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Peptide YY_3–36 and Glucagon-Like Peptide-1_7–36 Inhibit Food Intake Additively

Department of Metabolic Medicine, Hammersmith Hospital, Imperial College London, London W12 ONN, United Kingdom

Address all correspondence and requests for reprints to: Professor S. R. Bloom, Department of Metabolic Medicine, Hammersmith Hospital, Imperial College London, 6th Floor, Commonwealth Building, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom{at}imperial.ac.uk.

	Abstract

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Peptide YY (PYY) and glucagon like peptide (GLP)-1 are cosecreted from intestinal L cells, and plasma levels of both hormones rise after a meal. Peripheral administration of PYY_3–36 and GLP-1_7–36 inhibit food intake when administered alone. However, their combined effects on appetite are unknown. We studied the effects of peripheral coadministration of PYY_3–36 with GLP-1_7–36 in rodents and man. Whereas high-dose PYY_3–36 (100 nmol/kg) and high-dose GLP-1_7–36 (100 nmol/kg) inhibited feeding individually, their combination led to significantly greater feeding inhibition. Additive inhibition of feeding was also observed in the genetic obese models, ob/ob and db/db mice. At low doses of PYY_3–36 (1 nmol/kg) and GLP-1_7–36 (10 nmol/kg), which alone had no effect on food intake, coadministration led to significant reduction in food intake. To investigate potential mechanisms, c-fos immunoreactivity was quantified in the hypothalamus and brain stem. In the hypothalamic arcuate nucleus, no changes were observed after low-dose PYY_3–36 or GLP-1_7–36 individually, but there were significantly more fos-positive neurons after coadministration. In contrast, there was no evidence of additive fos-stimulation in the brain stem. Finally, we coadministered PYY_3–36 and GLP-1_7–36 in man. Ten lean fasted volunteers received 120-min infusions of saline, GLP-1_7–36 (0.4 pmol/kg·min), PYY_3–36 (0.4 pmol/kg·min), and PYY_3–36 (0.4 pmol/kg·min) + GLP-1_7–36 (0.4 pmol/kg·min) on four separate days. Energy intake from a buffet meal after combined PYY_3–36 + GLP-1_7–36 treatment was reduced by 27% and was significantly lower than that after either treatment alone. Thus, PYY_3–36 and GLP-1_7–36, cosecreted after a meal, may inhibit food intake additively.

	Introduction

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THE GUT HORMONES peptide YY (PYY) and glucagon-like peptide (GLP)-1 are cosecreted from endocrine L cells of the small and large bowel. Endogenous concentrations of both hormones are low in the fasting state and rise within 30 min of a meal (1). However, whereas these hormones have been found to inhibit food intake individually (2, 3), their combined effect has yet to be investigated.

It has been reported that peripheral administration of PYY_3–36 inhibits appetite in rodents (4, 5) and man (2). Peripheral administration of GLP-1_7–36 inhibits food intake in rodents (6, 7) and man (8). GLP-1_7–36 and the GLP-1 receptor agonist exendin-4 also stimulate insulin release and inhibit circulating glucagon (6). In contrast, no changes in insulin and glucagon concentrations occur after PYY_3–36 administration (9).

PYY and GLP-1 may mediate these peripheral effects by altering central nervous system appetite circuits. After ip administration of PYY_3–36, an increase in c-fos, a marker of neuronal activation, has been reported in the hypothalamic arcuate nucleus (ARC) (2). In contrast, GLP-1 is reported to act primarily on brain stem neurons. After iv exendin-4, c-fos activation was observed in the area postrema (AP) (10), which, like the ARC, has access to the peripheral circulation. c-fos immunoreactivity was also detected in the ARC and paraventricular nucleus (PVN). This hypothalamic activation could be mediated via neuronal projections from the brain stem to the PVN (11) or directly onto ARC neurons.

Both PYY_3–36 and GLP-1_7–36 have been administered to humans. Intravenous infusion of PYY_3–36 was associated with a 36% reduction in energy intake at a free buffet meal (2). PYY_3–36 has also been reported to inhibit food intake in obese subjects (12). A recent metaanalysis reported that infusion of GLP-1_7–36 was associated with a small dose-dependent reduction in energy intake in both lean and obese subjects (8). Interestingly, when PYY and GLP-1 were coadministered to man, an additive inhibitory effect on pentagastrin-induced gastric acid secretion was observed (13), although their combined effect on appetite was not examined.

Much work has been performed investigating the effect of exogenous administration of individual gut hormones on appetite to determine their potential physiological role in the control of food intake (4, 14, 15). However, after a meal not one but several gut hormones are elevated, and it may be that the interactions between the changes in levels of different hormones influence food intake more than each hormone individually.

The aim of our study was to investigate whether the combined administration of PYY_3–36 and GLP-1_7–36 was more effective in inhibiting food intake than either peptide alone. First, we examined the effects of coadministration of PYY_3–36 and GLP-1_7–36 in lean and obese rodents. Second, to investigate potential mechanisms, we quantified fos-like immunoreactivity (FLI) in the hypothalamus and brain stem after ip PYY_3–36 and GLP-1_7–36 alone and PYY_3–36 + GLP-1_7–36 coadministration. Finally, we performed a randomized, double-blind, placebo-controlled, crossover trial in healthy volunteers to compare the effects of PYY_3–36, GLP-1_7–36, and PYY_3–36 with GLP-1_7–36 on energy intake in man.

	Materials and Methods

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Materials
Synthetic human PYY_3–36 and GLP-1_7–36 were purchased from Phoenix Pharmaceuticals (Belmont, CA). Unless otherwise stated, all chemicals were purchased from Merck (Poole, Dorset, UK).

Animals
All animal procedures were approved by the British Home Office Animals (Scientific Procedures) Act 1986 (project license no. 70/5516 and 70/5281). Male mice and rats were maintained in individual cages under controlled temperature (21–23 C) and light (12-h light, 12-h dark cycle; lights on at 0700 h) with ad libitum access to standard chow (RM1 diet, SDS Ltd., Witham, Essex, UK) and water. All animals were handled daily for 7 d before the first study, particularly in the light of recent evidence that peripheral PYY_3–36 does not inhibit food intake in stressed animals unaccustomed to their experimental conditions but does so in well-handled, acclimatized animals (5). During the acclimatization period, each animal received two saline injections to minimize stress on the study days.

Intraperitoneal injections
Intraperitoneal injections were administered to mice via a 0.5 ml syringe with a 29-gauge needle (maximum injection volume, 0.1 ml) and rats via a 1 ml syringe with a 25-gauge needle (maximum volume, 0.5 ml). In each study animals received one injection only of equivalent volume for each treatment group. For the combination treatment, GLP-1_7–36 and PYY_3–36 were drawn up in a single syringe. In all feeding studies, food intake was measured 1, 2, 4, 8, and 24 h after injection as previously described (7).

Feeding studies in lean mice
C57BL/6 mice (20–25 g) were injected after a 20-h fast. To determine suitable doses for the combined study, dose-response studies were performed for PYY_3–36 and GLP-1_7–36 individually. For the PYY_3–36 study, mice were ip injected with 30, 100, 150, and 300 nmol/kg PYY_3–36 (n = 11–12). For the GLP-1_7–36 study, mice were injected with 30, 100, 300, and 900 nmol/kg GLP-1_7–36 (n = 10). For the combined study, mice were injected with saline, 100 nmol/kg PYY_3–36, 200 nmol/kg PYY_3–36, 100 nmol/kg GLP-1_7–36, 200 nmol/kg GLP-1_7–36, or 100 nmol/kg PYY_3–36 + 100 nmol/kg GLP-1_7–36 (n = 8–9).

Feeding studies in ob/ob and db/db mice
To investigate these effects in obese rodents, PYY_3–36 + and GLP-1_7–36 were coadministered to ob/ob and db/db mice (both on a C57BL/6 background) in a four-way crossover design. After a 20-h fast, ob/ob mice (mean weight 54.5 ± 1.4 g) and their wild-type (WT) littermates (32.5 ± 1.0 g) and db/db mice (41.8 ± 1.12 g) and their WT littermates (31.0 ± 0.5 g) were ip injected with saline, 200 nmol/kg PYY_3–36, 200 nmol/kg GLP-1_7–36, or 100 nmol/kg PYY_3–36 + 100 nmol/kg GLP-1_7–36. Four studies were performed with 72 h between each study so that each mouse received all four treatments (n = 10 per treatment group).

Feeding studies in rats
To examine the effect of PYY_3–36 and GLP-1_7–36 in another species and during another feeding phase, these peptides were coadministered in rats in the dark phase. Rodents are nocturnal feeders and eat the majority of their food during the night (16). Freely feeding rats (150–200 g) were injected in the hour before lights off. For the PYY_3–36 dose-response study, animals received ip saline or 1, 3, or 10 nmol/kg PYY_3–36 (n = 12–13). For GLP-1_7–36, rats received saline or 3, 10, 30, or 100 nmol/kg GLP-1_7–36 (n = 12–13). To investigate the combined effect of low doses, 1 nmol/kg PYY_3–36 and 10 nmol/kg GLP-1_7–36 were chosen for the joint study (see Results). On two evenings, 72 h apart, animals were randomized and injected ip with saline, 10 nmol/kg GLP-1_7–36, 20 nmol/kg GLP-1_7–36, 1 nmol/kg PYY_3–36, 2 nmol/kg PYY_3–36, or 1 nmol/kg PYY_3–36 + 10 nmol/kg GLP-1_7–36 (total n = 19–20 per group) before the dark phase.

Immunocytochemistry
The protocols for c-fos staining and quantification were similar to those described previously (17). Briefly, rats (240–265 g) were each handled for 5 min/d for 1 wk to minimize the stress of the procedure. After an overnight fast, rats received ip injections of saline, 10 nmol/kg GLP-1_7–36, 1 nmol/kg PYY_3–36, or 1 nmol/kg PYY_3–36 + 10 nmol/kg GLP-1_7–36 (n = 5–6 per group). Ninety minutes later, they were terminally anesthetized with ip sodium pentobarbitone (200 mg) (Rhône Mérieux, Harlow, UK) and transcardially perfused with 0.1 M PBS followed by 4% phosphate-buffered (PB)-formalin. The brains were removed and postfixed overnight in PB-formalin and then transferred to PB-sucrose (20% wt/vol) for 48 h. Serial 40-µm coronal brain sections were cut on a freezing microtome and stained for FLI using the avitin-biotin-peroxidase method. Fos-positive nuclei were counted using a light microscope (Eclipse E800; Nikon, Tokyo, Japan) by an experienced member of the research team with reference to the Rat Brain Atlas (18), who was unaware of which treatment had been given. The nuclei examined were the PVN, ARC, ventromedial and dorsomedial of the hypothalamus, and the nucleus of the solitary tract and the AP of the brain stem.

Behavioral studies
To determine whether alterations in behavior were associated with treatments of peptides used in the feeding and c-fos studies, an observation study was performed (19). After an overnight fast, rats (150–170 g) were injected ip with saline, 10 nmol/kg GLP-1_7–36, 1 nmol/kg PYY_3–36, 1 nmol/kg PYY_3–36 + 10 nmol/kg GLP-1_7–36, 10 nmol/kg exendin-4, or 1 M lithium chloride (positive control) (n = 10 per group) and their food replaced. Their behavior was assessed for the subsequent 60 min by two observers who were unaware of which treatment had been given. Each observer recorded behaviors of 30 animals (n = 5 per group per observer). Each rat was observed for 15 sec in every 5 min. The 15-sec periods were subdivided into three 5-sec periods. In each or these periods, the observer selected the behavior that most closely resembled what the rat was doing. These options were feeding, drinking, locomotion, grooming, head down, sleeping, and rearing.

In a further experiment, freely feeding rats were ip injected before the dark phase with saline, 10 nmol/kg GLP-1_7–36, 1 nmol/kg PYY_3–36, 1 nmol/kg PYY_3–36 + 10 nmol/kg GLP-1_7–36, or 10 nmol/kg exendin-4. Infrared beam breaks in two planes x-tot (horizontal movement) and z-tot (vertical movement) were counted for 1 h using an Opto-Max system (Columbus Instruments, Columbus, OH) to assess locomotion.

Human randomized, double-blind, controlled trial
The study was approved by the Hammersmith, Queen Charlotte’s, and Chelsea Ethics Committee (reference no. 2002/6261) and performed in accordance with the principles of the Declaration of Helsinki. Healthy lean volunteers were recruited through advertisement. Exclusion criteria were substance abuse, pregnancy, significant medical or psychiatric illness, or regular medication except the oral contraceptive pill. Twelve subjects were recruited, each of whom gave their informed consent to participate in the study. One subject was withdrawn due to violation of the protocol and another was withdrawn due to nausea on a study morning. Thus, 10 subjects (four men and six women) completed the study. Their ages ranged from 22 to 29 with a mean age of 25.6 ± 0.7 yr, and body mass index ranged from 20.5 to 26.4 with a mean of 23.0 ± 0.7 kg/m².

Each volunteer received four infusions, saline, GLP-1_7–36, PYY_3–36, or PYY_3–36 with GLP-1_7–36, each on a separate day at least 5 d apart. Subjects fasted from 2100 h on the evening before infusions. The infusion days were run as previously described (20).

Both PYY_3–36 and GLP-1_7–36 were sterile on culture, and limulus amoebocyte lysate assay tests for pyrogen were negative. Two vials were used for each infusion (saline + saline, saline + GLP-1_7–36, saline + PYY_3–36, and PYY_3–36 + GLP-1_7–36). Both vials were dissolved in 2.5 ml hemacel (Beacon, Tunbridge Wells, Kent, UK) to minimize peptide adsorption, diluted in 0.9% saline (Bayer, Newbury, Berkshire, UK) and drawn up in a single syringe (total volume 50 ml). The infusion rates chosen were based on the results of previous studies. PYY_3–36 at 0.8 pmol/kg·min was associated with a 36% reduction in food intake (2). The lowest rate of GLP-1_7–36 that has been shown to inhibit food intake is 0.75 pmol/kg·min (21). We aimed for a submaximal effect for each individual peptide and so selected rates of 0.4 pmol/kg·min for both GLP-1_7–36 and PYY_3–36 infusions and a rate of 0.4 pmol/kg·min PYY_3–36 + 0.4 pmol/kg·min GLP-1_7–36 for the combined infusion. Infusions were given for 120 min.

After 90 min infusion, a buffet lunch was served of preweighed food in excess consisting of chicken or vegetable curry, rice, fruit salad, and sweets. Thirty minutes later, the remaining food was removed and weighed and the infusion discontinued. Blood samples (10 ml) were taken 30 min before the start of the infusion, immediately before the infusion, and then every 30 min until 2 h after the meal. Blood was collected into heparin-coated tubes containing 2000 Kallikrein inhibitor units (0.2 ml) of aprotonin (Bayer). After centrifugation, plasma was separated and stored immediately at –70 C until RIA. Subjects were asked to score subjective nausea on a visual analog scales (0–100 mm) (22) at each time point. Pulse and blood pressure were also measured at these time points. Subjects were allowed to leave 2 h after the meal and instructed to eat ad libitum and complete a food diary for the following 24 h.

RIAs
All samples were assayed in duplicate and single assays to eliminate the effects of interassay variation. PYY, GLP-1, and insulin concentrations were quantified using established in-house RIAs and antibodies (23, 24, 25). Glucose concentrations were measured using a YSI-2300STAT analyzer (Yellow Springs Instruments, Yellow Springs, OH).

Statistical analysis
Results are shown as mean values ± SEM. Data from the animal feeding and immunocytochemistry and behavior studies were analyzed using a one-way ANOVA with a Dunnett’s two-sided post hoc test. For the human infusions, a random effect, repeated-measures ANOVA was performed and within-subject effect examined under a Greenhouse-Geisser correction. In all cases P < 0.05 was considered to be statistically significant.

	Results

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Coadministration of high-dose PYY_3–36 and GLP-1 in lean mice
In the PYY_3–36 dose-response study, food intake in the first hour was significantly inhibited by all doses of PYY_3–36 administered [food intake 0–1 h (grams): saline, 0.67 ± 0.05; PYY_3–36, 30 nmol/kg, 0.49 ± 0.04, 100 nmol/kg, 0.44 ± 0.03, 150 nmol/kg, 0.47 ± 0.02, 300 nmol/kg, 0.47 ± 0.04] (P < 0.05 for all doses vs. saline) (Fig. 1A). PYY appeared to cause maximal feeding inhibition from doses of 100 nmol/kg and above, and this dose was selected for the combined study. Inhibition in cumulative feeding was sustained with all doses for up to 8 h after injection (data not shown).

View larger version (29K): [in this window] [in a new window]   FIG. 1. The effect on food intake in fasted mice in the first hour of ip PYY3–36 (30, 100, 150, and 300 nmol/kg) (A) or GLP-17–36 (30, 100, 300, and 900 nmol/kg) (B). *, P < 0.05 vs. saline. , P < 0.05 for 100 vs. 30 nmol/kg GLP-17–36 and 300 vs. 100 nmol/kg GLP-17–36.

The doses for the coadministration study were as follows: saline, 100 nmol/kg PYY_3–36, 200 nmol/kg PYY_3–36, 100 nmol/kg GLP-1_7–36, 200 nmol/kg GLP-1_7–36 and 100 nmol/kg PYY_3–36 + 100 nmol/kg GLP-1_7–36. All peptides inhibited feeding in the first hour, compared with saline. Coadministration of PYY_3–36 with GLP-1_7–36 led to significantly greater feeding inhibition than all other groups [food intake 0–1 h (grams): saline, 0.74 ± 0.07; 100 nmol/kg GLP-1_7–36, 0.33 ± 0.05; 200 nmol/kg GLP-1_7–36, 0.33 ± 0.07; 100 nmol/kg PYY_3–36, 0.60 ± 0.03; 200 nmol/kg PYY_3–36, 0.51 ± 0.04; 100 nmol/kg PYY_3–36 + 100 nmol/kg GLP-1_7–36, 0.07 ± 0.07: P < 0.05 for combined groups vs. all other groups] (Fig. 2). Feeding inhibition after the combination treatment was sustained up to 24 h after injection [food intake 0–24 h (grams): saline, 7.0 ± 0.2; 100 nmol/kg GLP-1_7–36, 6.5 ± 0.2; 200 nmol/kg GLP-1_7–36, 7.2 ± 0.2; 100 nmol/kg PYY_3–36, 6.9 ± 0.2; 200 nmol/kg PYY_3–36, 6.8 ± 0.2; 100 nmol/kg PYY_3–36 + 100 nmol/kg GLP-1_7–36, 6.1 ± 0.3: P < 0.05 for combined group vs. saline].

View larger version (23K): [in this window] [in a new window]   FIG. 2. The effect of ip coadministration of PYY3–36 with GLP-17–36 in fasted lean mice on feeding in the first hour. Mice were injected with saline, P+G (100 nmol/kg PYY3–36 + 100 nmol/kg GLP-17–36), G (100 nmol/kg GLP-17–36), 2*G (200 nmol/kg GLP-17–36), P (100 nmol/kg PYY3–36), or 2*P (200 nmol/kg PYY3–36). *, P < 0.05 vs. saline. , P < 0.05, compared with all other groups.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Food intake in the first hour after ip coadministration of PYY3–36 with GLP-17–36 in fasted WT and ob/ob mice (A) and WT and db/db (B) mice. Results are presented as a percentage of the mean food intake on the saline day. Mice were injected with saline, 100 nmol/kg PYY3–36 + 100 nmol/kg GLP-17–36, 200 nmol/kg GLP-17–36, or 200 nmol/kg PYY3–36. *, P < 0.05 vs. saline. , P < 0.05, compared with all other groups. White column, Saline; hatched column, GLP-1; striped column, PYY; black column, PYY + GLP-1 (100 nmol/kg PYY3–36 + 100 nmol/kg GLP-17–36).

Coadministration of low-dose PYY_3–36 and GLP-1_7–36 in rats
PYY_3–36 and GLP-1_7–36 dose-response studies were performed in freely feeding rats in the dark phase. In the PYY_3–36 study, food intake in the first hour of the dark phase was significantly inhibited by 3 and 10 nmol/kg PYY_3–36 but not significantly by the 1 nmol/kg dose [food intake 0–1 h (grams): saline, 4.9 ± 0.2; PYY_3–36, 1 nmol/kg, 4.5 ± 0.5; 3 nmol/kg, 3.8 ± 0.3; 10 nmol/kg, 3.5 ± 0.3; P < 0.05 for 3 and 10 nmol/kg PYY_3–36 vs. saline] (Fig. 4A). In the GLP-1_7–36 study, food intake in the first hour was significantly inhibited by 30 and 100 nmol/kg GLP-1_7–36 [food intake 0–1 h (grams): saline, 5.0 ± 0.3; GLP-1_7–36, 3 nmol/kg 4.9 ± 0.5; 10 nmol/kg, 4.5 ± 0.3; 30 nmol/kg, 4.1 ± 0.3; 100 nmol/kg, 3.8 ± 0.3; P < 0.05 for 30 and 100 nmol/kg GLP-1_7–36 vs. saline] (Fig. 4B).

View larger version (43K): [in this window] [in a new window]   FIG. 4. The effect on food intake in the first hour of the dark phase in freely feeding rats of ip PYY3–36 (1, 3, and 10 nmol/kg) (A) or ip GLP-17–36 (3, 10, 30, and 100 nmol/kg) (B). *, P < 0.05 vs. saline.

View larger version (38K): [in this window] [in a new window]   FIG. 5. Food intake in the first hour of the dark phase in freely feeding rats after ip low-dose coadministration of PYY3–36 with GLP-17–36, compared with each peptide alone. Rats were injected with saline, P+G (1 nmol/kg PYY3–36 + 10 nmol/kg GLP-17–36), G (10 nmol/kg GLP-17–36), 2*G (20 nmol/kg GLP-17–36), P (1 nmol/kg PYY3–36), or 2*P (2 nmol/kg PYY3–36). , P < 0.05, compared with all other groups.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Expression of FLI in the ARC in response to ip saline, P+G (1 nmol/kg PYY3–36 + 10 nmol/kg GLP-17–36), G (10 nmol/kg GLP-17–36), and P (1 nmol/kg PYY3–36). *, P 0.05, compared with all other groups.

Behavioral studies
To determine whether behavioral alteration were associated with doses of PYY_3–36 and GLP-1_7–36 at which an additive feeding effect was observed, an observation study was performed. Fasted rats were ip injected with saline, 10 nmol/kg GLP-1_7–36, 1 nmol/kg PYY_3–36, 1 nmol/kg PYY_3–36 + 10 nmol/kg GLP-1_7–36, 10 nmol/kg exendin-4, or 1 M lithium chloride (positive control) (n = 10 per group). The groups treated with exendin-4 and lithium chloride had significantly fewer feeding episodes and significantly more head-down episodes, compared with saline (P < 0.05) (Fig. 7). However, no significant differences in behavior were observed after PYY_3–36, GLP-1_7–36, or PYY_3–36 + GLP-1_7–36.

View larger version (33K): [in this window] [in a new window]   FIG. 7. Number of the following behavior observations: feeding, drinking, locomotion, grooming, head down, sleeping, and rearing after ip saline, 10 nmol/kg GLP-17–36, 1 nmol/kg PYY3–36, 1 nmol/kg PYY3–36 + 10 nmol/kg GLP-17–36, 10 nmol/kg exendin-4 (ex-4), and 0.5 ml 1 M lithium chloride (LiCl). The animals were observed over 60 min after injection, and the total number of observations per animal was 36.

View larger version (15K): [in this window] [in a new window]   FIG. 8. Energy intake in human healthy volunteers from a free buffet meal with infusion of saline, G (0.4 pmol/kg·min GLP-17–36), P (0.4 pmol/kg·min PYY3–36), and P+G (0.4 pmol/kg·min PYY3–36 + 0.4 pmol/kg·min GLP-17–36). Results are presented as absolute difference in energy intake from the saline infusion day. , P < 0.05, compared with all other groups.

There were significant increases in plasma PYY levels with PYY_3–36 and PYY_3–36 + GLP-1_7–36 infusions and plasma GLP-1 levels with GLP-1_7–36 and PYY_3–36 + GLP-1_7–36 infusions (Tables 2 and 3). Interestingly, our GLP-1_7–36 infusion inhibited circulating PYY (PYY picomoles per liter at 90 min: saline infusion, 10.3 ± 1.2; GLP-1_7–36 infusion, 7.7 ± 0.8; P < 0.05). However, no significant changes in circulating GLP-1 were observed during PYY_3–36 infusion.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PYY_3–36 and GLP-1_7–36 inhibited food intake individually in mice and rats. PYY_3–36 appeared to cause maximal feeding inhibition at doses of 100 nmol/kg and above. In contrast, feeding inhibition associated with GLP-1_7–36 was similar to that seen with CCK-8, with increasing inhibition seen with increasing doses (14). In the dose-response studies with low doses, the minimum doses of PYY_3–36 and GLP-1_7–36 found to inhibit food intake were 3 and 30 nmol/kg, respectively. Both these results are consistent with our previously published data (2, 7). In both high- and low-dose coadministration studies, PYY_3–36 and GLP-1_7–36 administered together inhibited food intake significantly more than either peptide alone or more than double the dose of either peptide. Thus, PYY_3–36 and GLP-1_7–36 appeared to inhibit feeding additively.

PYY_3–36 and GLP-1_7–36 were coadministered in genetically obese mice. Examination of the food intake in the saline-injected groups revealed that the ob/ob and db/db mice ate less in the first hour of refeeding than their WT littermates. This somewhat surprising finding is consistent with previously published work (26, 27) and suggests that thermogenic adaptation to fasting reduced the subsequent energy requirement of the obese mouse models (28). Coadministration of PYY_3–36 and GLP-1_7–36 treatment led to significantly greater reduction in feeding than twice the dose of either peptide alone in the ob/ob and db/db and WT mice. Thus, PYY_3–36 and GLP-1_7–36 inhibited food intake additively in obese as well as lean rodents.

We quantified c-fos afterPYY_3–36, GLP-1_7–36, and PYY_3–36 + GLP-1_7–36 administration to determine a possible mechanism for their additive effect on feeding. There was a significant increase in c-fos in the ARC after low-dose combination PYY_3–36 + GLP-1_7–36 treatment but no changes in the other treatment groups. Therefore, PYY_3–36 and GLP-1_7–36 may inhibit food intake through additive stimulation of ARC neurons. However, c-fos mapping cannot identify the pathway(s) through which neuronal activation was mediated. The ARC neuronal activation observed in our study could have been mediated through the blood-brain barrier via the median eminence (29) or the vagus nerve and brain stem (30).

There were no significant differences in observed behavior in rats after PYY_3–36, GLP-1_7–36, or PYY_3–36 + GLP-1_7–36 at doses at which the combination inhibited feeding. In the locomotion study, no significant differences in horizontal beam breaks were observed after the combined PYY_3–36 + GLP-1_7–36 treatment, but there were marginally fewer vertical beam breaks. In their recent publication, Talsania et al. (31) suggested that low-dose exendin-4 together with PYY_3–36 may increase the suppression of food intake in rodents without inducing significant side effects. There were no differences in nausea scores between infusions in our human study. Taken together these results suggest that behavioral change is unlikely to account for the feeding inhibition observed with coadministration of PYY_3–36 and GLP-1_7–36. Thus, these peptides may have a specific effect on feeding independent of behavioral change.

In our human study, GLP-1_7–36 alone (0.4 pmol/kg·min) did not lead to a significant change in energy intake. In human subjects the lowest reported infusion rate of GLP-1_7–36 associated with a change in energy intake is 0.75 pmol/kg·min (21). However, incretin effects have been observed with a lower rate of GLP-1_7–36 of 0.5 pmol/kg·min (24). In the current study, we found a small but statistically significant reduction in fasting glucose levels and an increase in the fasting insulin concentration with GLP-1_7–36 infusion.

PYY_3–36 infusion (0.4 pmol/kg·min) was associated with a 15% reduction in energy intake, compared with saline infusion, although this did not reach statistical significance. The magnitude of this reduction is in keeping with the larger reduction of 36% reported after higher-rate PYY infusions (0.8 pmol/kg·min) (2).

Coadministration of PYY_3–36 with GLP-1_7–36 was associated with a 27% reduction in energy intake from the buffet meal. The combination was more effective in inhibiting appetite then either peptide alone. Cumulative energy intake over 24 h, including the buffet meal, was reduced by 13% after combined infusion, compared with the saline infusion, and was significantly less than that after all other treatment groups. In contrast with the additive inhibitory effect on energy intake, the incretin effect of GLP-1_7–36 appeared unchanged with the addition of PYY_3–36.

Our study involving coadministration of PYY_3–36 and GLP-1_7–36 suggests an additive inhibitory feeding effect. Interestingly, the GLP-1 receptor agonist, exendin-4, and PYY_3–36 have been reported to inhibit food intake synergistically in rodents (31). However, it appears that not all combinations of gut hormones behave in this way. Indeed, it has recently been reported that whereas infusion of CCK-33 or GLP-1_7–36 inhibited food intake when administered individually, no enhanced inhibition was observed when they were infused together (32). Further work is needed to determine whether the other combinations of gut hormones lead to additive alterations in food intake.

We observed a reduction of endogenous PYY levels with GLP-1_7–36 infusion. This has been previously demonstrated at a higher dose of peripheral GLP-1_7–36 administration (32). Inhibition of endogenous PYY could result in a reduction in the anorexic action of GLP-1. In contrast, as in previous work (12), no changes in endogenous GLP-1 concentrations were observed after PYY_3–36 infusion.

In summary, we have demonstrated that PYY_3–36 and GLP-1_7–36 may inhibit appetite additively in rodents and man. The mechanism may be through enhanced activation of hypothalamic arcuate neurons. Reduction in energy intake and increase in insulin secretion would make this combination a particularly attractive therapy for patients with type 2 diabetes.

	Acknowledgments

 
We thank Dr. Adam Kennedy, Ms. Tammy Ng, Professor Malcolm Alison, Dr. Chrystalla Orphanides, the human volunteers, and the Sir John McMichael Centre for Clinical Investigation and Research, Hammersmith Hospital.

	Footnotes

 
This work was supported by European Union FP6 Integrated Project Grant (contract LSHM-CT-2003-503041). This publication reflects the authors’ views and not necessarily those of the European Union. The information in this document is provided as is, and no guarantee of warranty is given that the information is fit for any particular purpose. The user thereof uses the information at his/her sole risk and liability. The authors also thank the Medical Research Council and the Wellcome Trust for program grant support. N.M.N., A.J.P., and M.R.D. are Wellcome Trust Clinical Research Training Fellows. C.L.D. was a Diabetes United Kingdom student.

First Published Online September 8, 2005

Abbreviations: AP, Area postrema; ARC, arcuate nucleus; FLI, fos-like immunoreactivity; GLP, glucagon like peptide-1; PB, phosphate buffered; PVN, paraventricular nucleus; PYY, peptide YY; WT, wild type.

Received February 25, 2005.

Accepted for publication August 22, 2005.

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