- Email: [email protected]
Neuropeptide Y-Related Compounds and Feeding
Physiology & Behavior, Vol. 65, Nos. 4/5, pp. 901–905, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99 $–see front matter
PII S0031-9384(98)00220-0
Neuropeptide Y-Related Compounds and Feeding MARK C. FLYNN,* CARLOS R. PLATA-SALAMÁN*1 AND JARLATH M. H. FFRENCH–MULLEN† *Division of Molecular Biology, School of Life and Health Sciences, University of Delaware, Newark, DE 19716-2590 †RIN Bioscience, Zeneca Pharmaceuticals, Zeneca Inc., Wilmington, DE 19850-5437 Received 9 April 1998; Accepted 31 July 1998 FLYNN, M. C., C. R. PLATA–SALAMÁN AND J. M. H. FFRENCH–MULLEN. Neuropeptide Y-related compounds and feeding. PHYSIOL BEHAV 65(4/5) 901–905, 1999.—Neuropeptide Y (NPY) and related compounds increase shortterm feeding. Previous studies have used different animal models, feeding schedules, sources of the compounds, and time and routes of administration. These differences in methodology are important in the variability reported on the potency of NPYrelated compounds. To obtain reliable data on the relative efficacy, we tested NPY, NPY 3–36, and pancreatic polypeptide (PP) using an identical protocol and the same commercial source. These three NPY-related compounds were tested using the intracerebroventricular (i.c.v., into the third ventricle) administration, and the profile of the feeding enhancement including the dose response and potency was determined. Compounds were tested in parallel on at least 2 successive days. NPY, NPY 3–36, and PP exhibited different potencies in enhancing 2-h food intake. Comparison of their dose responses (using 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg/rat) demonstrated an overall potency of NPY 3–36 . NPY . PP for the high doses. To study ligand interactions, we examined the effects of various combinations of NPY-related compounds administered concomitantly. These combinations were justified based on the data obtained from the individual dose responses. The data show that the effects of NPY plus NPY 3–36 or NPY 3–36 plus PP were less than additive. When compared to the individual responses, the effects of NPY 3–36 were almost identical to those induced by the combinations using low doses of NPY plus NPY 3–36, or low and high doses of PP plus NPY 3–36. The results support the notion that NPY and its analogues induce a short-term feeding response by activating multiple receptor subtypes. © 1999 Elsevier Science Inc. NPY Peptide Cerebrospinal fluid
Receptor Behavior Food intake Ingestion Central nervous system Brain Hypothalamus
pounds. To obtain reliable data on the relative efficacy, we tested NPY, NPY 3–36, and PP using the same protocol and commercial source. These three NPY-related compounds were tested in parallel, and the profile of the feeding enhancement including the dose response and potency was determined. Various Y-receptor subtypes have been proposed to be involved in mediating the feeding response by NPY-related compounds [e.g., (7,13)], and NPY and its analogs can bind to several of these receptors (2,8,28,30). Thus, the study of ligand interactions can provide information on the relative contribution of each receptor. To study potential interactions, we examined the effects of various combinations of NPY-related compounds administered concomitantly. These combinations were justified based on the data obtained from the individual dose responses.
NEUROPEPTIDE Y (NPY) is a 36-amino acid peptide found in the hypothalamus. The intracerebroventricular (i.c.v.) administration of NPY increases the short-term (2-h) food intake (7,11,17). There are several other NPY-related compounds that have been reported to produce similar effects including NPY 3–36 (9,22) and pancreatic polypeptide (PP) (4,7). These ligands bind to the same group of receptors called the Y receptor family (2,12,28,30). Previous studies have reported on the feeding response induced by individual NPY-related compounds using different animal models, feeding schedules, sources of the compounds, time, and routes of administration [e.g., (4,10,11,13,15,16,20, 21,27)]. These differences in methodology are important in the variability reported on the potency of NPY-related com1To
Intracerebroventricular administration Rat
whom requests for reprints should be addressed. E-mail: [email protected]
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FLYNN, PLATA-SALAMÁN AND FFRENCH-MULLEN MATERIALS AND METHODS
Subjects and Maintenance Male Wistar (VAF) rats, weighing between 250 and 275 g at the beginning of the experiments, were used. Rats were randomly assigned into groups and placed in individual cages. They were maintained ad lib on powdered rat food (Labdiet, PMI Feeds, Inc., St. Louis, MO) and tap water as previously described (25). Artificial light illumination was from 0600 to 1800 h, and room temperature was kept at 21 6 28C. All rats were handled daily. After several days of adaptation of the rats to their home cages, brain cannulas were implanted. Test solutions were administered following a recovery period of at least 10 days after surgery. Powdered Food Consumption The measurement of powdered food intake was the same as in previous studies (24). In all cases food intake was measured to within 0.1 g. Before and after the cannula implantation, the rats were fed ad lib daily, except between 1630 and 1800 h when food was removed to be measured and replaced and rats were handled. Premeasured food was presented at 1800 h, and food intake was measured at 2000 (2-h consumption). Thus, we assessed the feeding response to the i.c.v. administration of test compounds during the initial interval of the nighttime period or period of eating in rats. Implantation of Brain Cannulas Under intraperitoneal (i.p.) ketamine (100 mg/kg) plus xylazine (5 mg/kg) anesthesia, a 23-gauge stainless steel guide cannula was implanted into the third ventricle at stereotaxic coordinates: 22.1 anteroposterior and 0.0 lateral with respect to the bregma, and 7.5–8.0 dorsoventral from the brain surface as previously described (23). The location of the cannula tip into the third ventricle was verified by the free outflow of CSF through the guide cannula. A sterile 29-gauge stainless steel obturator was used to ensure the cannula remained patent.
specific adsorption of the compounds on the experimental tools, all such materials were siliconized. Data Analyses All results are expressed as means 6 SE. Statistical analyses compared: (a) the preinfusion levels (values for the day before infusion) to those obtained after infusion of test solutions; and (b) the values among different groups following test solutions. Analyses were performed with the paired and twosample Student’s t-tests when data passed the normality (Kolmogorov–Smirnov) and equal variance (Levene Median) tests; otherwise, the data were analyzed with the Mann–Whitney rank sum or Wilcoxon tests. The two-way ANOVA was also performed when indicated. Differences were considered to be significant only for p , 0.05. RESULTS
Individual Treatments The dose response to the i.c.v. administration of NPY, NPY 3–36, and PP is shown in Fig. 1. All three compounds enhanced feeding significantly. However, NPY, NPY 3–36, and PP exhibited different potencies. The effects of NPY ranged from 20% (for 0.1 mg) to 59% (for 5.0 mg) increase in food intake from baseline, while NPY 3–36 increased feeding by about 20% for 0.1 mg and 72% for 5.0 mg. [All groups shown in Fig. 1 had similar baseline food intake. The range of baseline food intake was from 6.06 6 0.6 g to 6.42 6 0.5 g for all groups treated with different doses of NPY; 6.09 6 0.7 g to 6.60 6 0.6 g for all groups treated with NPY 3–36; and 5.96 6
Intracerebroventricular Administration Microinfusions were made into the third ventricle because of the importance of hypothalamic regions in the regulation of feeding. Intracerebroventricular microinfusions (10 mL/rat, into unrestrained, undisturbed animals) were at the rate of 1 mL per 60 s using a Harvard infusion pump (Harvard Apparatus, South Natick, MA). Each animal was infused between 1715 and 1800 h, i.e., before the nighttime period. Randomly assigned groups received either NPY, NPY 3–36, or pancreatic polypeptide (PP) (Research Biochemicals International, Natick, MA) at doses 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg/rat. NPY-related compounds were also administered concomitantly in the following combinations: NPY 1 NPY 3–36: 0.1, 0.25, 0.5, 2.5, and 5.0 mg; NPY 3–36 1 PP: 0.5 1 0.1, 0.5 1 5.0, or 2.5 1 2.5 mg. All test compounds were dissolved in sterile 0.15 M NaCl. Test solutions were administered in parallel with NPY 5.0 mg/rat, which was used as a reference for consistency. Groups of animals selected for testing had a similar baseline food intake. In addition, all treatments and doses were tested on at least two successive days on different groups of rats to ensure that there were no variations in the test substances or infusion procedure. In some cases, we repeated an infusion in other complete group of rats, and therefore, some of the groups have a larger number of animals. To avoid non-
FIG. 1. Dose response for NPY, NPY 3–36, and PP effects on food intake. All compounds were administered i.c.v. at 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg/rat. Values represent mean 6 SE increase in 2-h food intake (g) from baseline. Numbers for each group tested are shown next to the symbols. All three dose responses are significantly different from each other (ANOVA, see text).
NEUROPEPTIDE Y AND FEEDING
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0.9 g to 6.34 6 0.5 g for all groups treated with PP. In addition, vehicle alone had no effect on food intake (not shown).] At each dose from 1 mg/rat, NPY 3–36 was at least as effective as NPY; in fact, at 2.5 mg, NPY 3–36 was significantly more potent than NPY in stimulating 2-h food intake (p , 0.05). In addition, comparison of NPY and NPY 3–36 dose responses from 1.0 mg to 5.0 mg showed that NPY 3–36 was more effective than NPY in increasing feeding, F(2, 51) 5 4.57, p , 0.04, two-way ANOVA. Pancreatic polypeptide also induced an increase in 2-h feeding (Fig. 1). However, PP was less effective relative to NPY or NPY 3–36. The efficacy of PP in increasing food intake ranged from 7% (for 0.1 mg) to 44% (for 5.0 mg). Overall, the dose response of NPY-induced feeding enhancement was significantly different from that of PP, F(4, 67) 5 10.532, p , 0.00001, two-way ANOVA. Thus, all three dose responses were significantly different from each other; this indicates an overall effectiveness of NPY 3–36 . NPY . PP. Concomitant Treatments The effects of the i.c.v. administration of selected combinations of NPY-related compounds are shown in Figs. 2 and 3. The effects of NPY plus NPY 3–36 (Fig. 2) or NPY 3–36 plus PP (Fig. 3) were less than additive. When compared to the individual responses, the effects of NPY 3–36 are almost identical to those induced by the combinations using low doses (0.1 and 0.25 mg/rat) of NPY plus NPY 3–36 (Fig. 4), or low and high doses of PP plus NPY 3–36 (Fig. 5). Moreover, the feeding response to a combination of
FIG. 3. Effects of the i.c.v. administration of NPY 3–36 plus PP on 2-h food intake. Other explanations are as for Fig. 2.
5.0 mg PP/rat plus 0.5 mg NPY 3–36/rat was actually less than for PP alone, that is, the increase in feeding was intermediate between the individual feeding responses to PP and NPY 3–36 (2.8 g for 5.0 mg PP, 1.3 g for 0.5 mg NPY 3–36, and 2.5 g for the combination 5.0 mg PP/rat plus 0.5 mg NPY 3–36/rat). DISCUSSION
FIG. 2. Effects of the i.c.v. administration of NPY plus NPY 3–36 on 2-h food intake. Filled bars represent the observed increase in feeding by various doses of NPY plus NPY 3–36. Open bars represent the increase expected from adding the individual data obtained from the dose responses at the doses indicated. The number of animals in each group is shown above filled bars.
Neuropeptide Y, NPY 3–36, and PP, when administered i.c.v., stimulated short-term (2-h) food intake. The higher doses of NPY 3–36 were more effective than NPY or PP in increasing feeding. For the overall profile, the rank order of potency was NPY 3–36 . NPY . PP when tested in parallel and identical experimental conditions. Our results using the individual administration of NPYrelated compounds compare well with those of other investigators (3,4,7,9,10,11,13,17,22). We found, as have others, that NPY was very potent in stimulating short-term food intake. Our data with NPY 3–36 is also consistent with that of Gerald et al. (1996), in that PYY 3–36 (a molecule similar to NPY 3–36) was more effective than NPY itself. However, O’Shea et al. (1997) found that NPY and NPY 3–36 were equipotent for the entire dose response. The shape of their dose response was similar to ours, though; there was an increase in the feeding response with an increase in the dosage (0.1–3.0 mg) and a plateau at the highest dose for both NPY and NPY 3–36. The results of Clark et al., 1984 were in agreement with ours; that is, NPY was more effective than PP. Neuropeptide Y 3–36 is a high affinity agonist for the Y2 receptor (but also for the Y5), PP for the Y4 receptor, and NPY for the Y1, Y2, and Y5 receptors (1,2,7–9,14,26,30). Thus, the involvement of multiple receptor subtypes in the NPY-induced feeding response is supported by the present
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FIG. 4. Mean 2-h food intake in response to the i.c.v. administration of two doses of NPY and NPY 3–36 and their combination. Data obtained from Figs. 1 and 2. The effect of the combination was almost identical to the effect induced by NPY 3–36 alone.
data and by previous reports (7,9,22). Previous studies have focused on the Y1 and Y5 as “feeding receptors” (7,13). The present data, however, also suggest an involvement of the Y2 [the predominant Y-receptor subtype found in the hypothalamus; (5,6,29)] and Y4 receptors in the feeding enhancement induced by NPY-related compounds. When NPY and NPY 3–36, or NPY 3–36 and PP were given in combination, the effect obtained was less than additive. These data also suggest the involvement of the Y2 and Y4 receptors, the use of similar receptor mechanisms, and/or modulation of transducer (G-protein)- or intracellular mediators-associated processes. The feeding response to a combination of NPY 3–36 plus PP was less than for PP alone, also suggesting competition for similar receptor mechanisms. The combination of NPY and NPY 3–36 may be especially relevant because both of these compounds may be found together (9). NPY 3–36 is generated from NPY, and accounts for 35% of NPY-like immunoreactivity (9). It is possible that
FIG. 5. Mean 2-h food intake in response to the i.c.v. administration of two doses of PP and NPY 3–36 and their combination. Data obtained from Figs. 1 and 3. The effect of the combination was almost identical to the effect induced by NPY 3–36 alone.
this may be an endogenous feeding regulatory mechanism. By modulating the activity of dipeptidyl peptidase IV (18,19), the enzyme that cleaves the C-terminal tyr-pro from NPY, this could alter the ratio of NPY:NPY 3–36 and thus modify the feeding response. Our data suggest that this may be plausible, because the individual responses to NPY at 2.5 mg and NPY 3–36 at 1.0 mg (i.e., 40% of the NPY 2.5 mg dose) produce the same increase in 2-h feeding (mean of 3.01 and 2.99 g, respectively). In conclusion, although the selectivity of the various ligands used is not complete, the data suggest that NPY and its analogues produce a dramatic increase in short-term feeding by activating multiple receptor subtypes. ACKNOWLEDGEMENTS
This work was supported by Zeneca Pharmaceuticals.
REFERENCES 1. Bard, J. A.; Walker, M. W.; Branchek, T. A.; Weinshank, R. L.: Cloning and functional expression of a human Y4 subtype receptor for pancreatic polypeptide, neuropeptide Y, and Peptide YY. J. Biol. Chem. 270:26762–26765; 1995. 2. Blomqvist, A. G.; Herzog, H.: Y-receptor subtypes—How many more? Trends Neurosci. 20:294–298; 1997. 3. Broqua, P.; Wettstein, J. G.; Rocher, M. N.; Gauthier–Martin, B.; Riviere, P. J. M.; Junien, J. L.; Dahl, S. G.: Antinociceptive effects of neuropeptide Y and related peptides in mice. Brain Res. 724:25–32; 1996. 4. Clark, J. T.; Kalra, P. S.; Crowley, W. R.; Kalra, S. P.: Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115:427–429; 1984.
5. Dumont, Y.; Fournier, A.; St-Pierre, S.; Quirion, R.: Comparative characterization and autoradiographic distribution of neuropeptide Y receptor subtypes in the rat brain. J. Neurosci. 13:73–86; 1993. 6. Gehlert, D. R.; Gackenheimer, S. L.; Schober, D. A.: [Leu31Pro34] Neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain. Neurochem. Int. 21:45–67; 1992. 7. Gerald, C.; Walker, M. W.; Criscione, L.; Gustafson, E. L.; BatzlHartmann, C.; Smith, K. E.; Vaysse, P.; Durkin, M. M.; Laz, T. M.; Linemeyer, D. L.; Schaffhauser, A. O.; Whitebread, S.; Hofbauer, K. G.; Taber, R. I.; Branchek, T. A.; Weinshank, R. L.: A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382:168–171; 1996.
NEUROPEPTIDE Y AND FEEDING 8. Gerald, C.; Walker, M. W.; Vaysse, P. J.; Chaogang, H.; Branchek, T. A.; Weinshank, R. L.: Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype. J. Biol. Chem. 270:26758–26761; 1995. 9. Grandt, D.; Schimiczek, M.; Rascher, W.; Feth, F.; Shively, J.; Lee, T. D.; Davis, M. T.; Reeve, J. R., Jr.; Michel, M. C.: Neuropeptide Y 3-36 is an endogenous ligand selective for Y2 receptors. Regul. Pept. 67:33–37; 1996. 10. Jewett, D. C.; Cleary, J.; Schaal, D. W.; Thompson, T.; Levine, A. S.: [Leu31,Pro34] Neuropeptide Y (NPY), but not NPY 20-36, produces discriminative stimulus effects similar to NPY and induces food intake. Brain Res. 631:129–132; 1993. 11. Kalra, S. P.; Dube, M. G.; Fournier, A.; Kalra, P. S.: Structure– function analysis of stimulation of food intake by Neuropeptide Y: Effects of receptor agonists. Physiol. Behav. 50:5–9; 1991. 12. Larhammar, D.: Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide. Regul. Pept. 65:165– 174; 1996. 13. Leibowitz, S. F.; Alexander, J. T.: Analysis of neuropeptide Y-induced feeding: Dissociation of Y1 and Y2 receptor effects on natural meal patterns. Peptides 12:1251–1260; 1991. 14. Lundell, I.; Blomqvist, A. G.; Berglund, M. M.; Schober, D. A.; Johnson, D.; Statnick, M. A.; Gadski, R. A.; Gehlert, D. R.; Larhammar, D.: Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY. J. Biol. Chem. 270:29123–29128; 1995. 15. Matos, F. F.; Guss, V.; Korpinen, C.: Effects of neuropeptide Y (NPY) and [D-Trp32]NPY on monoamine and metabolite levels in dialysates from rat hypothalamus during feeding behavior. Neuropeptides 30:391–398; 1996. 16. McCarthy, H. D.; McKibbin, P. E.; Holloway, B.; Mayers, R.; Williams, G.: Hypothalamic neuropeptide Y receptor characteristics and NPY-induced feeding responses in lean and obese Zucker rats. Life Sci. 49:1491–1497; 1991. 17. McLaughlin, C. L.; Tou, J. S.; Rogan, G. J.; Baile, C. A.: Full amino acid sequence of centrally administered NPY required for maximal food intake response. Physiol. Behav. 49:521–526; 1991. 18. Medeiros, M. S.; Turner, A. J.: Post-secretory processing of regulatory peptides: The pancreatic polypeptide family as a model example. Biochimie 76:283–287; 1994. 19. Mentlein, R.; Dahms, P.; Grandt, D.; Kruger, R.: Proteolytic pro-
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cessing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul. Pept. 49:133–144; 1993. Miner, J. L.; Della–Fera, M. A.; Paterson, J. A.; Baile, C. A.: Lateral cerebroventricular injection of neuropeptide Y stimulates feeding in sheep. Am. J. Physiol. 257:R383–R387; 1989. Morley, J. E.; Hernandez, E. N.; Flood, J. F.: Neuropeptide Y increases food intake in mice. Am. J. Physiol. 253:R516–R522; 1987. O’Shea, D.; Morgan, D. G. A.; Meeran, K.; Edwards, C. M. B.; Turton, M. D.; Choi, S. J.; Heath, M. M.; Gunn, I.; Taylor, G. M.; Howards, J. K.; Bloom, C. I.; Small, C. J.; Haddo, O.; Ma, J. J.; Callinan, W.; Smith, D. M.; Ghatei, M. A.; Bloom, S. R.: Neuropeptide Y induced feeding in the rat is mediated by a novel receptor. Endocrinology 138:196–202; 1997. Plata–Salamán, C. R.: Interferons and central regulation of feeding. Am. J. Physiol. 263:R1222–R1227; 1992. Plata–Salamán, C. R.; Borkoski, J. P.: Interleukin-8 modulates feeding by direct action in the central nervous system. Am. J. Physiol. 265:R877–R882; 1993. Plata–Salamán, C. R.; Sonti, G.; Borkoski, J. P.: Modulation of feeding by b2-microglobulin, a marker of immune activation. Am. J. Physiol. 268:R1513–R1519; 1995. Small, C. J.; Morgan, D. G. A.; Meeran, K.; Heath, M. M.; Gunn, I.; Edwards, C. M. B.; Gardiner, J.; Taylor, G. M.; Hurley, J. D.; Rossi, M.; Goldstone, A. P.; O’Shea, D.; Smith, D. M.; Ghatei, M. A.; Bloom, S. R.: Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release. Proc. Natl. Acad. Sci. USA 94:11686–11691; 1997. Stanley, B. G.; Leibowitz, S. F.: Neuropeptide Y injected in the paraventricular hypothalamus: A powerful stimulant of feeding behavior. Proc. Natl. Acad. Sci. USA 82:3940–3943; 1985. Weinberg, D. H.; Sirinathsinghji, D. J. S.; Tan, C. P.; Shiao, L. L.; Morin, N.; Rigby, M. R.; Heavens, R. H.; Rapoport, D. R.; Bayne, M. L.; Cascieri, M. A.; Strader, C. D.; Linemeyer, D. L.; MacNeil, D. J.: Cloning and expression of a novel neuropeptide Y receptor. J. Biol. Chem. 271:16435–16438; 1996. Widdowson, P. S.: Quantitative receptor autoradiography demonstrates a differential distribution of neuropeptide-Y Y1 and Y2 receptor subtypes in human and rat brain. Brain Res. 631:27–38; 1993. Wieland, H. A.; Willim, K.; Doods, H. N.: Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines. Peptides 16:1389–1394; 1995.
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Neuropeptide Y-Related Compounds and Feeding MARK C. FLYNN,* CARLOS R. PLATA-SALAMÁN*1 AND JARLATH M. H. FFRENCH–MULLEN† *Division of Molecular Biology, School of Life and Health Sciences, University of Delaware, Newark, DE 19716-2590 †RIN Bioscience, Zeneca Pharmaceuticals, Zeneca Inc., Wilmington, DE 19850-5437 Received 9 April 1998; Accepted 31 July 1998 FLYNN, M. C., C. R. PLATA–SALAMÁN AND J. M. H. FFRENCH–MULLEN. Neuropeptide Y-related compounds and feeding. PHYSIOL BEHAV 65(4/5) 901–905, 1999.—Neuropeptide Y (NPY) and related compounds increase shortterm feeding. Previous studies have used different animal models, feeding schedules, sources of the compounds, and time and routes of administration. These differences in methodology are important in the variability reported on the potency of NPYrelated compounds. To obtain reliable data on the relative efficacy, we tested NPY, NPY 3–36, and pancreatic polypeptide (PP) using an identical protocol and the same commercial source. These three NPY-related compounds were tested using the intracerebroventricular (i.c.v., into the third ventricle) administration, and the profile of the feeding enhancement including the dose response and potency was determined. Compounds were tested in parallel on at least 2 successive days. NPY, NPY 3–36, and PP exhibited different potencies in enhancing 2-h food intake. Comparison of their dose responses (using 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg/rat) demonstrated an overall potency of NPY 3–36 . NPY . PP for the high doses. To study ligand interactions, we examined the effects of various combinations of NPY-related compounds administered concomitantly. These combinations were justified based on the data obtained from the individual dose responses. The data show that the effects of NPY plus NPY 3–36 or NPY 3–36 plus PP were less than additive. When compared to the individual responses, the effects of NPY 3–36 were almost identical to those induced by the combinations using low doses of NPY plus NPY 3–36, or low and high doses of PP plus NPY 3–36. The results support the notion that NPY and its analogues induce a short-term feeding response by activating multiple receptor subtypes. © 1999 Elsevier Science Inc. NPY Peptide Cerebrospinal fluid
Receptor Behavior Food intake Ingestion Central nervous system Brain Hypothalamus
pounds. To obtain reliable data on the relative efficacy, we tested NPY, NPY 3–36, and PP using the same protocol and commercial source. These three NPY-related compounds were tested in parallel, and the profile of the feeding enhancement including the dose response and potency was determined. Various Y-receptor subtypes have been proposed to be involved in mediating the feeding response by NPY-related compounds [e.g., (7,13)], and NPY and its analogs can bind to several of these receptors (2,8,28,30). Thus, the study of ligand interactions can provide information on the relative contribution of each receptor. To study potential interactions, we examined the effects of various combinations of NPY-related compounds administered concomitantly. These combinations were justified based on the data obtained from the individual dose responses.
NEUROPEPTIDE Y (NPY) is a 36-amino acid peptide found in the hypothalamus. The intracerebroventricular (i.c.v.) administration of NPY increases the short-term (2-h) food intake (7,11,17). There are several other NPY-related compounds that have been reported to produce similar effects including NPY 3–36 (9,22) and pancreatic polypeptide (PP) (4,7). These ligands bind to the same group of receptors called the Y receptor family (2,12,28,30). Previous studies have reported on the feeding response induced by individual NPY-related compounds using different animal models, feeding schedules, sources of the compounds, time, and routes of administration [e.g., (4,10,11,13,15,16,20, 21,27)]. These differences in methodology are important in the variability reported on the potency of NPY-related com1To
Intracerebroventricular administration Rat
whom requests for reprints should be addressed. E-mail: [email protected]
901
902
FLYNN, PLATA-SALAMÁN AND FFRENCH-MULLEN MATERIALS AND METHODS
Subjects and Maintenance Male Wistar (VAF) rats, weighing between 250 and 275 g at the beginning of the experiments, were used. Rats were randomly assigned into groups and placed in individual cages. They were maintained ad lib on powdered rat food (Labdiet, PMI Feeds, Inc., St. Louis, MO) and tap water as previously described (25). Artificial light illumination was from 0600 to 1800 h, and room temperature was kept at 21 6 28C. All rats were handled daily. After several days of adaptation of the rats to their home cages, brain cannulas were implanted. Test solutions were administered following a recovery period of at least 10 days after surgery. Powdered Food Consumption The measurement of powdered food intake was the same as in previous studies (24). In all cases food intake was measured to within 0.1 g. Before and after the cannula implantation, the rats were fed ad lib daily, except between 1630 and 1800 h when food was removed to be measured and replaced and rats were handled. Premeasured food was presented at 1800 h, and food intake was measured at 2000 (2-h consumption). Thus, we assessed the feeding response to the i.c.v. administration of test compounds during the initial interval of the nighttime period or period of eating in rats. Implantation of Brain Cannulas Under intraperitoneal (i.p.) ketamine (100 mg/kg) plus xylazine (5 mg/kg) anesthesia, a 23-gauge stainless steel guide cannula was implanted into the third ventricle at stereotaxic coordinates: 22.1 anteroposterior and 0.0 lateral with respect to the bregma, and 7.5–8.0 dorsoventral from the brain surface as previously described (23). The location of the cannula tip into the third ventricle was verified by the free outflow of CSF through the guide cannula. A sterile 29-gauge stainless steel obturator was used to ensure the cannula remained patent.
specific adsorption of the compounds on the experimental tools, all such materials were siliconized. Data Analyses All results are expressed as means 6 SE. Statistical analyses compared: (a) the preinfusion levels (values for the day before infusion) to those obtained after infusion of test solutions; and (b) the values among different groups following test solutions. Analyses were performed with the paired and twosample Student’s t-tests when data passed the normality (Kolmogorov–Smirnov) and equal variance (Levene Median) tests; otherwise, the data were analyzed with the Mann–Whitney rank sum or Wilcoxon tests. The two-way ANOVA was also performed when indicated. Differences were considered to be significant only for p , 0.05. RESULTS
Individual Treatments The dose response to the i.c.v. administration of NPY, NPY 3–36, and PP is shown in Fig. 1. All three compounds enhanced feeding significantly. However, NPY, NPY 3–36, and PP exhibited different potencies. The effects of NPY ranged from 20% (for 0.1 mg) to 59% (for 5.0 mg) increase in food intake from baseline, while NPY 3–36 increased feeding by about 20% for 0.1 mg and 72% for 5.0 mg. [All groups shown in Fig. 1 had similar baseline food intake. The range of baseline food intake was from 6.06 6 0.6 g to 6.42 6 0.5 g for all groups treated with different doses of NPY; 6.09 6 0.7 g to 6.60 6 0.6 g for all groups treated with NPY 3–36; and 5.96 6
Intracerebroventricular Administration Microinfusions were made into the third ventricle because of the importance of hypothalamic regions in the regulation of feeding. Intracerebroventricular microinfusions (10 mL/rat, into unrestrained, undisturbed animals) were at the rate of 1 mL per 60 s using a Harvard infusion pump (Harvard Apparatus, South Natick, MA). Each animal was infused between 1715 and 1800 h, i.e., before the nighttime period. Randomly assigned groups received either NPY, NPY 3–36, or pancreatic polypeptide (PP) (Research Biochemicals International, Natick, MA) at doses 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg/rat. NPY-related compounds were also administered concomitantly in the following combinations: NPY 1 NPY 3–36: 0.1, 0.25, 0.5, 2.5, and 5.0 mg; NPY 3–36 1 PP: 0.5 1 0.1, 0.5 1 5.0, or 2.5 1 2.5 mg. All test compounds were dissolved in sterile 0.15 M NaCl. Test solutions were administered in parallel with NPY 5.0 mg/rat, which was used as a reference for consistency. Groups of animals selected for testing had a similar baseline food intake. In addition, all treatments and doses were tested on at least two successive days on different groups of rats to ensure that there were no variations in the test substances or infusion procedure. In some cases, we repeated an infusion in other complete group of rats, and therefore, some of the groups have a larger number of animals. To avoid non-
FIG. 1. Dose response for NPY, NPY 3–36, and PP effects on food intake. All compounds were administered i.c.v. at 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg/rat. Values represent mean 6 SE increase in 2-h food intake (g) from baseline. Numbers for each group tested are shown next to the symbols. All three dose responses are significantly different from each other (ANOVA, see text).
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0.9 g to 6.34 6 0.5 g for all groups treated with PP. In addition, vehicle alone had no effect on food intake (not shown).] At each dose from 1 mg/rat, NPY 3–36 was at least as effective as NPY; in fact, at 2.5 mg, NPY 3–36 was significantly more potent than NPY in stimulating 2-h food intake (p , 0.05). In addition, comparison of NPY and NPY 3–36 dose responses from 1.0 mg to 5.0 mg showed that NPY 3–36 was more effective than NPY in increasing feeding, F(2, 51) 5 4.57, p , 0.04, two-way ANOVA. Pancreatic polypeptide also induced an increase in 2-h feeding (Fig. 1). However, PP was less effective relative to NPY or NPY 3–36. The efficacy of PP in increasing food intake ranged from 7% (for 0.1 mg) to 44% (for 5.0 mg). Overall, the dose response of NPY-induced feeding enhancement was significantly different from that of PP, F(4, 67) 5 10.532, p , 0.00001, two-way ANOVA. Thus, all three dose responses were significantly different from each other; this indicates an overall effectiveness of NPY 3–36 . NPY . PP. Concomitant Treatments The effects of the i.c.v. administration of selected combinations of NPY-related compounds are shown in Figs. 2 and 3. The effects of NPY plus NPY 3–36 (Fig. 2) or NPY 3–36 plus PP (Fig. 3) were less than additive. When compared to the individual responses, the effects of NPY 3–36 are almost identical to those induced by the combinations using low doses (0.1 and 0.25 mg/rat) of NPY plus NPY 3–36 (Fig. 4), or low and high doses of PP plus NPY 3–36 (Fig. 5). Moreover, the feeding response to a combination of
FIG. 3. Effects of the i.c.v. administration of NPY 3–36 plus PP on 2-h food intake. Other explanations are as for Fig. 2.
5.0 mg PP/rat plus 0.5 mg NPY 3–36/rat was actually less than for PP alone, that is, the increase in feeding was intermediate between the individual feeding responses to PP and NPY 3–36 (2.8 g for 5.0 mg PP, 1.3 g for 0.5 mg NPY 3–36, and 2.5 g for the combination 5.0 mg PP/rat plus 0.5 mg NPY 3–36/rat). DISCUSSION
FIG. 2. Effects of the i.c.v. administration of NPY plus NPY 3–36 on 2-h food intake. Filled bars represent the observed increase in feeding by various doses of NPY plus NPY 3–36. Open bars represent the increase expected from adding the individual data obtained from the dose responses at the doses indicated. The number of animals in each group is shown above filled bars.
Neuropeptide Y, NPY 3–36, and PP, when administered i.c.v., stimulated short-term (2-h) food intake. The higher doses of NPY 3–36 were more effective than NPY or PP in increasing feeding. For the overall profile, the rank order of potency was NPY 3–36 . NPY . PP when tested in parallel and identical experimental conditions. Our results using the individual administration of NPYrelated compounds compare well with those of other investigators (3,4,7,9,10,11,13,17,22). We found, as have others, that NPY was very potent in stimulating short-term food intake. Our data with NPY 3–36 is also consistent with that of Gerald et al. (1996), in that PYY 3–36 (a molecule similar to NPY 3–36) was more effective than NPY itself. However, O’Shea et al. (1997) found that NPY and NPY 3–36 were equipotent for the entire dose response. The shape of their dose response was similar to ours, though; there was an increase in the feeding response with an increase in the dosage (0.1–3.0 mg) and a plateau at the highest dose for both NPY and NPY 3–36. The results of Clark et al., 1984 were in agreement with ours; that is, NPY was more effective than PP. Neuropeptide Y 3–36 is a high affinity agonist for the Y2 receptor (but also for the Y5), PP for the Y4 receptor, and NPY for the Y1, Y2, and Y5 receptors (1,2,7–9,14,26,30). Thus, the involvement of multiple receptor subtypes in the NPY-induced feeding response is supported by the present
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FIG. 4. Mean 2-h food intake in response to the i.c.v. administration of two doses of NPY and NPY 3–36 and their combination. Data obtained from Figs. 1 and 2. The effect of the combination was almost identical to the effect induced by NPY 3–36 alone.
data and by previous reports (7,9,22). Previous studies have focused on the Y1 and Y5 as “feeding receptors” (7,13). The present data, however, also suggest an involvement of the Y2 [the predominant Y-receptor subtype found in the hypothalamus; (5,6,29)] and Y4 receptors in the feeding enhancement induced by NPY-related compounds. When NPY and NPY 3–36, or NPY 3–36 and PP were given in combination, the effect obtained was less than additive. These data also suggest the involvement of the Y2 and Y4 receptors, the use of similar receptor mechanisms, and/or modulation of transducer (G-protein)- or intracellular mediators-associated processes. The feeding response to a combination of NPY 3–36 plus PP was less than for PP alone, also suggesting competition for similar receptor mechanisms. The combination of NPY and NPY 3–36 may be especially relevant because both of these compounds may be found together (9). NPY 3–36 is generated from NPY, and accounts for 35% of NPY-like immunoreactivity (9). It is possible that
FIG. 5. Mean 2-h food intake in response to the i.c.v. administration of two doses of PP and NPY 3–36 and their combination. Data obtained from Figs. 1 and 3. The effect of the combination was almost identical to the effect induced by NPY 3–36 alone.
this may be an endogenous feeding regulatory mechanism. By modulating the activity of dipeptidyl peptidase IV (18,19), the enzyme that cleaves the C-terminal tyr-pro from NPY, this could alter the ratio of NPY:NPY 3–36 and thus modify the feeding response. Our data suggest that this may be plausible, because the individual responses to NPY at 2.5 mg and NPY 3–36 at 1.0 mg (i.e., 40% of the NPY 2.5 mg dose) produce the same increase in 2-h feeding (mean of 3.01 and 2.99 g, respectively). In conclusion, although the selectivity of the various ligands used is not complete, the data suggest that NPY and its analogues produce a dramatic increase in short-term feeding by activating multiple receptor subtypes. ACKNOWLEDGEMENTS
This work was supported by Zeneca Pharmaceuticals.
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