide Y from adrenal-medullary phaechromocytoma tissue, Biochem. J., 219 (1984) 669-706. 13 Balasubramaniam, A., Grupp, I., Srivastava, L., Tatemoto, K., Murphy, R.F., Joffe, S.N. and Fischer, J.E., Synthesis of Neuropeptide Y, Int. J. Peptide Protein Res., in press. 14 Blundell, J.E., Bio-grammar of feeding: pharmacological manipulations and their interpretations. In: S.J. Cooper (Ed.), Theory in Psychopharmacology, Vol. 1, Academic, New York, pp. 233-250. 15 Morley, J.E., Levine, A.S., Grace, M. and Kneip, J., Peptide YY (PYY), a potent orexigenic agent, Brain Res., 341 (1985) 200-203. 16 Stanley, B.G., Daniel, D.R., Chin, A.S. and Leibowitz, S.F., Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion, Peptides, in press. 17 Kalra, S.P., Allen, L.G., Clark, J.T., Crowley, W.R. and Kalra, P.S., Neuropeptide Y - an integrator of reproductive and appetitive functions. In: T.W. Moody (Ed.), Neural and Endocrine Peptides and Receptors, Plenum, New York, 1986, pp. 353-366. 18 Lundberg, J., Terenius, L., H6kfelt, T., Martling, C., Tatemoto, K., Polack, J., Bloom, S. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Scand., 116 (1982) 477--480. 19 Emson, P. and deQuidt, M., NPY - a new member of the pancreatic polypeptide family, Trends Neurosci., 7 (1984) 31-36. 20 O'Donohue, T., Chronwall, B., Pruss, R., Mezey, E., Kiss,. J., Eiden, L., Massari, V., Tessel, R., Pickel, V., DiMaggio, D., Hotchkiss, A., Crowley, W. and Zukowska-Grojec, L., Neuropeptide Y and peptide YY neuronal and endocrine systems, Peptides, 6 (1985) 755-768. 21 Saria, A., Theorlorsson-Norheim, E. and Lundberg, J.M., Evidence for specific neuropeptide Y binding sites in rat brain synaptosomes, Eur. J. Pharmacol., 107 (1985) 105-107. 22 Lundberg, J., Terenius, L., Hokfelt, T. and Tatemoto, K., Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons, J. Neurosci., 4 (1984) 2376-2386. 23 Chang, R., Lotti, V., Chen, T.-B., Cerino, D. and Kling, P., Neuropeptide Y (NPY) binding sites in rat brain labeled with ~25I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens, Life Sci., 37 (1985) 2111-2122. 24 Inui, A., Mizuno, N., Ooya, M., Suenaga, K., Morioka, H., Ogawa, T., Ishida, M. and Baba, S., Cross-reactivities of neuropeptide Y and peptide YY with pancreatic polypeptide antisera: evidence for the existence of pancreatic polypeptide in the brain, Brain Res., 330 (1985) 386-390.
31
RPT 00561
Neuropeptide Y (NPY)-induced feeding behavior in female rats: comparison with human NPY ([Met 17]NPY), NPY analog ([norLeu4]NpY) and peptide YY* John T. Clark T M , A b h i r a m Sahu 1, Pushpa S. Kalra 1, A m b i k a i p a k a n Balasubramaniam 2 and Satya P. Kalra 1 1Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. and 2Department of Surgery, University of Cincinnati Medical Center, Cincinnati, OH 45267, U.S.A. (Received 22 August 1986; revised version received 24 November 1986; accepted 24 November 1986)
Summary Porcine neuropeptide Y (pNPY) administered into the third ventricle of the brain is known to elicit a powerful feeding response in steroid-treated ovariectomized and intact male rats. The present study compared the effects of pNPY and 3 structurally related peptides, human NPY (hNPY), an analog of NPY (NPY-A, [norLeu4]NPY) and peptide YY (PYY) on feeding behavior in intact female rats. Intraventricular administration of pNPY, hNPY, NPY-A and PYY over a dose range of 0.5 to 10 #g evoked feeding behavior to a varying extent. Cumulative food intake during 60 and 120 min was increased in a dose-related fashion at 0.5 and 2.0 /~g for the 4 peptides. Whereas the 10-#g dose of pNPY evoked a feeding response smaller than that seen after 2/ag, the responses to either 10 #g hNPY or 10/~g PYY were similar to that seen after 2 #g. The effects of these peptides on the time spent eating were quite different: while pNPY increased the time spent eating, this effect was not dose-related, whereas hNPY, NPY-A and PYY produced dose-related increments in the time spent eating. The most dramatic increment in local eating rate was observed
*Preliminary data were presented at the Society for Neuroscience annual meeting in Dallas, TX, 1985. **Present address: Department of Physiology, Meharry Medical College, 1-5 D.B. Todd Blvd., Nashville, TN 37208, U.S.A. Correspondence: Satya P. Kalra, Ph.D., Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. 0167-0115/87/$03.50 O 1987 Elsevier Science Publishers B.V. (Biomedical Division)
32
after 2.0/~g pNPY, with lesser increments seen after 2.0/~g hNPY and NPY-A. This increased local eating was apparently responsible for the highest cumulative food intake observed. These results demonstrate that (a) 2 #g pNPY is equally effective in stimulating feeding behavior in intact female rats as it is in steroid-primed ovariectomized female and intact male rats; (b) like the other members of the pancreatic polypeptide family, the PYY-stimulated feeding response was smaller than seen after pNPY; (c) in general, the cumulative food intake response, when compared with that evoked by pNPY, is reduced after substitution of either single, as in hNPY and NPY-A, or multiple amino acids as in the case of the members of the pancreatic polypeptide family, including PYY and (d) surprisingly, unlike pNPY, the other peptides tested facilitate the time spent eating response in a dose-related fashion. Further studies are needed to identify those active moieties in pNPY and other polypeptides of the pancreatic polypeptide family that impart differential effects on components of the feeding behavior in rats. Food intake; Neuropeptide; Intraventricular; Grooming; Behavior; Latency
Introduction
For over a decade attention has been focused on the role of neuropeptides in the stimulation of feeding behavior in the rat. Endogenous opioid peptides, opiates and their analogs increase food intake under a variety of experimental conditions [1,2]. Recently the members of the pancreatic polypeptide family were shown to stimulate various aspects of ingestive behavior in the rat (Table I). Neuropeptide Y, isolated and chemically characterized from porcine brain by Tatemoto [3], is structurally related to the pancreatic polypeptide family of hormones [4]. Administration of porcine NPY (pNPY) intracerebroventricularly (i.v.t.) stimulated food intake in satiated TABLE I Amino acid sequences of pNPY. hNPY and other members of the pancreatic polypeptide family of horlYIOl'leS
(Homology 4
8
12
16_
20
24
28
pNPY:
Y P S K P D N P G E D A P A E D L A R Y Y S A L R H Y I
hNPY:
.
rPP:
A
P L E -
M Y -
-
D Y -
T H -
Q R -
Q -
E T Q -
-
hPP-
A P L E -
V Y -
-
D N -
T P -
Q M -
Q - A A D -
-
PYY:
-
E A . . . . .
S P -
E -
.
.
-
.
A
.
~
.
-
.
.
.
.
.
.
.
.
.
.
M .
.
.
S -
.
-
.
-
.
A
.
.
.
.
.
N L
I
T R Q R Y .
.
.
to NH 2
NPYL
100%
.
.
R -
-
-
T
L
-
-
P -
-
46%
R -
-
- M L
-
-
P -
-
46%
L
-
-
.
36
.
S . . . .
.
32
97%
V . . . . .
69%
[Norleu4]NPY is a synthetic analog differing from NPY only by substitution of a norleucine for lysine at the 4 position, rPP, rat pancreatic polypeptide; hPP, human pancreatic polypeptide. One-letter codes for amino acids are used. Dashes indicate identity with NPY (sources: refs. I, 2, I 1, 13-15).
33 steroid-pretreated ovariectomized rats, and we observed a dose-related stimulation of feeding in intact male rats in both diurnal and nocturnal tests [5-7]. Interestingly, the NPY-induced feeding responses seen in our studies [5-7] and subsequently corroborated by others [8,9] were reliably more robust as compared to those reported for opiates and endogenous opioid peptides and growth hormone-releasing factor [1,2,10]. Similarly, members of the pancreatic polypeptide (PP) family, which show considerable structural homologies with pNPY, stimulate feeding in satiated rats (Table I [4,5]). We found that human pancreatic peptide and rat pancreatic peptide stimulated feeding response in a manner quite different from that induced by pNPY [6,7]. In an attempt to further understand the structure-function relationships among the members of PP, we have now compared the effects of pNPY with those of a gut peptide (PYY [11]) and two related peptides, human NPY (hNPY) and a NPY analog (NPY-A) on key parameters of ingestive behavior in the female rat. The two peptides, hNPY and NPY-A, are of interest because they differ from pNPY in only one amino acid, methionine replaced leucine at the 17 position in hNPY [12] and norleucine replaced lysine at the 4 position in NPY-A (Table I [12]).
Materials and Methods
Adult female rats (CRL:CDtR~(SD)BR, Charles River Breeding Laboratories, Wilmington, MA) were maintained in individual wire-bottom cages under controlled temperature (22-23°C) and light (lights on 05.00-19.00 h) with ad libitum access to food and water. Rats were implanted with permanent cannulae in the third cerebral ventricle under pentobarbital anesthesia (40 mg/kg [6]). Ten to 12 days after surgery, food and water were removed (12.00-14.00 h) and patency of the i.v.t, cannula was checked by observing the efflux of cerebrospinal fluid. Only those females exhibiting efflux of fluid were used in this study. One to two hours later, the effects on food intake of i.v.t, injection of the saline vehicle (3/~1, n = 17), pNPY (0.5, n = 6; 2, n = 6; 10 #g, n = 9), hNPY (0.5, n = 6; 2, n = 6; 10 #g, n = 7) or PYY (0.5, n = 8; 2, n = 6; 10 #g, n = 5) or NPY-A (0.5, n = 6; 2/~g, n = 6) were assessed. NPY-A was not tested at the 10-#g dose due to a supply problem as well as the observation of a decreased response after 10/~g pNPY and hNPY. Water was not available during the test. Each rat was used only once. The peptides (NPY, hNPY, PYY) were obtained commercially from Peninsula Laboratories (Belmont, CA) and NPY-A was synthesized as described [13]. Immediately after the i.v.t, injection, rats were returned to their home cages which contained a known amount of standard rat chow and continuously observed for 60 min [14]. The behavior was recorded in 3 categories: eating, grooming and yawning. At the end of 1 h the remaining food was carefully collected and weighed on a Mettler balance to the nearest 0.01 g. From the record, together with the food weighing, the following measurements were derived: total food intake (g); latency to first feeding response (min); duration of time spent eating (min); latency to first grooming response (min); time spent grooming (min) and local eating rate (g/min). Local eating rate is an index of the amount of food consumed per unit
34 time spent eating [14]. Following the food weighing, the remaining food was replaced in the cage with the animal and food intake was recorded for the period of 60-120 min after treatment. Thus, the number of rats displaying feeding behavior during the initial 60 min after injection and in the subsequent 60-120-min period and cumulative 2-h food intake was recorded. Data are presented as mean + S.E.M. The number of rats initiating behavior was evaluated using the Fisher exact probability test (comparing to saline-treated rats) and the parameters of feeding behavior were evaluated by Dunnett's test (comparison with a common control) followed by Mann-Whitney U-tests, where appropriate. Comparisons of food intake were made for the two discrete intervals after i.v.t, administration as well as for cumulative 2-h food intake.
Results
Effects of pNPY, hNPY, NPY-A and P Y Y on cumulative food intake I.v.t. pNPY at doses of 0.5 and 2.0 #g stimulated a dose-related increase in cumulative food intake for 60 min, with the most dramatic increase seen after the 2#g (0.47-nmol) dose. However, a higher dose of 10 #g pNPY was only slightly effective; in fact, the response was drastically lower than that seen after the 2-#g dose. NPY-A enhanced food intake in a manner similar to pNPY at 0.5- and 2-#g doses. All doses of hNPY and PYY also increased food intake. However, the responses were different from that seen after pNPY. As in the case of pNPY, the maximal response was seen after a 2-#g dose, but higher 10 pg doses of hNPY or PYY, unlike pNPY, produced an equivalent feeding response. A comparative analysis of the results indicates that whereas 2 #g of these peptides produced maximal responses, the quantitative responses were different among the peptides; pNPY and NPY-A were the most effective followed by hNPY and PYY, in that order. The cumulative 2-h food intake was also increased by these peptides (Fig. 1). However, there were additional quantitative differences that were not apparent when 60-min cumulative food intake was analyzed. After 0.5 #g, the cumulative food intake was greater after hNPY or NPY-A than after pNPY or PYY (P < 0.05). However, the response was reversed somewhat with the 2-pg dose: pNPY now elicited food intake equivalent to NPY-A but significantly greater than hNPY and PYY (P < 0.05). Further, 10 #g hNPY produced the greatest food intake which was higher than pNPY or PYY. Effects of pNP Y, hNP Y, NP Y-A and P Y Y on parameters of ingestive behavior The latency to the initial feeding response was variable, but was usually less than 20 min after 2 #g i.v.t, administration of the 4 peptides (Table II). The lowest latency of 7.4 min to first feeding response was seen after 2 #g pNPY, which increased dramatically to 38.8 4- 7.6 after 10.0 pg injection. Thus, all peptides tested are similar with respect to their effect on the latency to initiate feeding. The time spent eating increased in a dose-related manner after administration of hNPY, NPY-A and PYY (Fig. 2). In the case of pNPY, the time spent eating re-
5/6 a 5/6" 4/6 a 5/8 b
2.0 2.0 2.0 2.0 10.0 10.0 10.0
pNPY hNPY NPY-A PYY
pNPY hNPY PYY
1.6d 3.0 a,, 8.4 a 3.5 a
11.4a 7.1
7.7 a
4.4 1.8a
7.6 13.1 4- 2.2 a,, 10.4 4- 2.6 d,c
44+ 4-
+ 44+ 4-
38.8 4-
7.4 14.9 18.4 13.1
53.2 18.9 24.0 24.6 40.0
Latency to initial feeding response (minI
" P < 0.01 compared to saline-treated females using the Fisher Exact Probability Test. b p < 0.02 compared to saline-treated females using the Fisher Exact Probability Test. c M a x i m u m latency of 60 rain used for those females not feeding. d p < 0.01 compared to saline-treated females using the Man-Whitney U-test. P < 0.05 compared to NPY-treated females using the M a n n - W h i t n e y U-test.
5/9 5/5" 5/5"
6/6 a 5/5" 5/6" 7/7"
2/17
0.5 0.5 0.5 0.5
Proportion of females exhibiting feeding within 60 min o f treatment
-
Dose (#g)
Saline pNPY hNPY NPY-A PYY
Treatment
4+ 4+
+ + + ± 42.6 4.0 6.3 0.4
1.4 3.1 1.0 2.2 1.5
8.7 4- 2.1 5.2 + 1.6 16.1 4- 11.0
9.5 10.5 10.1 3.7
6.8 9.3 3.3 6.7 6.6
Latency to initial grooming response (min)
44+ 4-
+ + 44+
0.2 a 2.0 0.6 d 2.3
0.8 0.8 1.5 1.6 1.7
5.3 4- 0.8 2.9 4- 1.9 1.0 4- 0.4 a
0.6 3.5 1.9 6.9
5.0 3.0 5.1 3.5 8.0
Time spent grooming in the 60 min after treatment (min)
Effects o f pNPY, hNPY, N P Y - A (synthetic) and P Y Y (porcine) on aspects o f feeding and grooming behavior after intraventricular administration
T A B L E II
36 o) Cumulohve 6 0
If
minutefoodintake:
•e5
1"25
2hfoodintake:
' 3"0 b) Cumulohve
3"0
b
L
b
ose:
t
~
0
i
1.5
Treatment:
[]
~ hNPY
• NPY-A
[] P Y Y
~ to.o,,.,q q
Fig. I. Sixty-min and 2-h food intake after i.v.t, administration of pNPY, hNPY, N P Y - A and PYY. a, P < 0.05 compared to saline-treated female rats; b, P < 0.01 compared to saline-treated female rats; c, P < 0.05 compared to pNPY-treated female rats. For saline, n = 17 and for the other treatments, 5-9.
mained unaltered at doses of 0.5-10 #g. In fact, NPY-A, PYY at 2 #g and hNPY and PYY at 10 #g dosage significantly stimulated the time spent eating over that seen after pNPY administration. Local eating rate (g/min) was increased over the controls by 2 #g of pNPY, NPY-A and hNPY. pNPY also dramatically increased the local eating rate at this dose over that produced by either of the 3 peptides or 0.5 #g pNPY ( x 10 compared to 0.5 #g). Grooming activity was significantly reduced only after 2 #g NPY, 2 #g NPY-A and 10 #g PYY (Table II) in association with the largest increments in food intake (2 #g pNPY and NPY-A) or time spent eating (PYY and NPY-A). 30
a) T~me spent eatmq..
ob
l
3O
3. 15
o
2"2
o
;I
FE'jhNPY
b) Local eotmq r a t e
~ 0.4[
Dose."
--
O
k
~-O'SflJq ~
Tree tmen t.
~PYY 0.2
F2.0pQ-~
Fig. 2. Effects of i.v.t, administration of pNPY, hNPY, N P Y - A and P Y Y on parameters of feeding behavior in the 60 min after treatment, a, P < 0.01 compared to saline-treated female rats; b, P < 0.01 compared to pNPY-treated rats.
37 Discussion We have previously shown that pNPY was more effective than human pancreatic peptide or rat pancreatic peptide in stimulating food intake in ovariectomized steroid-primed female, and intact male rats, respectively [7,8]. We have now extended these comparative studies to show that pNPY is equally effective in intact female rats, and in terms of maximal cumulative food intake, it was more effective than either hNPY or PYY. These observations are of considerable interest because they reveal, for the first time, that substitution occurring naturally either of a single amino acid at the 17 position as in hNPY [11] or of multiple amino acids, as in the case of PYY, hPP or rPP [5,14] (Table I), attenuated the cumulative food intake response compared to that produced by pNPY. In the case of NPY-A [12], an experimentally produced analog which has norleucine substituted for lysine at the 4 position, the food intake response was comparable to that seen after pNPY. We noted that the gut peptide PYY, which has identical amino acid residues in 25 positions [14] (Table I), was only about half as effective as pNPY in increasing food intake in satiated female rats. This observation is interesting because other studies have found PYY more potent than pNPY in male rats [15,16]. While it is possible that PYY-induced response may be different in the two sexes; there are other important variables between our studies and those of others that may explain these differences in feeding responses. These include: route of administration [11,12], the composition of food [10,16], prescreening of rats for feeding response tests [10,16] and the availability of water during the test period [9,10,16]. In our study water was not available during the experiment. This may be an important procedural difference because we have seen that the dose response for the elicitation of feeding behavior in satiated males is shifted to the left if the water is unavailable [7] compared to when water is available [8]. Despite these differences, our findings together with previous reports on hPP [6] and rPP [7] suggest that structural modifications of the pNPY molecule, as those occurring in nature in PP and hNPY may result in diminution of cumulative food intake as compared to pNPY. Interestingly, the 3 naturally occurring peptides also differ considerably when other parameters related to ingestive behavior were monitored. Whereas pNPY evoked similar increments in time spent eating after 0.5 or 2 pg, the 2-/~g dose evinced a dramatic increase in the local eating rate suggesting that the dramatic increment in food intake seen after 2 pg pNPY was probably due to the increased local rate. On the other hand, the two naturally occurring peptides, hNPY and PYY, and the experimentally produced analog NPY-A, enhanced the time spent eating in a doserelated fashion. Further, it is interesting to note that the higher 10-#g dose of pNPY was less effective than 2 pg, an observation suggestive of a bell-shaped response typically observed after central administration of peptides. On the other hand, hNPY and PYY produced similar food intake and the time spent eating responses after the 2 or 10 #g dose. Thus, we suggest that the mode of action of these peptides in enhancing food intake may be different from that produced by pNPY. In addition, our data suggest that the stimulation of feeding is behaviorally specific and not due to a general activation of arousal since opposite effects are seen on grooming and sexual behavior [7].
38
Although these findings consistently reveal that structural modifications of the pNPY molecule have profound effects on feeding behavior in rats, the precise physiological role of pNPY and other related members of the pancreatic polypeptide family remains to be clarified [17]. A number of studies suggest that pNPY or a related peptide in the rat may be the endogenous neurochemical signal in the hypothalamic circuitry involved in the regulation of feeding behavior [18-24]. PYY, a gut hormone of non-neural origin, has been localized in small amounts in the rat brain [20,24] and therefore its role in the neural control of food intake remains to be defined. On the other hand, it is tempting to infer from our studies that hNPY, isolated from human adrenal medullary pheochromocytoma tissue [12], may play an important role in the neural regulation of feeding in man. In summary, our comparative studies have consistently shown that pNPY evokes a robust feeding response, primarily by increasing the local eating rate and that other members of the pancreatic polypeptide family stimulate feeding to variable, and usually lesser, degrees, primarily by increasing the time spent eating.
Acknowledgements This research was supported by NIH DK 37273 and J.T.C. was supported by Postdoctoral Fellowship, HD 06660. The technical assistance of D.L. Heaton-Jones and secretarial assistance of Sally McDonell are gratefully acknowledged.
References 1 Grandison, L. and Guidotti, A., Stimulation of food intake by muscimol and beta endorphin, Neuropharmacology, 16 (1977) 533-536. 2 Morley, J., Levine, A., Yim, L. and Lowy, M., Opioid modulation of appetite, Neurosci. Behav. Rev., 7 (1983) 281-305. 3 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5485-5489. 4 Lin, T.M., Pancreatic polypeptides: isolation, chemistry and biological functions. In: G.B.J. Glass (Ed.), Gastrointestinal Hormones, Raven, New York, 1980, pp. 275-306. 5 Clark, J.T., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology, I 15 (1984) 427--429. 6 Clark, J.T., Kalra, P.S. and Kalra, S.P., Neuropeptide Y stimulates feeding but inhibits sexual behavior in rats, Endocrinology, 117 (1985) 2435-2442. 7 Allen, L.G., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Comparison of the effects of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats. Life Sci,, 37 (1985) 617623. 8 Levine, A.S. and Morley, J.E., Neuropeptide Y: a potent inducer of consummatory behavior in rats, Peptides, 5 (1984) 1025-1029. 9 Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci., 35 (1984) 2635-2642. 10 Vaccarino, F.J., Bloom, F.E., Rivier, J., Vale, W. and Koob, G.F., Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor, Nature (London), 314 (1985) 167-168.
39 11 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London), 296 (1982) 659~560. 12 Cordor, R., Emson, P. and Lowry, P., Purification and characterization of human neuropeptide Y from adrenal-medullary phaechromocytoma tissue, Biochem. J., 219 (1984) 669-706. 13 Balasubramaniam, A., Grupp, I., Srivastava, L., Tatemoto, K., Murphy, R.F., Joffe, S.N. and Fischer, J.E., Synthesis of Neuropeptide Y, Int. J. Peptide Protein Res., in press. 14 Blundell, J.E., Bio-grammar of feeding: pharmacological manipulations and their interpretations. In: S.J. Cooper (Ed.), Theory in Psychopharmacology, Vol. 1, Academic, New York, pp. 233-250. 15 Morley, J.E., Levine, A.S., Grace, M. and Kneip, J., Peptide YY (PYY), a potent orexigenic agent, Brain Res., 341 (1985) 200-203. 16 Stanley, B.G., Daniel, D.R., Chin, A.S. and Leibowitz, S.F., Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion, Peptides, in press. 17 Kalra, S.P., Allen, L.G., Clark, J.T., Crowley, W.R. and Kalra, P.S., Neuropeptide Y - an integrator of reproductive and appetitive functions. In: T.W. Moody (Ed.), Neural and Endocrine Peptides and Receptors, Plenum, New York, 1986, pp. 353-366. 18 Lundberg, J., Terenius, L., H6kfelt, T., Martling, C., Tatemoto, K., Polack, J., Bloom, S. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Scand., 116 (1982) 477--480. 19 Emson, P. and deQuidt, M., NPY - a new member of the pancreatic polypeptide family, Trends Neurosci., 7 (1984) 31-36. 20 O'Donohue, T., Chronwall, B., Pruss, R., Mezey, E., Kiss,. J., Eiden, L., Massari, V., Tessel, R., Pickel, V., DiMaggio, D., Hotchkiss, A., Crowley, W. and Zukowska-Grojec, L., Neuropeptide Y and peptide YY neuronal and endocrine systems, Peptides, 6 (1985) 755-768. 21 Saria, A., Theorlorsson-Norheim, E. and Lundberg, J.M., Evidence for specific neuropeptide Y binding sites in rat brain synaptosomes, Eur. J. Pharmacol., 107 (1985) 105-107. 22 Lundberg, J., Terenius, L., Hokfelt, T. and Tatemoto, K., Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons, J. Neurosci., 4 (1984) 2376-2386. 23 Chang, R., Lotti, V., Chen, T.-B., Cerino, D. and Kling, P., Neuropeptide Y (NPY) binding sites in rat brain labeled with ~25I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens, Life Sci., 37 (1985) 2111-2122. 24 Inui, A., Mizuno, N., Ooya, M., Suenaga, K., Morioka, H., Ogawa, T., Ishida, M. and Baba, S., Cross-reactivities of neuropeptide Y and peptide YY with pancreatic polypeptide antisera: evidence for the existence of pancreatic polypeptide in the brain, Brain Res., 330 (1985) 386-390.