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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 10 3781-3791
Copyright © 1999 by The Endocrine Society

Original Studies

Activation of Central Neuropeptide Y Y1 Receptors Potently Stimulates Food Intake in Male Rhesus Monkeys1

P. J. Larsen, M. Tang-Christensen, C. E. Stidsen, K. Madsen, M. S. Smith and J. L. Cameron2

Departments of Anatomy (P.J.L., M.T.-C.) and Neurology (P.J.L.), University of Copenhagen, 2200 Copenhagen N, Denmark; Novo Nordisk (C.E.S., K.M.), Målov, Denmark; and the Departments of Reproductive Sciences (J.L.C.) and Molecular Biology (M.S.S.), Oregon Regional Primate Research Center, Beaverton, Oregon 95616

Address all correspondence and requests for reprints to: Philip Just Larsen, M.D., Ph.D., Department of Anatomy, The Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark. E-mail: p.larsen{at}mai.ku.dk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The orexigenic role of central neuropeptide Y (NPY) in nonhuman primates has been questioned. Therefore, we have studied the effect of central NPY on feeding in ad libitum-fed male rhesus macaques. NPY dose-dependently increased food intake, with the maximal effect obtained by 50 µg (960 min food intake ± SEM, 104 ± 5 to 188 ± 11 g; vehicle vs. NPY; n = 6). Blood glucose levels were unaffected by intracerebroventricular administration of NPY, but animals receiving either 20 or 50 µg displayed increased plasma levels of insulin and cortisol at few time points. To assess the pharmacological specificity of this response, a novel Y1 antagonist, [(Ile,Glu,Pro,Daba,Tyr, Arg,Leu,Arg,Tyr-NH2)2 cyclic (2,4'),(2',4)-diamide] (Y1ANT), was synthesized. Receptor binding experiments demonstrated that Y1ANT preferentially binds to Y1 and Y4 receptors (pKi 10.12 ± 0.06 and 9.11 ± 0.05 nmol/L, respectively). Functional analysis revealed that Y1ANT is a Y1 antagonist and a partial Y4 agonist. Central administration of Y1ANT blocked NPY-induced feeding. In food-deprived monkeys, Y1ANT attenuated the feeding response. However, Y1ANT had no effect on food intake in satiated monkeys. Thus, endogenous NPY is likely to be involved in the regulation of food intake in the nonhuman primate, and this effect is at least partially mediated via Y1-like receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SINCE its discovery in 1982, neuropeptide Y (NPY) has been shown to exert a plethora of effects on behaviors regulated by central neural pathways. Thus, NPY has been implicated in learning and memory, epileptic seizures, anxiety, circadian rhythms, thermoregulation, anterior pituitary release of ACTH, LH, and GH (for review, see Ref. 1). One of the most elaborately studied central functions of NPY is its effect on body energy homeostasis, including regulation of feeding behavior, insulin secretion, hepatic glucose output, lipoprotein lipase activity, and thermogenesis (2, 3, 4, 5, 6). In most species studied to date centrally applied NPY potently stimulates feeding behavior via a hypothalamic target localized in the area of the paraventricular nucleus or neighboring perifornical area (7, 8, 9). NPY stimulates food intake even in satiated animals, as evidenced by the persistent hyperphagia and obesity after chronic central administration of NPY (10, 11). Furthermore, central application of antibodies and antisense oligonucleotides to NPY significantly reduce food intake (12, 13). Food deprivation and exercise-induced energy deficits are potent stimulatory drives, resulting in increased NPY synthesis in arcuate neurons (14). Hypothalamic NPY is also believed to partly trigger the final common pathway responsible for overeating in a number of monogenetic rodent obesity models such as ob/ob and db/db mice and fa/fa Zucker rats (15, 16, 17). All of these models are characterized by defective leptin signaling, and given the inhibitory role of leptin on NPY synthesis in the arcuate nucleus, it is hardly surprising that NPY has been ascribed partially responsibility for the obesity of these animals (18). Furthermore, cross-breeding of leptin-deficient ob/ob mice and NPY knockout mice gives rise to offspring that are less obese than ob/ob mice (19), further supporting that leptin exerts an inhibitory role on hypothalamic NPY-containing neurons involved in feeding. Neuropeptide Y- expressing neurons of the arcuate nucleus projecting to the paraventricular nucleus are believed to constitute the central pathway mediating NPY-induced food intake (20, 21). Nevertheless, it is debated which receptor subtype(s) mediates the orexigenic effect of NPY (for review, see Ref. 1). Currently available pharmacological data suggest that both Y1 and Y5 receptors are capable of stimulating food intake, whereas most Y1 antagonists have been shown to inhibit food intake; this has nourished the speculation that yet another NPY receptor subtype constitutes the much sought for feeding receptor (22). Both of the receptors are expressed in hypothalamic nuclei centrally involved in feeding behavior (23, 24), and treatment with Y5 antisense oligoribonucleotides induces long lasting anorexia (25, 26). However, with the advent of Y1 and Y5 receptor knockout mice, some of the functional consequences of life without either of these receptors have become clearer (27, 28). Neither Y1 nor Y5 knockout mice display overt disturbances of body weight and food intake, but given the redundancy of central control of feeding, this finding is hardly surprising. However, fasting-induced feeding is severely affected in Y1-deficient mice and maximal NPY-induced food intake is much lower in Y5-deficient mice, supporting the broadly held view that both Y1 and Y5 receptors are involved in NPY-mediated food intake (26, 27, 28). Thus, the possibility that the combined effect of both Y1 and Y5 receptor activation influences feeding behavior should be considered. The possible involvement of more than one NPY receptor subtype in body weight regulation may also explain why nonpeptidic Y5 antagonists have no long term effects on the regulation of appetite in genetically obese mice (29).

Preliminary experiments have demonstrated that primates also respond to fasting with increased hypothalamic NPY synthesis in the arcuate nucleus, and this effect is rapidly and potently reversed by refeeding, suggesting that endogenous NPY drives feeding in primates (30). Given these experiments, it is surprising that a recent in vivo experiment with male baboons was unable to demonstrate an effect of central neuropeptide Y administration on food intake (31). To further examine the role of NPY on feeding behavior in primates, we performed an experiment on freely moving male rhesus monkeys with ad libitum access to food and water. Simultaneous collection of blood samples made possible a thorough analysis of endocrine parameters before, during, and after NPY infusion. Furthermore, to determine the pharmacological characteristics of the receptor(s) mediating the orexigenic effect of NPY, the capacity of a synthetic Y1 peptide antagonist Y1ANT to block the endogenous as well as exogenous effects of NPY on feeding was tested in both satiated and food-deprived monkeys.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides and drugs

The peptides human NPY and the NPY antagonist Y1ANT [(Ile, Glu,Pro,Daba,Tyr,Arg,Leu,Arg,Tyr-NH2)2 cyclic (2,4'),(2',4)-diamide] was synthesized by the Department of Medicinal Chemistry at Novo Nordisk A/S (Copenhagen, Denmark). NNC 58–0036 was synthesized according to the Boc strategy on an PE Applied Biosystems 430A peptide synthesizer (Foster City, CA). The following protected amino acid derivatives were used: Boc-Tyr(Br-Z), Boc-Arg(Tos), Boc-Leu, Boc-Dap(F-moc), Boc-Dab(F-moc), Boc-Pro, Boc-Glu(OFm), and Boc-Ile (Bachem AG, Bubendorf, Switzerland). The peptide was cyclized on the resin, deprotected and cleaved from the resin at 0 C for 75 min, and precipitated with diethyl ether. The crude peptide was purified by semipreparative high performance liquid chromatography on a C18 reverse phase column that was eluted with a gradient of acetonitrile in 0.05 mol/L (NH4)2SO4, pH 2.5. The peptide-containing fractions were collected, diluted with water, and applied to a Sep-Pak C18 cartridge (Waters-Millipore Corp., Milford, MA). After removing buffer salts by washing with water, the peptide was eluted with 70% acetonitrile-0.1% trifluoroacetic acid and lyophilized. The final product obtained was characterized by amino acid analysis, analytical reverse phase high performance liquid chromatography, and plasma desorption mass spectrometry. Morphine sulfate was purchased from the local pharmacy, and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Characterization of the NPY antagonist: NPY receptor complementary DNA (cDNA)

The human neuropeptide Y receptor cDNAs for Y1, Y2, Y4, and Y5 were cloned by PCR (Perkin Elmer Corp., Norwalk, CT) from human cDNA libraries as previously described (23, 32, 33, 34).

Radioligands

[125I]Tyr1-porcine (p) NPY and [125I]Tyr36-human pancreatic polypeptide (hPP) were synthesized and purified at Novo Nordisk A/S at a specific activity of 2000 Ci/mmol. Di-[125I]Tyr1,36-pPYY was purchased from Amersham Pharmacia Biotech (IM259, Piscataway, NJ) at a specific activity of 4000 Ci/mmol.

Cell culture

Baby hamster kidney cells (BHK tk- ts13, CRL-1632) and human embryonic kidney cells (HEK 293, CRL-1573) were obtained from American Type Culture Collection (Manassas, VA). All media and reagents for cell culture were purchased from Life Technologies, Inc. (Roskilde, Denmark).

Radioligand binding assays

Radioligand binding assays were performed with human NPY receptor subtypes individually and stably expressed in BHK cells (Y1, Y2, and Y4) or HEK 293 cells (Y5). Cells were grown in DMEM with Glutamax (DMEM) supplemented with 100 U/mL penicillin G, 100 µg/mL streptomycin, and 10% FBS in a humidified atmosphere of 5% CO2 at 37 C. Cells were harvested mechanically in growth medium and centrifuged at 20,000 x g for 10 min. The pellet was homogenized in buffer [25 mmol/L potassium-HEPES and 2.5 mmol/L CaCl2 (pH 7.4) for Y1; 25 mmol/L sodium-HEPES, 2.5 mmol/L CaCl2, and 1.2 mmol/L MgCl2 (pH 7.4) for Y2 and Y4; and 10 mmol/L NaCl, 20 mmol/L HEPES, 0.22 mmol/L KH2PO4, 1.26 mmol/L CaCl2, and 0.81 mmol/L MgSO4 (pH 7.4) for Y5] using an Ultra Turrax homogenizer for 30 s. The homogenate was centrifuged at 48,000 x g at 4 C for 10 min. The pellet was resuspended by homogenization in buffer, and the protein concentration was determined by use of the Bio-Rad Laboratories, Inc. protein assay kit (Richmond, CA).

Binding assays using 20 µg protein were conducted in a 0.5-mL (Y1, Y2, and Y4) or 0.25-mL (Y5) assay volume of buffer supplemented with 0.1% BSA, 0.1% bacitracin, and either [125I]Tyr1-pNPY (Y1 and Y2) and [125I]Tyr36-hPP (Y4) at a final assay concentration of 25 pmol/L or di-[125I]Tyr1,36-pPYY (Y5) at a final assay concentration of 50 pmol/L. Nonspecific binding was defined in the presence of 1 µmol/L NPY (Y1, Y2, and Y5) or 100 nmol/L hPP (Y4). Competition binding curves were generated with 10 concentrations of the competing ligands diluted in buffer containing 0.1% BSA. Assays were incubated at 30 C for 90 min (Y1, Y4, and Y5) or 30 min (Y2) and terminated by rapid filtration through Whatman GF/C filters (Clifton, NJ) presoaked in 0.5% polyethyleneimine using a 48-channel Brandel cell harvester. Filters were washed (3 times, 4.5 mL each time) with ice-cold 0.9% saline, and bound radioactivity was determined using a Packard {gamma}-counter. Competition data were transformed to affinity constants using nonlinear regression (Prism 2.01, GraphPad Software, Inc., San Diego, CA).

cAMP assay

Cells expressing human NPY receptors were seeded in 24-well tissue culture multidishes at 200,000 cells/well and grown for 16–20 h as mentioned in the previous section. The medium was removed, and fresh DMEM medium supplemented with 1) 3-isobutyl-1-methylxanthine, 2) forskolin or medium, and 3) medium, NPY, PYY or PP, or compound was added. The plates were incubated for 15–30 min at 37 C, the reaction medium was removed, and the cells were lysed with 0.1 mol/L sodium hydroxide. After neutralization with 0.1 mol/L hydrochloric acid, an aliquot was removed for cAMP determination using an Amersham Pharmacia Biotech scintillation proximity RIA (RPA 538).

Animals

Animal studies were reviewed and approved by the institutional animal care and use committee of the Oregon Regional Primate Research Center. Initially, 10 adult male rhesus monkeys (Macaca mulatta), weighing 10–14 kg, housed in individual cages were enrolled in this study. Animals were exposed to a 12-h light, 12-h dark lighting schedule, with lights on at 0700 h, and the animal room was temperature and humidity controlled. Before the initiation of experiments, monkeys had been adapted for at least 6 weeks to an ad libitum feeding schedule, and only six animals with a stable food intake and reliable feeding behavior (not throwing biscuits all over the place) were used for further experimentation.

Catheterization procedure

Monkeys had a central venous catheter and a third cerebroventricular cannula implanted at least 3 weeks before these experiments. The venous catheter was implanted using standard sterile surgical procedures, as described previously (35). The distal end of the venous catheter was tunnelled to the midscapular region of the back, exteriorized via a small cutaneous incision, and threaded through a flexible metal tether to attach to a swivel (three-port fluid swivel, Alice King Chatham, Inc., Los Angeles, CA) on the top of the monkey’s cage. Monkeys wore jackets to protect the catheters. Catheters were maintained as previously described (35).

Before placement of the third ventricular cannula, monkeys were placed in a Kopf stereotaxic frame (Tujunga, CA), and a radiograph was taken to determine the proper length of the cannula. At the time of surgery, monkeys were anesthetized (with ~20 mg/kg pentobarbital, Nembutal, Abbott Laboratories, North Chicago, IL) and placed in the stereotaxic frame, and a midline scalp incision was made to expose the cranium. The ventricular system was visualized by radiograph after injection of 300 µL of a radioopaque solution (Omnipaque, Winthrop Pharmaceuticals, New York, NY) into the lateral ventricle through a 20-gauge spinal needle inserted through a small hole in the skull. Based on this radiograph, the third ventricular cannula was lowered into position through a second hole in the skull, so that the tip of the cannula was in the lower third of the third ventricle, above the median eminence. Placement of the cannula was confirmed by radiograph subsequent to an injection of 50 µL Omnipaque (Fig. 1Go). The top portion of the cannula was adhered to the skull with dental acrylic, and a catheter of polyethylene tubing (PE 50, Becton Dickinson and Co., Parsippany, NJ) filled with artificial cerebrospinal fluid (aCSF; composed of 0.166 g/L CaCl2, 7.014 g/L NaCl, 0.298 g/L KCl, 0.203 g/L MgCl2·6H2O, and 2.10 g/L NaHCO3; Life Technologies, Inc., Grand Island, NY) was attached to the cannula and tunnelled sc to exit with the venous catheter between the scapulae. The ventricular cannula was passed through the same tether as the venous catheter, threaded through the three-channel fluid swivel, and attached to a miniature swivel mounted above the three-channel swivel. Tubing was extended from the top of the miniature swivel to a digital peristaltic pump (Minipuls 3, Gilson, Middleton, WI), and the catheter was kept patent with a constant infusion of aCSF at a rate of approximately 600 µL/24 h.



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  		Figure 1. X-Ray demonstrating the placement of a third ventricular cannula used for central infusion of NPY and related peptides. After surgery, the ventricular system was visualized by injecting 50 µL Omnipaque immediately before the x-ray radiograph was taken. The contrast-filled third ventricle is clearly seen together with the cerebral aqueduct and the fourth ventricle.

 
The placement of the cannula and the dead space of the third ventricular infusion system were verified by testing the capacity of a small bolus of morphine to increase plasma PRL. Twenty microliters of morphine sulfate (1.5 µg/µL) were loaded into the infusion system and delivered into the third ventricle in a series of pulses (60 µL every 30 min in a 5-min pulse). Blood samples were taken every 10 min in a 2-h time window around the expected arrival of the morphine bolus. Plasma PRL levels increased from approximately 0.5 ng/mL before arrival of the morphine bolus to more than 100 ng/mL after arrival of morphine into the third ventricle.

Diets and ad libitum feeding assay

Monkeys were fed twice daily by offering 20 biscuits of Purina high protein monkey diet, jumbo size (no. 5047, Ralston Purina Co., St. Louis, MO), per meal. Each meal equals approximately 1100 Cal. The morning meal was given between 0830–0900 h, and the afternoon meal was provided between 1500–1600 h. The morning meal was supplemented with fresh fruit. Before offering new biscuits, all biscuits from the previous feeding session were counted and removed. On the day of experimentation, cages were emptied of food at 1530 h (t-30). Twenty biscuits were offered at 1600 h (t0), and food consumption was evaluated every 30 min by counting biscuits. At 1800 h (t240), remaining biscuits were taken out of the cage, counted and weighed, and finally put back into the cage. The following morning, food consumption was evaluated once again at 0800 h (t960) by emptying the cages and weighing and counting the biscuits. To determine whether the introduced ad libitum feeding assay was able to detect increased food intake in response to a known orexigenic stimulus, the effect of an im injection of clonidine (0.1 mg/kg; Sigma Chemical Co.) on food intake was initially tested. The {alpha}2-agonist clonidine, which has been shown to stimulate food intake in primates (36), was injected at t-30, and food intake monitored as described.

Blood-sampling procedures

When needed, blood samples were collected via indwelling venous catheters into sterile heparinized syringes. Samples for measurement of hormones were transferred into sterile glass tubes (5 mL/sample) and kept cold until centrifugation, whereas blood glucose was measured by transferring a single drop of blood to a glucose test strip (Roche Molecular Biochemicals, Indianapolis, IN). All samples were centrifuged at 2400 rpm for 10 min, and the plasma was subsequently transferred into Eppendorf vials containing 20 µL heparin-citrate solution (50:50 solution of 1000 U/mL sodium heparin and 38% sodium citrate) to prevent clotting of plasma proteins and stored at -20°C. The erythrocytes from this sample were resuspended in sterile saline and reinfused through the indwelling venous catheters. The animals were regularly monitored for potential anemia, and the reinfusion methodology ensured that none of the animals used for experimentation displayed hematocrit values lower than 0.4.

NPY infusions

In these experiments, vehicle or varying doses of NPY (1, 5, 20, and 50 µg in aCSF and 1.0% BSA) were pulse injected into the third ventricle. The intracerebroventricular (icv) infusion system was loaded with 20 µL NPY and 40 µL vehicle separated by a small diaphragm of air, which corresponded to the first 60-µL pulse. Subsequently, a series of 5-min 60-µL pulses was given every 30 min until arrival of the NPY bolus to the third ventricle. The arrival of NPY into the third ventricle was timed to coincide with the onset of the afternoon feeding session (t0). Food intake was monitored as described, and blood samples were taken at t-30, t0, t10, t20, t30, t40, t50, t60, t70, t80, t90, t100, t110, t120, t150, t180, and t240.

Antagonist experiments

The effect of concomitant administration of a modified peptide NPY antagonist, Y1ANT, on food intake was studied by loading the icv infusion system with 50 µg antagonist (dissolved in 20 µL aCSF and 1% BSA) and 40 µL vehicle separated by a small diaphragm 30 min before introduction of a 5-µg NPY bolus into the infusion system. Both the Y1ANT bolus and the following NPY bolus were delivered into the third ventricle using the same pulse infusion pattern as that used for NPY alone, ensuring that Y1ANT arrived at the third ventricle 30 min before NPY. Blood samples were collected as described for the NPY infusion experiments.

The effect of Y1ANT on food intake in food-deprived animals was examined in monkeys that had been deprived of their morning meal. No blood samples were taken in this series of experiments. Two doses of Y1ANT (100 and 200 µg in 20 µL) were loaded into the third ventricular infusion system and pulse infused into the third ventricle as described for the NPY infusions. The arrival of Y1ANT to the third ventricle was timed to coincide with presentation of the afternoon meal at 1600 h. The same paradigm was also used to test the effect of Y1ANT on food intake in freely fed (satiated monkeys). Again, no blood samples were taken from these animals.

Assays

Blood glucose was measured simultaneously with sampling using a standard blood glucose monitor based on a colorimetric glucose oxidation technique (Roche Molecular Biochemicals). Plasma cortisol and insulin levels were measured at the core facility Biochemistry Laboratory at the Oregon Regional Primate Center using a standard RIA kit (Diagnostic Products, San Diego, CA).

Data analyses

In each experiment, food intake, plasma substrate, and hormone responses of the monkeys were compared at each time point using paired Student’s t test, and differences between treatments were evaluated using ANOVA followed by Fisher’s post-hoc analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The ability of the ad libitum feeding assay to detect increased appetite and food intake was tested with the orexigenic agent clonidine, which has previously been shown to stimulate food intake in primates (36). As shown in Fig. 2Go, clonidine significantly increased food intake both 120 min (mean ± SEM, 125 ± 11 vs. 81 ± 10 g; n = 6) and 960 min (102 ± 10 vs. 160 ± 14 g; n = 6) after administration.



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  		Figure 2. Effect of im injection of the {alpha}2-agonist clonidine (0.1 mg/kg) on food intake (mean ± SEM; n = 6) in male rhesus monkeys. Food intake was assessed by counting the number of biscuits eaten 120 and 960 min after initiation of the feeding session. *, P < 0.05, as determined by ANOVA followed by Fisher’s post-hoc analysis.

 
The effect of centrally administered NPY was followed across time (Figs. 3Go and 4Go). The highest doses of NPY (20 and 50 µg) significantly increased food intake at all time points, whereas 1- and 5-µg doses of NPY first produced significantly higher food intake after 60 min. After 120 min, maximal food intake was obtained using a dose of 50 µg NPY (number of biscuits ± SEM, 11.2 ± 1.1 vs. 5.5 ± 0.6). To minimize the disturbance of animals, which could cause interference with the feeding assay, the weight of consumed food was evaluated only at two time points (t120 and t960). As shown in Fig. 3Go, all of the employed doses of NPY significantly increased food intake after 120 and 960 min. Also, it is evident that the majority of food intake during an overnight feeding session took place during the initial 120 min.



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  		Figure 3. Effects of increasing doses of centrally administered NPY on food intake (mean ± SEM; n = 6 in all groups) in male rhesus monkeys. Food intake was assessed by counting the number of biscuits eaten 30, 60, 90, and 120 min after initiation of the feeding session. Animals were fed 20 biscuits at 1600 h, which coincided with the arrival of the NPY bolus to the third ventricle. Statistical analysis: *, P < 0.05 vs. vehicle; a, P < 0.05 vs. 1 µg NPY (as determined by ANOVA followed by Fisher’s post-hoc analysis).

 


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  		Figure 4. Effects of increasing doses of centrally administered NPY on cumulated food intake (mean ± SEM; n = 6 in all groups) in male rhesus monkeys. Food intake was calculated by subtracting the weight of the remaining biscuits at t120 and t960 from that of the biscuits initially offered. Statistical analysis: *, P < 0.05 vs. vehicle (as determined by ANOVA followed by Fisher’s post-hoc analysis).

 
Antagonist experiments using Y1ANT

To evaluate the involvement of the receptor subtype(s) involved in mediating the effect of central NPY on feeding behavior, we made use of a novel modified peptide NPY antagonist, Y1ANT. This peptide antagonist shares similarities with a previously characterized Y-1 antagonist, BW1229U91 (37). Competition binding experiments demonstrated a concentration-related displacement of specifically bound radioligand to membrane receptors prepared from cells individually expressing the human Y1, Y2, Y4, and Y5 receptors (Fig. 5Go, A–D). These studies demonstrate high affinities for Y1 and Y4 receptors (mean ± SEM pKi, 10.12 ± 0.06 and 9.11 ± 0.05, respectively; n = 3), whereas the affinities for Y2 and Y5 were lower (pKi, 7.04 ± 0.05 and 6.83 ± 0.08, respectively; n = 6). The affinity of Y1ANT for Y1 was not significantly different (P > 0.2, by unpaired t test) from the affinity of BW1229U91 (pKi, 10.25 ± 0.06; n = 3), whereas the affinities of Y1ANT for Y2, Y4, and Y5 were significantly lower (P < 0.05, by unpaired t test) than those of BW1229U91 [pKi, 7.65 ± 0.08 (n = 4), 9.71 ± 0.07 (n = 3), and 7.14 ± 0.05 (n = 4), respectively]. It has previously been reported that BW1229U91 is a potent antagonist of NPY on Y1 receptors, as evidenced by the inhibition of NPY-induced increase in intracellular Ca2+ in CHO cells expressing human Y1 receptors (38). On the contrary, BW1229U91 showed NPY agonistic effects on the other NPY receptor subtypes, Y2, Y4, and Y5. Similarly, we observed antagonistic effects of Y1ANT on human Y1 receptors expressed in BHK cells, abolishing NPY inhibition of forskolin-induced cAMP accumulation, and agonistic properties on the other receptor subtypes, Y2, Y4, and Y5 (data not shown).



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  		Figure 5. Displacement by NNC 58–0036 (•) and BW1229U91 ({circ}) of [125I]Tyr1-pNPY from human Y1 (A) and human Y2 receptors (B) and of [125I]Tyr36-hPP from human Y4 receptors (C), all expressed in BHK cells, and of di-[125I]Tyr1,36-pPYY from human Y5 receptors (D) expressed in HEK 293 cells. Binding assays were performed as described in Materials and Methods.

 
Feeding induced by central administration of NPY (5 µg) was completely abolished by prior administration of Y1ANT (50 µg; Fig. 6Go). No effect on feeding was seen after administration of Y1ANT alone (Fig. 9Go). To assess the role of endogenous NPY as a mediator of feeding, the effect of centrally administered Y1ANT on food intake in an overnight feeding session was studied in monkeys that had been deprived of their morning meal. No blood samples were collected in these experiments. Monkeys that had been without food for 8 h ate considerably more over the first 120-min observation period than animals given their morning meal (mean ± SEM, 195 ± 15 vs. 86 ± 5 g; n = 6). The resulting overnight food intake was more than twice the normal level in food-deprived animals (t960, 253 ± 14 vs. 104 ± 5 g; n = 6). When examined across time, food intake in food-deprived animals was significantly lower for the first 60 min in animals given 200 µg Y1ANT (Fig. 7Go). However, when examined at t120 and t960, food intake was significantly lower only at t960 and only for the group that had been given 100 µg Y1ANT (Fig. 8Go). Finally, the effect of centrally administered Y1ANT (25 and 100 µg) on food intake in satiated monkeys was examined and compared to the effect of central administration of 20 µg NPY. When given alone, neither of the Y1ANT doses affected food intake in satiated monkeys, whereas 20 µg NPY had a profound effect on feeding in monkeys not being blood sampled (Fig. 9Go).



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  		Figure 6. Effect of central administration of Y1ANT (50 µg) 30 min before the induction of feeding by central administration of NPY (5 µg) in male rhesus monkeys compared to the feeding response after either central NPY or vehicle (mean ± SEM; n = 6). Food intake was assessed by counting the number of biscuits eaten 30, 60, 90, and 120 min after initiation of the feeding session. Animals were fed 20 biscuits at 1600 h, which coincided with the arrival of the NPY bolus to the third ventricle (t0), and Y1ANT arrived at the third ventricle 30 min before NPY. Blood samples were collected from all animals as described in Materials and Methods, beginning at t-30 and finishing at t240. Statistical analysis: *, P < 0.05 vs. vehicle (as determined by ANOVA followed by Fisher’s post-hoc analysis).

 


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  		Figure 9. Effect of Y1ANT on food intake in satiated male rhesus monkeys (mean ± SEM; n = 6) compared to that obtained after infusion of either vehicle or NPY (20 µg). Animals were fed 20 biscuits at 1600 h, which coincided with the bolus arrival of either NPY (20 µg) or Y1ANT (25 and 100 µg) to the third ventricle. Food intake was calculated by subtracting the weight of the remaining biscuits at t120 and t960 from that of the biscuits initially offered. No blood samples were collected from these animals. Statistical analysis: a, P < 0.05 vs. vehicle; b, P < 0.05 vs. Y1ANT (25 µg); c, P < 0.05 vs. Y1ANT (100 µg; as determined by ANOVA followed by Fisher’s post-hoc analysis).

 


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  		Figure 7. Effect of central administration of Y1ANT (100 and 200 µg) on feeding behavior induced by food deprivation (skipping the morning meal) in male rhesus monkeys compared to food intake in vehicle-treated animals (mean ± SEM; n = 6). Animals were deprived of their morning meal and fed 20 biscuits at 1600 h, which coincided with the arrival of the Y1ANT bolus to the third ventricle. Two doses of Y1ANT were given (100 and 200 µg). Food intake was assessed by counting the number of biscuits eaten 30, 60, 90, and 120 minutes after initiation of the feeding session. No blood samples were taken. Statistical analysis: *, P < 0.05 vs. vehicle (as determined by ANOVA followed by Fisher’s post-hoc analysis).

 


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  		Figure 8. Effect of central administration of Y1ANT (100 or 200 µg) on cumulated food intake induced by food deprivation (skipping the morning meal) in male rhesus monkeys compared to food intake in vehicle-treated animals (mean ± SEM; n = 6). Food intake was calculated by subtracting the weight of the remaining biscuits at t120 and t960 from that of the biscuits initially offered. Animals were fed 20 biscuits at 1600 h, which coincided with the arrival of the Y1ANT bolus to the third ventricle. No blood samples were collected from these animals. Statistical analysis: *, P < 0.05 vs. vehicle (as determined by ANOVA followed by Fisher’s post-hoc analysis).

 
The effect of NPY on blood glucose was examined across time. Neither of the NPY doses affected blood glucose, which remained constant around 65 mg/dL (Fig. 10Go). The effect of centrally administered NPY on circulating insulin and cortisol was evaluated for vehicle and 20- and 50-µg NPY doses. Central infusion of both 20 and 50 µg NPY markedly elevated plasma insulin levels from 40 min postinjection on (Fig. 11Go). However, low sample numbers and profound variance in the measured hormonal levels rendered this difference nonsignificant at most time points with group statistical analysis. Only at 40 min after icv infusion did 50 µg NPY significantly elevate plasma insulin levels (mean ± SEM, 50.0 ± 15.7 vs. 135.3 ± 21.7 pmol/L). Plasma cortisol levels also rose after icv infusion of NPY into the third ventricle. Levels steadily increased from 20–30 min after the bolus was timed to arrive in the CSF and remained elevated throughout the observation period. However, as with insulin values, this increase did not reach statistical significance at most observation time points (Fig. 11Go). Thus, icv administration of 20 µg NPY significantly elevated plasma cortisol levels 50 and 60 min after arrival of the peptide to the third ventricle (t50, 90.8 ± 12.1 vs. 244.7 ± 53.9 ng/mL; t60, 89.2 ± 15.8 vs. 236.5 ± 55.6 pg/mL).



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  		Figure 10. Blood glucose levels before and after central administration of aCSF (vehicle) or NPY (20 µg) to male rhesus monkeys (mean ± SEM; n = 6). Food was removed from the cages at t-30, and animals were fed 20 biscuits at 1600 h, which coincided with the arrival of the NPY bolus to the third ventricle. Neither this nor any other dose of centrally applied NPY (1, 5, or 50 µg) induced fluctuations in blood glucose.

 


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  		Figure 11. Plasma insulin levels before and after central administration of aCSF (vehicle) or NPY (20 or 50 µg) to male rhesus monkeys (mean ± SEM; n = 4–6; upper panel). Food was removed from the cages at t-30, and animals were fed 20 biscuits at 1600 h, which coincided with the arrival of the NPY bolus to the third ventricle. *, Statistical significance vs. the vehicle-treated group (P < 0.05), as determined by ANOVA followed by Fisher’s post-hoc analysis. Plasma cortisol levels were determined before and after central administration of aCSF (vehicle) or NPY (20 or 50 µg) to male rhesus monkeys (mean ± SEM; n = 4–6; lower panel). Food was removed from the cages at t-30, and animals were fed 20 biscuits at 1600 h, which coincided with the arrival of the NPY bolus to the third ventricle. *, Statistical significance vs. vehicle-treated group (P < 0.05), as determined by ANOVA followed by Fisher’s post-hoc analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major aim of the present series of experiments was to determine a possible role for NPY as an activator of feeding in nonhuman primates. To accomplish this, we have developed an ad libitum feeding assay for tethered monkeys. We have previously used this assay successfully in the study of anorectic compounds (e.g. leptin) (39). However, the sensitivity of the assay to reliably monitor an orexigenic response was tested with a clonidine challenge because previous experiments have shown a potent feeding response after systemic administration of {alpha}2-adrenergic agonists to nonhuman primates (36). Some feeding assays used to assess primate feeding behavior employ liquid food and the possible involvement of a psychological reward mechanism inevitably connected to the use of feeding automats, whereas other experimental set-ups make use of restrained animals (40, 41). By presenting solid neutral food items in excess at each meal, we have reduced possible interference with the pure feeding response from such diverse factors as drinking, positive reward, and possible stress from being restrained.

The data presented clearly demonstrate that central infusion of NPY into the third ventricle potently stimulates food intake in nonhuman primates. It is surprising that doses as low as 1 µg NPY significantly increased food intake to levels about 50% higher than those in vehicle-infused animals, because most studies performed in rodents have shown maximal feeding responses to NPY injected into lateral ventricle at doses of about 10 µg. The volume of the cerebral ventricles in the rhesus macaque has previously been assessed to be about 1/10th of that in the human (42), suggesting that the infused NPY would be dissolved in maximally 7.5 mL CSF. However, there is reason to believe that by choosing the third ventricle as the site of injection, the concentration of NPY reaching potential hypothalamic targets is higher than or similar to those obtained by infusion into the lateral ventricles of other species (e.g. rodents) with more extensive communication between the lateral and third ventricles. In a previous study, the effect of exogenous NPY on feeding behavior in another nonhuman primate, the male baboon, was investigated by infusing NPY into the lateral ventricle (31). In contrast to our data, no effect of NPY on feeding was observed after lateral ventricular application of the peptide, and the different sites of injection may account for the discrepant results. The lateral ventricle is situated in close apposition to both basal ganglia and central cortical structures, including the hippocampus. Therefore, it is possible that NPY infusions directed into the lateral ventricle may have triggered complex behaviors that might counteract its tendency to stimulate feeding behavior.

The feeding response elicited by central NPY infusion was not accompanied by increased blood glucose levels, suggesting that compensatory mechanisms are fully capable of maintaining constant glucose levels. Presumably, the high fiber content of monkey pellets gives rise to relatively slow gastrointestinal absorption rates with resulting moderate incretin release. Thus, plasma glucose values accompanying increased food intake are unaffected due to increased insulin secretion, but it is not possible with the current experimental design to assess whether the tendency toward increased insulin secretion is directly affected by central NPY or is simply caused by larger food intake per se. In rats, central administration of NPY causes sustained and dose-dependent insulin secretion regardless of feeding status (6, 43), suggesting that at least part of the insulin surge was caused by the NPY infusion. Yet another neuroendocrine effect of central NPY is a direct stimulatory action on hypothalamic CRH neurons orchestrating the activity of the hypothalamo-pituitary-adrenocortical axis (44). The observed activation of the hypothalamo-pituitary-adrenocortical axis gives further evidence that icv infused NPY actually reaches target neurons within the paraventricular nucleus (PVN).

Chronic NPY administration to rodents induces an overall shift of the autonomic balance of energy homeostasis toward a parasympathetic dominance, resulting in increased energy intake, reduced energy expenditure, and increased insulin secretion resulting in obesity (15). The hypothalamic target responsible for mediating the orexigenic effect of NPY is still subject to debate, but site-directed microinjections of NPY into various hypothalamic nuclei suggest that the perifornical area and the PVN constitute the nuclei most sensitive to NPY (4, 9). Starvation increases and refeeding reduces local NPY release in the PVN (45). Possible sources of endogenous NPY in the PVN are afferents originating in the arcuate nucleus, brain stem catecholaminergic areas, and hypothalamic interneurons (46, 47). However, it is the NPY-containing arcuate-paraventricular pathway that has drawn the most attention as the neuronal pathway responsible for regulating increased appetite for food intake (for review, see Refs. 1, 48, 49, 50, 51). Food deprivation potently stimulates NPY synthesis in arcuate neurons (19, 52, 53, 54, 55), and most rodent models of metabolic dysfunctions characterized by increased food intake and obesity also display grossly elevated NPY messenger ribonucleic acid levels in arcuate neurons (10, 11, 12, 56, 57). It is not fully understood which NPY-sensitive neuronal pathways emerging from the PVN are responsible for the diverse behavioral and metabolic changes seen in response to central NPY administration. However, the increased insulin secretion and lowered energy consumption suggest that descending pathways to autonomic nuclei of the brain stem and intermediolateral cell column of the spinal cord regulating the pancreas and adipose tissue are involved. The circuitry responsible for food-seeking behavior is still very speculative, but given the fact that the PVN send very few projections to cortical structures, multisynaptic pathway(s) involving other limbic areas seem the most likely explanation.

Although most studies concerned with the verification of NPY as an endogenous regulator of feeding and energy homeostasis have been carried out in rodents, it is important to emphasize that the hypothalamus of both humans and nonhuman primates is densely innervated by NPY-immunoreactive nerve fibers (58, 59, 60). Thus, both the PVN and the tuberoinfundibular area, which corresponds to the rodent arcuate nucleus, are densely targeted by NPY-containing nerve terminals. In situ hybridization histochemistry has demonstrated high numbers of NPY messenger ribonucleic acid-containing neurons in the tuberoinfundibular area of female rhesus macaques, and the level of expression is rapidly elevated in response to short term food deprivation (30).

At present, six different NPY receptors have been characterized in mammals, but it is still uncertain which of these, if any, mediate the orexigenic effect of NPY (61). The announcement of the recently cloned Y5 receptor subtype as an alleged feeding receptor (23) raised brief hope that understanding of feeding physiology was within our grasp, but it has become increasingly clear that explaining NPY-induced feeding behavior in narrow receptor phrenological terms has not proven fruitful. Both Y1 and Y5 receptors are expressed in hypothalamic nuclei centrally involved in feeding behavior (23, 24), and treatment with Y5 antisense oligodeoxyribonucleotides induces long lasting anorexia (25, 26). With the advent of Y1 and Y5 receptor knockout mice, the involvement of either of these receptors on feeding behavior has not become any clearer (27, 28). It is uncertain why some receptor knockout constructs become phenotypically altered and others do not, but it seems evident that control of feeding behavior and body weight homeostasis are highly redundant features. However, fasting-induced feeding is severely affected in Y1-deficient mice, and maximal NPY-induced food intake is much lower in Y5-deficient mice, supporting the broadly held view that both Y1 and Y5 receptors are involved in NPY-mediated food intake. Hypothalamic NPY synthesis and release are inversely correlated to circulating levels of leptin (18), but cross-breeds of Y5-deficient mice with leptin-deficient ob/ob mice displayed the characteristic ob/ob phenotype, suggesting that hypothalamic NPY in the ob/ob mouse mediates its actions via other NPY receptors (62). If the predominant stimulus to eat during states of energy deficiency (starvation) is exerted by NPY, this is either mediated via combined effects on both Y1 and Y5 receptors or via interaction with a nonidentified receptor. Surprisingly, both Y1- and Y5-deficient mice grow larger fat deposits than their wild-type companions, but different mechanisms may be responsible for this effect, because Y1 knockout mice have a lower metabolic rate during activity and consequently increase their fat deposits, whereas Y5 knockout mice appear hyperphagic.

Very few of the recently developed and publicly available selective NPY receptor antagonists lend themselves to pharmacological in vivo experiments, because of poor bioavailability and toxic side-effects. Using truncated peptide agonists it has become evident that both Y1- and Y5-preferring ligands are capable of stimulating food intake, whereas most Y1 antagonist inhibit food intake (22). A recent report has shown that ip administration of a small nonpeptide molecule with Y5 antagonistic properties inhibits food intake induced by either icv NPY or energy deprivation (63). However, this report contains no information about pharmacokinetics and bioavailability, leaving it impossible to assess the anatomical site of this pharmacological target (central nervous system or peripheral). In the rat, the peptidergic Y1 antagonist BW1229U91 has been shown to inhibit feeding behavior induced by food deprivation as well as by NPY (37, 64). However, this high affinity Y1 receptor antagonist also displays partial agonism on Y4 and Y5 receptors, making it less suited for pharmacological experiments (38). We therefore tried to synthesize a more specific Y1 antagonist by substituting the Dpr moiety in BW1229U91 with diaminobutyric acid. We found that Y1ANT is a highly potent antagonist of NPY on human Y1 receptors, and in our hands, it is even more selective for Y1 than BW1229U91 compared to Y2, Y4, and Y5 binding. As for BW1229U91, Y1ANT is a weak agonist on Y4 and Y5 receptors. In agreement with this observation, we have shown that Y1ANT inhibits feeding in monkeys induced by central administration of NPY, and this effect is probably mediated via Y1 receptors. In the rat, however, classical Y1 agonists, such as [Pro34]NPY, have much lower efficacy on feeding behavior than either NPY or the Y5 agonist [Pro34]NPY-(3–36), which has inspired the hypothesis that a novel feeding receptor may exist in the rat (22). Given the constraints of in vivo agonist physiology, it is still possible that more than one receptor subtype may participate in mediating NPY-induced feeding. Both doses of Y1ANT exerted short term effects on feeding in response to food deprivation (skipping of the morning meal), confirming that endogenous NPY mediates the hunger-induced feeding response in nonhuman primates. In satiated monkeys, Y1ANT had no effect on feeding when administered alone, suggesting that well fed monkeys have virtually no release of endogenous NPY acting on receptors responsible for mediating the orexigenic effect of NPY.

Obesity, defined as increased body fat content, is a growing problem in Western societies, and it is estimated that more than 30% of the population of the United States is obese, as defined by a body mass index exceeding 27 kg/m2 (65). The association of increased body mass index with hypertension, ischemic heart disease, hyperlipidemia, and most certainly noninsulin-dependent diabetes makes pharmacological agents counteracting severe obesity high in demand, and it has been speculated that NPY antagonists could subserve such a role. In both humans and nonhuman primates, leptin is synthesized and released from adipose tissue and conveyed via the systemic circulation to act upon target neurons in the central nervous system. It is well known that circulating leptin levels constitute a reliable reflection of body adipose tissue mass, with increased concentrations during weight gain and decreased levels during weight loss (66, 67), but the constantly elevated plasma levels render obese subjects less sensitive to leptin and make leptin a poor candidate as a pharmacological tool to reduce body weight. Profound evidence exists to suggest that leptin partly mediates its effect on appetite both via inhibition of NPY-containing neurons of the arcuate nucleus and via postsynaptic abolition of NPY-stimulated food intake (18, 68), presenting the possibility that NPY antagonist may constitute pharmacological useful tools in the treatment of obesity. Counteracting the central actions of NPY on feeding and energy metabolism will not only reduce food intake, but will also lead to lowered fat deposition and increased resting metabolic rate, ultimately lowering the tendency toward hyperinsulinemia, insulin resistance, glucose intolerance, dyslipidemia, and hypertension that otherwise prevails in obese patients.

Conclusion

We have presently developed an ad libitum feeding assay capable of monitoring physiologically relevant feeding behavior in nonhuman primates. Using this assay, we have demonstrated that NPY is a powerful orexigenic agent in male rhesus monkeys as in other mammals. Using the putative Y1 antagonist Y1ANT, we were able to completely block the feeding response induced by central NPY infusions, suggesting that Y1-like receptors mediate the orexigenic effect of exogenous NPY. Furthermore, feeding induced by food deprivation was partially inhibited by Y1ANT, suggesting that endogenous NPY may constitute a physiologically relevant regulator of feeding behavior. However, the participation of other receptor subtypes in mediating NPY-induced feeding behavior during states characterized by negative energy balance cannot be ruled out.


   Acknowledgments
 
The technical assistance of Weimin Zhang, Stephanie Kauk, the surgical staff, and the Laboratory Animal Medicine Staff at the Oregon Regional Primate Research Center is greatly appreciated.


   Footnotes
 
1 This work was supported by Novo Nordisk A/S (Malov, Denmark), Karl Thomae AG (Biberach, Germany), and supporting grants from the Danish Diabetes Association, the Novo Nordisk Foundation, the Foundation for the Advancement of Medical Science, the Danish Medical Association, the Danish Medical Research Council (J12–1642), the NIH (HD-26888, DK-50129, and RR-00169). Back

2 Supported by a Research Scientist Development Award from the NIH. Back

Received February 3, 1999.

Revised April 28, 1999.

Accepted May 4, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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