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Departments of * Pharmacology, In Vivo Modeling, and ** Cell and Molecular Biology, Amylin Pharmaceuticals, San Diego, CA
2 To whom correspondence should be addressed. E-mail: sadams{at}whnrc.usda.gov.
ABSTRACT
In rodents, weight reduction after peptide YY[3-36] (PYY[3-36]) administration may be due largely to decreased food consumption. Effects on other processes affecting energy balance (energy expenditure, fuel partitioning, gut nutrient uptake) remain poorly understood. We examined whether s.c. infusion of 1 mg/(kg·d) PYY[3-36] (for up to 7 d) increased metabolic rate, fat combustion, and/or fecal energy loss in obese mice fed a high-fat diet. PYY[3-36] transiently reduced food intake (e.g., 25–43% lower at d 2 relative to pretreatment baseline) and decreased body weight (e.g., 9–10% reduction at d 2 vs. baseline) in 3 separate studies. Mass-specific metabolic rate in kJ/(kg·h) in PYY[3-36]–treated mice did not differ from controls. The dark cycle respiratory quotient (RQ) was transiently decreased. On d 2, it was 0.747 ± 0.008 compared with 0.786 ± 0.004 for controls (P < 0.001); light cycle RQ was reduced throughout the study in PYY[3-36]–treated mice (0.730 ± 0.006) compared with controls (0.750 ± 0.009; P < 0.001). Epididymal fat pad weight in PYY[3-36]–treated mice was 
50% lower than in controls (P < 0.01). Fat pad lipolysis ex vivo was not stimulated by PYY[3-36]. PYY[3-36] decreased basal gallbladder emptying in nonobese mice. Fecal energy loss was negligible (2% of ingested energy) and did not differ between PYY[3-36]–treated mice and controls. Thus, negative energy balance after PYY[3-36] administration in diet-induced obese mice results from reduced food intake with a relative maintenance of mass-specific energy expenditure. Fat loss and reduced RQ highlight the potential for PYY[3-36] to drive increased mobilization of fat stores to help meet energy requirements in this model.
KEY WORDS: • obesity • metabolic syndrome • gut peptide • lipid • ß-oxidation
Gut-derived hormones such as the peptide YY (PYY)4 cleavage product PYY[3-36] may play a role in modulating energy balance and adiposity through actions that regulate food intake, the efficiency of energy uptake, and tissue metabolism of nutrients. Interestingly, circulating total PYY concentration increases after food ingestion (especially fat-rich meals) (1,2), and has been reported to be low in obese humans (3,4). PYY[3-36] was recently implicated in the regulation of food intake because its peripheral infusion resulted in reduced hunger in humans (4,5). In rodent models, peripheral administration of PYY[3-36] was shown to reduce food intake and/or body weight gain (5–13). At least one study suggested that the peptide's anorexigenic properties in rodents are influenced by factors such as daily handling and acclimation to injection (10). However, food intake reduction was also observed after PYY[3-36] administration in naïve mouse models [e.g., (7,11) and data herein].
It remains to be established whether PYY[3-36] influences other processes that affect energy balance such as gut nutrient uptake, metabolic rate, and fuel partitioning. Several lines of evidence indicate that PYY regulates gut function, and it was proposed that the hormone participates in the ileal brake mechanism in which unabsorbed nutrients reaching the distal intestine trigger signals that slow gastrointestinal motility and reduce gastric/pancreatic secretion (14). Peripheral administration of full-length PYY was reported to decrease the cephalic phase of gallbladder emptying in humans (15). This effect may be species specific because it was not observed in dogs (16), and activation of gallbladder contraction was reported for anesthetized guinea pigs (17) given PYY. Full-length PYY decreased gastric acid secretion, gastric emptying, and exocrine pancreas function, and increased orocecal transit time [see (18)]. Further, PYY[3-36] was recently reported to inhibit gastric emptying in primates (19). In addition to potential gastrointestinal tract motility and secretory effects, PYY[3-36] administration reduced feed efficiency (body weight gain relative to energy intake) and adiposity in high-fat diet–induced obese (DIO) mice (11), suggesting that PYY[3-36] regulates intestinal energy uptake and/or tissue-level metabolism.
We used DIO mice to test the hypothesis that increased metabolic rate and higher fat combustion contribute to the reductions in body weight and adiposity after pharmacologic administration of PYY[3-36]. Furthermore, we examined whether the peptide reduces net energy uptake in the gastrointestinal tract as part of its mechanism of action.
MATERIALS AND METHODS
Animals and peptide information. All studies were approved by the Institutional Animal Care and Use Committee at Amylin Pharmaceuticals in accordance with Animal Welfare Act guidelines. Obese male C57BL/6J mice (Jackson Laboratories) were used in studies examining PYY[3-36] effects on energy balance. Obesity was induced by high-fat feeding (58% of energy from fat, 16% from protein, 26% from carbohydrate, D12331, Research Diets) starting at 4 wk of age (20). The diet composition was as follows (g/kg): casein, 228; DL-methionine, 2; maltodextrin, 170; sucrose, 175; soybean oil, 25; hydrogenated coconut oil, 333.5; mineral mix S10001, 40; sodium bicarbonate, 10.5; potassium citrate, 4; vitamin mix V10001, 10; and choline bitartrate, 2. Mice were fed the pelleted diet for 6 wk before treatment (or 4 wk for the long-term study), and continued to be fed this diet in powdered form throughout the treatment period unless otherwise noted. Continuous, s.c. administration of vehicle [50% dimethyl sulfoxide in water] or PYY[3-36] was achieved using Alzet® osmotic pumps (Durect; Models 1003D, 2001, and 2004 for the 3-, 7-, and 28-d studies, respectively) placed in the intrascapular region under isoflurane anesthesia. Previously, it was reported that continuous infusion of PYY[3-36] via s.c. osmotic pump in DIO mice resulted in reduced weight gain and adiposity, with the most robust effect seen at the highest dose employed, 1 mg/(kg·d) (11). Thus, this dosing regimen was utilized in the 3- and 7-d experiments described below (Studies A–C). All mice were housed under a 12-h light:dark cycle (lights on at 0600) at ambient temperatures of 21–23°C, and consumed food ad libitum both pre- and post-treatment. Studies examining gallbladder emptying utilized nonobese 8-wk-old male NIH Swiss mice (HarlanTeklad) fed a standard diet (Teklad LM7012). The trifluoroacetic acid salt of human PYY[3-36] (>98% purity) was synthesized using standard methods (Peptisyntha) and its identity confirmed using MS.
Study A: effects of PYY[3-36] on metabolic rate and respiratory quotient (RQ).
Mice were housed singly 1 wk before calorimetric measurements, and acclimated to calorimetry cages for 4 d before measurement of post-treatment metabolic rate and RQ by indirect calorimetry (Oxymax; software version 2.52; Columbus Instruments). Considering the mean values for each mouse for the 2-d pretreatment baseline, within-animal CV% was 4.6 ± 0.8 and 4.0 ± 0.8% for light and dark cycle energy expenditure, respectively, thus indicating adequate acclimation. After implantation of the osmotic pump [vehicle controls, n = 13; PYY[3-36] at 1 mg/(kg·d), n = 12], calorimetric measurements were made continuously over 7 d. Heat production was calculated by the instrument software [based on (21)] and is reported relative to body mass measured on each treatment day. Food intake and body weight were determined each morning (0900) during the baseline period and thereafter. At the end of the study in the morning, mice that had been deprived of food for 2–4 h were anesthetized with isoflurane and blood was collected into Na-heparin–flushed syringes by cardiac puncture; plasma was immediately frozen. After the isoflurane overdose, bilateral epididymal fat pads and the intrascapular brown adipose tissue (BAT) depots were dissected and weights determined. Excised liver samples were placed in RNALater (Ambion) and stored at –20°C.
Study B: effects of PYY[3-36] on body composition and net energy uptake across the gut.
Mice were housed singly starting 1 wk before treatment. To enable daily feces collection, mice were placed in metabolic cages (Diuresis Cages, Cat. 650–0322; Nalge Nunc International) and acclimated to caging and the powdered high-fat food for 4 d before treatment. Food intake and body weight were determined each morning (0900) during a 2-d pretreatment baseline period and thereafter. A total of 18 mice [vehicle controls, n = 9; PYY[3-36] at 1 mg/(kg·d), n = 9] were used. Blood collection was carried out as in Study A. Before fat pad dissection, staples and pumps were removed from mice that had been killed, and body composition was determined using dual-energy X-ray absorptiometry (DEXA) analysis (PixiMus, GE Lunar). The energy content of feces was determined at Covance Labs using bomb calorimetry (as little as 50 mg of material could be analyzed). To ensure that adequate material was available, a pooling strategy was utilized for each mouse; pooled samples from an individual's 2-d baseline period, early treatment period (d 1, 2, 3), and late treatment period (d 5, 6, 7) were compared.
Study C: short-term effects of PYY[3-36] on food intake, body weight, and adiposity. One week before treatment, mice were housed singly in standard caging with a raised flooring to prevent coprophagy and were fed the high-fat diet. As in Studies A and B, body weight and food intake were monitored during a baseline period and after osmotic pump administration of vehicle (n = 10) or 1 mg/(kg·d) PYY[3-36] (n = 10). After 3 d of treatment, DEXA measurement of body composition was determined, and tissues and blood were collected and processed as in Study A. Fecal energy content was determined by bomb calorimetry (Covance Labs) in samples collected over the final 24 h from cage bottoms lined with absorbent paper.
Effects of PYY[3-36] on gallbladder emptying. Postabsorptive (food-deprived for 3 h) nonobese mice were injected s.c. in the morning with saline (n = 14) or PYY[3-36] at 1, 10, or 100 µg/kg (n = 6, 11, 8, respectively). Mice were killed by cervical dislocation at 30 min postinjection, and gallbladders were removed and weighed as a measure of gallbladder emptying rate.
Long-term effects of PYY[3-36] on body weight in DIO mice. DIO mice were fitted with Alzet osmotic pumps for continuous delivery of vehicle (n = 18) or PYY[3-36] [n = 24; given at 300 µg/(kg·d), the estimated ED50 (dose yielding 50% of maximal response) for weight change in an earlier study in this model (11)]. At 28 d, pumps were replaced; the controls continued to receive vehicle, and one PYY[3-36] group (n = 12) continued to receive the peptide to enable a longer evaluation of the peptide's effects. The third group, which had received PYY[3-36] for the initial treatment period, received new pumps containing vehicle to test the effect of peptide withdrawal (n = 12). Mice were fed pelleted high-fat diet and body weights and food intake were recorded weekly. For statistical analyses relating to the first 28 d period, data from all PYY[3-36]–treated mice were used to compare with control values. Thereafter, control, PYY[3-36]–treated, and PYY[3-36]–withdrawal groups were considered independently.
Biochemical assays and gene expression analyses.
Plasma ß-hydroxybutyrate (Cat. 2440, STANBIO Laboratory), glycerol (Cat. TR0100, Sigma Chemical), and nonesterified fatty acids (NEFA C, Cat. 994–75409, Wako Chemicals) were measured using standard colorimetric assays scaled down to accommodate 96-well plates and using 5–10 µL of plasma/well. Total PYY immunoreactivity in plasma was determined by Linco Diagnostic Services using a human PYY RIA displaying <0.1% cross-reactivity to mouse or rat PYY[3-36], and averaged 39303 pg/mL (10 nmol/L) in mice treated with 1 mg/(kg·d) PYY[3-36].
Using conditions mimicking those of Heffernan et al. (22), ex vivo lipolysis (glycerol release over 1 h) was measured in nonobese female mouse retroperitoneal fat pad preparations incubated with PYY[3-36] at concentrations ranging from the upper physiologic to pharmacologic plasma levels [0.05, 0.5, and 10 nmol/L; the latter value was similar to plasma levels determined from mice treated with 1 mg/(kg·d) PYY[3-36]]. Values were compared with basal rates from untreated adipose tissue.
DNAse I-treated total mRNA for gene expression analyses was obtained from a subset of tissues according to the manufacturer's instructions (RiboPure kit 1924; Ambion). One-step quantitative real-time RT-PCR analyses employing standard reagents (Cat. 4309169; Applied Biosystems) and an ABI 7900HT instrument were used to measure mRNA abundance. The 50-µL reaction conditions were: 2.5 µL Assay-on-Demand® primer/probe mix, 1x Master Mix, 1x Multiscribe/RNAse inhibitor mix, and 50 ng RNA. RT-PCR conditions were: 48°C for 30 min, 95°C for 10 min, then 40 cycles (95°C 15 s/60°C 1 min). For each gene, cycle numbers were corrected for loading variation by simultaneously assaying 18S RNA abundance using a commercially available primer/probe set (ABI). The relative abundance of mRNAs corresponding to the following genes was determined using ABI Assay-on-Demand primer/probe sets: liver-type carnitine palmitoyltransferase 1 (L-CPT1 or CPT1a; Mm00550438_m1), acetyl-CoA carboxylase 1 (ACC1; Mm01304257_m1), ACC2 (Mm01204677_m1), mitochondrial hydroxymethylglutaryl-CoA synthase (HMGCS2; Mm00550050_m1), malonyl-CoA decarboxylase (MCD or MLYCD; Mm01245664_m1), and uncoupling protein 1 (UCP1; Mm00494069_m1).
Statistics. Differences between treatment group means for variables determined over time were analyzed using a repeated-measures ANOVA examining the effects of time, treatment, and the time x treatment interaction. Differences between PYY[3-36]–treated mice and controls were tested for simple effects within time points using pooled SE whenever time x treatment effects were significant (SPSS version 13.0). A repeated-measures ANOVA was also used for 3-group comparisons (e.g., control vs. PYY[3-36]–treated vs. PYY[3-36]–withdrawal groups), but because the time x treatment interactions were not significant for body weight or food intake in the period after hormone withdrawal, post hoc tests were not performed. Simple 2-group comparisons were carried out using a Student's t test, whereas gallbladder emptying dose-response data were evaluated using a 1-way ANOVA and Dunnett's test comparing PYY[3-36] responses with controls. Data are presented as means ± SEM, with differences considered to be significant at P < 0.05.
RESULTS
Body weight, food intake, and adiposity in DIO mice administered PYY[3-36] for 1 wk (Studies A and B). Mice administered 1 mg/(kg·d) PYY[3-36] continuously via s.c. osmotic pump for 1 wk displayed significant weight loss and a transient reduction in food intake relative to vehicle-treated controls. In Studies A and B (7-d calorimetry and gut energy uptake studies, respectively), PYY[3-36]–treated mice lost up to 10% of their baseline body weight by d 3, compared with only 3–4% loss in controls (Fig. 1). We suspect that weight loss in controls likely reflects postsurgical stress in these cohorts because their body weight returned to baseline levels by d 7. Compared with controls, food intake was significantly lower at least through d 3–4 in PYY[3-36]–treated mice (Fig. 1).
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Adipose tissue loss is rapid in mice administered 1 mg/(kg·d) PYY[3-36] (Study C).
To address the time frame in which PYY[3-36] lowers body fat in DIO mice, an additional cohort was administered the peptide for only 3 d. The results were consistent with the 7-d studies in that food intake was reduced significantly by PYY[3-36] administration: intake was decreased to 49.7 ± 7.1, 57.0 ± 7.5, and 76.2 ± 7.3% of baseline values on d 1–3, respectively. These values were significantly different from those of time-matched controls, which were 108.0 ± 24.7 (P < 0.05), 109.9 ± 9.4 (P < 0.001), and 99.2 ± 7.5% (P < 0.05) of baseline intake on d 1–3, respectively (the lack of reduced food intake in controls contrasts with Studies A and B, potentially due to cohort-specific differences in surgical recovery or other unknown factors that differed between studies). Compared with baseline values, body weight decreased by 7.7 ± 1.2, 9.3 ± 1.0, and 7.4 ± 1.2% on d 1–3, respectively, in PYY[3-36]–treated mice. These changes differed from controls (P < 0.001), whose body weight remained stable (
1% change from baseline). Relative to controls (0.58 ± 0.08 g), epididymal fat pad weights were reduced by 55% in the peptide-treated group (0.26 ± 0.03 g; P < 0.01), with DEXA indicating a 30% lower whole-animal fat mass (4.58 ± 0.34 and 3.19 ± 0.19 g in controls and PYY[3-36]–treated mice, respectively; P < 0.01). Again, lean mass did not differ between controls (19.65 ± 0.23 g) and PYY[3-36]–treated mice (18.93 ± 0.47 g). Intrascapular BAT weight was lower (P = 0.01) in PYY[3-36]–treated mice (81 ± 5 mg) than in controls (108 ± 9 mg).
Metabolic rate and RQ (Study A). Metabolic rate in kJ/(kg·h) during the light or dark cycle after initiation of treatment did not differ significantly between controls and PYY[3-36]–treated mice (Fig. 2). In contrast, PYY[3-36] elicited a significant decrease in RQ (indicative of increased fatty acid combustion) during the dark cycle through 3 d of treatment, which normalized and then increased slightly relative to controls at d 6–7 (Fig. 3). Light cycle RQ was persistently reduced throughout the study period in PYY[3-36]–treated mice (Fig. 3).
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Expression of genes encoding metabolically relevant proteins. The mRNA levels for genes encoding several components that regulate fat metabolism were determined at d 3 and 7 in the liver. Few significant differences were noted. mRNA levels for ACC1 and ACC2 were increased slightly in PYY[3-36]–treated mice at d 7 (fold of control, 1.36 ± 0.13 and 1.47 ± 0.17, respectively; P < 0.05), and HMGCS was modestly reduced at d 3 (to 68 ± 10% of controls; P < 0.05). Expressions of L-CPT-1 and MCD did not differ between the groups (results not shown). BAT UCP1 mRNA abundance (a marker correlated with BAT activation) determined after 3 d did not differ in PYY[3-36]–treated mice (82 ± 9% of controls, n = 10).
Gallbladder emptying and net energy uptake across the gut. A pharmacologic dose of 100 µg/kg PYY[3-36] in nonobese mice increased basal gallbladder weight (9.77 ± 2.03 mg) compared with controls (3.64 ± 0.59 mg) (P < 0.01), consistent with reduced gallbladder emptying rate. Gallbladder weights in mice given lower doses of PYY[3-36] did not differ significantly from controls: 3.45 ± 0.77 and 6.02 ± 1.32 mg at 1 and 10 µg/kg, respectively. In DIO mice administered PYY[3-36] over 3 d (Study C), fecal energy density was increased by 23% relative to controls in samples derived from the final 24 h of treatment (1188 ± 59 vs. 933 ± 50 kJ/100 g; P < 0.01). Fecal energy density in PYY[3-36]–treated mice in Study B samples pooled over d 1–3 (1322 ± 79 kJ/100 g) tended to be higher (10%) than in controls (1197 ± 46 kJ/100 g ), but this difference was not significant. In the latter study, in samples pooled over d 5–7, fecal energy density did not differ between controls (1033 ± 42 kJ/100 g) and PYY[3-36] mice (1021 ± 33 kJ/100 g). As a percentage of energy ingested, fecal energy was a negligible route of energy loss in both groups in Study C, i.e., controls, 1.75 ± 0.23% and PYY[3-36]–treated mice, 2.18 ± 0.38%.
Long-term body weight effects and effect of PYY[3-36] withdrawal.
Body weight in PYY[3-36]–treated mice was lower than that of controls in our 7-d paradigm, and in another study in which this variable was monitored for 28 d in DIO mice administered several doses (11). To explore whether relatively reduced body weight persists for a longer period and is reversed by treatment termination, DIO mice were treated with PYY[3-36] [300 µg/(kg·d)] in a more chronic setting (Fig. 4). Administration of PYY[3-36] significantly lowered body weight compared with controls (Fig. 4A), although to a lesser extent than in studies using 1 mg/(kg·d) (see above). This treatment effect (P < 0.001) remained significant throughout the study, and no time x treatment interaction was observed. For food intake, the time, treatment, and time x treatment interactions were all significant (P < 0.001) comparing PYY[3-36]–treated and control mice during the first 4 wk of the study. PYY[3-36] reduced food intake per mouse during wk 1 (296.2 ± 3.3 kJ/wk) compared with controls (331.8 ± 6.7 kJ/wk); P 0.001), but intake in PYY[3-36]–treated mice did not differ from controls for the remainder of the study (Fig. 4B). After the 4-wk time point, there were time and treatment effects (P < 0.001) for body weight among control, PYY[3-36]–treated, and PYY[3-36]–withdrawal groups, but there was no time x treatment interaction. PYY[3-36] withdrawal after 28 d accelerated weight gain, which resulted in a body weight equaling that of controls by 35 d (Fig. 4A), and greater than that of mice continuing treatment with PYY[3-36] (P 0.001). Comparing food intake among the control, PYY[3-36]–treated, and PYY[3-36]–withdrawal groups, there were time (P < 0.001) and treatment (P 0.05) effects, but no time x treatment interaction. The treatment withdrawal group had a higher intake than mice continuing to receive PYY[3-36] (P < 0.05). Food intakes did not differ between PYY[3-36]–withdrawal mice and controls during wk 1 of withdrawal (Fig. 4B). During wk 1 of withdrawal, energy intake was higher in the treatment-withdrawal group (422.6 ± 8.8 kJ/wk) than in the PYY[3-36]–treated mice (393.3 ± 11.3 kJ/wk). Controls consumed 397.5 ± 9.6 kJ/wk at that time (Fig. 4B).
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DISCUSSION
The rising incidence of excess weight and obesity necessitates the pursuit of new strategies to reverse or prevent inordinate body weight gain. A better understanding of the physiology and pharmacology of endocrine factors implicated in metabolism will benefit these efforts. Interest in PYY[3-36] as a potential obesity therapeutic grew after initial reports of its anorexigenic properties (4,5) and the reductions in weight gain or adiposity after PYY[3-36] administration in rodent models (11). Diverse laboratories examining food intake in rodents (5) (6–13) and primates (19) further established that PYY[3-36] treatment can decrease energy intake. We extended these findings by illustrating that in DIO mice, PYY[3-36] treatment maintains the body mass–specific metabolic rate in a setting of reduced food intake, and drives marked fat loss coupled with increased whole-animal fat combustion. These metabolic effects, together with the anorexigenic properties of the peptide, contributed to an energy deficit and, hence, reductions in weight and adiposity.
Reduced food intake is expected to decrease metabolic rate. Despite lower energy intake over the first several days of treatment, mass-specific metabolic rate [kJ/(kg·h)] in PYY[3-36]–treated mice did not differ from vehicle-treated controls. We found no evidence that metabolism was driven by increased BAT sympathetic tone because BAT UCP1 mRNA expression (a marker of BAT activation) was unchanged in PYY[3-36]–treated mice relative to controls. After the initial weight loss, PYY[3-36]–treated mice ate a similar amount of food as controls, yet maintained a relatively lower body weight for an extended period (see Figs. 1 and 4). We suspect that this outcome resulted from subtle treatment-related differences in energy balance (food intake, fecal energy loss, and/or energy expenditure) that were not detectable within the limits of our analytical methods. Evidence for this emerged from determination of the effects of PYY[3-36] withdrawal on food intake, i.e., compared with mice continuing to receive the peptide, mice withdrawn from PYY[3-36] ate significantly more food over wk 1 after withdrawal, gaining weight to a level equal to that of controls. These results indicate that an attenuating effect of PYY[3-36] on food intake remained throughout the study, despite gross measures of food intake that did not detect significant differences between control and peptide-treated mice beyond 1 wk of administration. Furthermore, should there be a treatment-related difference in nonprotein RQ (which would not be detected under our experimental conditions), modest changes in the calculation of energy expenditure would result (21).
Oxidative utilization of fat was increased substantially by PYY[3-36] in DIO mice, illustrated by the reduction in RQ after PYY[3-36] treatment (Fig. 3). These effects may be secondary to reduced food intake, and/or reflect a direct action of PYY[3-36] on fat-mobilizing or fat-utilizing tissues. First, diminished food intake in response to PYY[3-36] treatment is expected to contribute to lowered RQ because this nutritional state results in hormonal and biochemical shifts promoting NEFA release and ß-oxidation in liver and skeletal muscle [reviewed in (23,24)]. Food restriction lowers the insulin:glucagon ratio, increases lipolysis, decreases de novo lipogenesis, reduces tissue malonyl-CoA levels (likely via AMP-kinase action on ACC and MCD), and thus activates CPT-1, ß-oxidation, and liver ketogenesis. We did not observe differences in plasma ß-hydroxybutryate levels at 3 or 7 d of PYY[3-36] administration (data not shown), but it is not known whether ketone body flux rate was changed or whether differences may have been present on an earlier treatment day. Consistent with a possible relation between food intake and RQ, temporal changes in 24-h food intake between controls and PYY[3-36]–treated mice correlated with dark-cycle RQ patterns, and a majority of intake typically occurs during this period in mice [e.g., 68 ± 5% of daily intake took place during the dark cycle in a separate cohort of untreated DIO mice (P < 0.001 vs. light cycle; n = 10); J. L. Piercey and S. H. Adams, unpublished results]. It was notable that dark-cycle RQ was slightly increased in PYY[3-36]–treated mice on d 6–7 despite 24-h food intake equal to controls (Study A). Theoretically, this outcome could be driven by subtle differences in diurnal feeding patterns, changes in tissue-level metabolic regulation, or other factors not directly addressed in the current study. Because interpretation of metabolic data in pair-fed or food-restricted mice is confounded by torpor-like events (25,26), alternative models will be required in future studies to determine the specific contribution of reduced food intake per se to PYY[3-36]–induced fat loss.
Second, it is intriguing to postulate that PYY[3-36] drives food intake–independent biochemical and molecular changes that promote fat combustion. Consistent with this idea, light-cycle RQ remained persistently reduced in PYY[3-36] mice relative to controls (Fig. 3), despite similar daily food intake by d 5 of treatment (Fig. 1). We postulated that the reduced RQ observed with PYY[3-36] treatment may be associated with upregulation of genes associated with fat oxidation (e.g., CPT1, MCD, and HMGCS), whereas genes related to the generation of the ß-oxidation inhibitor malonyl-CoA (ACC isoforms) would be downregulated. However, our initial studies in liver revealed that PYY[3-36] treatment did not induce hepatic genes involved with fatty acid catabolism, and did not reduce the expression of ACC1 or ACC2 (see Results). Thus, the underlying molecular mechanisms regulating fat combustion after PYY[3-36] treatment remain to be characterized. Studies exploring gene changes at earlier treatment times and in other metabolically relevant tissues such as skeletal muscle and white adipose tissue (WAT), plus determinations of the hormone's effects on the activity and phosphorylation states of relevant enzymes involved with fat metabolism will be of future interest.
In addition to decreased RQ, PYY[3-36] treatment led to a striking and rapid fat loss. These coincident events are consistent with a model in which accelerated WAT lipolysis is coupled with increased oxidative flux of liberated NEFA; hypothetically, however, reduced adipocyte triglyceride synthesis could also contribute to diminished WAT depot mass. Several groups indicated that in vitro, full-length PYY and neuropeptide Y (potentially via Y1 receptors in rat or human adipocytes, or Y2 receptors in dog adipose tissue), actually inhibit lipolysis (27–29); to our knowledge, however, there have been no published data concerning PYY[3-36] (a Y2-preferring ligand) in this regard. PYY[3-36] did not have an effect on ex vivo lipolysis in murine fat pad preparations (see Results), and plasma lipolysis markers (glycerol, NEFA) did not differ between controls and PYY[3-36]–treated mice at 3 or 7 d (data not shown). It was reported recently that a short-term 3- to 4-h infusion of PYY[3-36] in high-fat fed C57BL/6J mice did not alter whole-animal NEFA flux rate (30), but effects on metabolite flux over longer-term administration remain unknown. Therefore, results to date indicate that the adipose tissue loss in response to PYY[3-36] in vivo is not explained by a direct, independent lipolytic effect of the peptide in WAT. Of course, the influence of PYY[3-36] on adipose depot size may simply be related to reduced food intake, which promotes lipid mobilization and reduces lipogenesis through alterations in multiple hormone activities and changes in nervous system signaling [see (24)].
At pharmacologic doses, PYY[3-36] increased gallbladder weight in nonobese mice, suggesting a decrease in gallbladder emptying rate as reported for full-length PYY in humans (15). We hypothesized that if the findings from nonobese mice manifest in the high-fat fed DIO mouse model, one could expect to see enhanced energy loss as lipid via the feces because bile acid secretion would be compromised. Consistent with this idea, PYY[3-36] treatment modestly increased fecal energy density in DIO mice (see Results). Notably, in a separate study, fecal energy density was increased 14% in DIO rats [1782 ± 33 kJ/100 g (n = 6)] treated with 1 mg/(kg·d) PYY[3-36] via continuous s.c. infusion for 4 wk compared with controls [1561 ± 25 kJ/100 g (n = 15); P < 0.001]. On balance, a small amount of energy was lost via the feces in our DIO mice (1–2% of ingested energy), suggesting that net lipid digestion is high as reported in other studies of mice fed high-fat diets (31,32). Therefore, the majority of initial weight loss after PYY[3-36] treatment in DIO mice cannot be ascribed to attenuated energy uptake across the gut. It will be interesting in future studies to determine the degree to which lipids contribute to the energy content of feces, and to analyze in more detail the dynamics of lipid balance across the gut after PYY[3-36] administration.
In summary, the current results extend previous reports of the effects of PYY[3-36] on ingestive behavior (4–13,19) by characterizing other components of the energy balance equation. It appears that reduced food intake coupled with maintenance of body mass–specific metabolic rate were the primary processes leading to energy deficit and hence rapid weight loss in DIO mice. Recent results from studies using continuous intracerebroventricular administration of a specific Y2 receptor agonist in mice are consistent with this idea (33). Interestingly, body weight differences between control and PYY[3-36]–treated DIO mice persisted through at least 56 d (data herein) and fat mass remained lower through at least 28 d (11), illustrating that the hormone retains activity for long periods of time. Should the effects of PYY[3-36] observed in DIO mice, i.e., persistently reduced body weight, fat loss, and maintenance of lean mass, translate to a clinical setting, activation of PYY[3-36]–related pathways may prove to be a new approach to treating metabolic disorders.
ACKNOWLEDGMENTS
The authors thank Amy Bloom and Scott Barnhill (Comparative Medicine Department) for valuable technical assistance, Jay Hu (Biometrics Department) and Raywin Huang for statistical help, Liz Rinehart (Linco Diagnostics, Incorporated) for conducting the PYY assays, Jaime Piercey for input regarding diurnal feeding in mice, and Jon Roth, Diane Hargrove, James Paterniti, Jr., Alain Baron, David Parkes, and Christen Anderson for helpful discussions and editing.
FOOTNOTES
1 Presented in part at The Endocrine Society's 87th Annual Meeting, June 2005 San Diego, CA [Lei C, Jodka CM, Hoyt JA, Adams SH. Effects of PYY[3-36] on energy expenditure (EE), respiratory quotient (RQ), and adiposity in diet-induced obese (DIO) mice. (Poster P3-12)]. 
3 Present address: USDA-ARS Western Human Nutrition Research Center, University of California, Davis CA 95616.
4 Abbreviations used: ACC, acetyl-CoA carboxylase; BAT, brown adipose tissue; CPT, carnitine palmitoyltransferase; DEXA, dual-energy X-ray absorptiometry; DIO, diet-induced obese; ED50, dose yielding 50% of maximal response; HMGCS, hydroxymethylglutaryl-CoA synthase; MCD or MLYCD, malonyl-CoA decarboxylase; NEFA, nonesterified fatty acid; PYY, peptide YY; RQ, respiratory quotient; WAT, white adipose tissue.
Manuscript received 12 August 2005. Initial review completed 19 September 2005. Revision accepted 18 October 2005.
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