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Pharmacology Department, Amylin Pharmaceuticals, Inc. (S.H.A., W.B.W., J.R.P.), San Diego, California 92121; and Division of Gastroenterology, Tufts-New England Medical Center (S.E.S., A.B.L.), Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Dr. Sean H. Adams, Pharmacology Department, Amylin Pharmaceuticals, Inc., 9360 Town Centre Drive, San Diego, California 92121. E-mail: sadams{at}amylin.com.
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Abstract |
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Introduction |
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MSH derived from proopiomelanocortin (POMC) neurons (1). There is a growing appreciation that gut-derived hormones also play a role in modulating energy balance through actions that regulate food intake, gastrointestinal function, and the efficiency of nutrient uptake and metabolism. For example, the gut-derived hormones ghrelin (2, 3, 4) and a cleavage product of peptide YY (PYY[3–36]) (5) have recently been implicated in the regulation of food intake and nutrient partitioning. Ghrelin is a 28-amino acid peptide highly expressed in the oxyntic X/A cells of the stomach (6), although expression has also been reported in hypothalamus (6, 7, 8), kidney (9), and elsewhere (10). The stomach appears to be the primary source of circulating ghrelin (11, 12). The peptide, processed from prepro-ghrelin, displays a unique octanoylation at Ser3, conferring the ability to activate the GH secretagogue receptor 1a (GHSR1a) (6). Interestingly, desacyl-ghrelin (lacking Ser3 acylation) is the predominant form of the peptide in blood (data herein) (9, 10, 13, 14) and may have physiological functions distinct from those of acyl-ghrelin. A role for acylated ghrelin as an anabolic, orexigenic hormone has been postulated based on several lines of evidence. First, administration of the peptide to rodents (2, 3, 15, 16, 17) and humans (18) increased short-term food intake coupled to increased hunger ratings in the human studies. Long-term administration in animals increased weight gain, food intake, and adiposity (2, 15, 19). Rodents treated with ghrelin also displayed a higher respiratory quotient (2). Second, circulating levels of total ghrelin increased with fasting (2, 15, 20), dropped with nutrient ingestion (2, 21, 22, 23), and rose before expected meal times in humans and meal-entrained sheep (21, 23). Third, ghrelin administration in rodents activates hypothalamic NPY/AgRP neurons, and functionally antagonizes POMC neurons (4, 7, 16, 24, 25). No effects on food intake were observed upon administration of ghrelin to GHSR1a knockout mice (17). These results have led to the proposal that circulating, stomach-derived ghrelin represents an important hunger-inducing signal, participating in meal initiation and long-term fat accretion via activation of GHSR1a in the arcuate nucleus (ARC) of the hypothalamus (1, 2, 4, 26).
PYY[1–36], expressed predominantly in endocrine cells of the lower small intestine and the colon (reviewed in Ref. 27), is posttranslationally processed by dipeptidyl-dipeptidase IV to the 34-amino acid PYY[3–36] (28). PYY[3–36] represents approximately half the total postprandial circulating PYY in humans (29). PYY[1–36] binds to all known NPY receptor subtypes, whereas PYY[3–36] is more discriminating, with high binding affinity to the Y2 and Y5 receptor subtypes (30). Recent findings suggest that PYY[3–36] has several properties that contrast with those of ghrelin. First, peripheral or intra-ARC administration of PYY[3–36] lowered acute food intake in rodents (5, 31, 32, 33) and when given iv to humans (5, 34). The Y2 receptor may mediate at least in part the anorectic effects of PYY[3–36], because Y2 receptor knockout mice failed to respond to the peptide (5). Second, the plasma concentration of total PYY decreases with fasting and increases after food ingestion (especially fat-rich meals) (29, 35). Interestingly, total PYY immunoreactivity is significantly decreased in human obesity (34, 36). Third, administration of PYY[3–36] may inactivate NPY/AgRP neurons and thus activate POMC neurons (5, 31). Based on these findings, it has been hypothesized that peripherally produced PYY[3–36] acts as a postmeal satiety factor, interacting with Y2 receptors in the ARC to down-regulate NPY/AgRP neurons and activate POMC pathways (1, 5, 26).
Thus, prevailing models would predict that PYY[3–36] and ghrelin influence similar hypothalamic pathways, albeit with opposing actions in terms of energy balance. This raises the possibility that the peptides have counterregulatory properties in vivo with respect to hormone activity at key brain feeding centers and/or at the level of secretion. To explore these possibilities, we conducted pharmacological studies to evaluate interactions between PYY[3–36] and ghrelin in mice. Because plasma levels of total ghrelin decreased upon short-term infusion of PYY[3–36] in humans (34), the hypothesis that PYY[3–36] decreases plasma ghrelin concentration as part of its anorexigenic mechanism of action was evaluated in rodents. Furthermore, the hypothesis that genetic ablation of PYY results in elevated circulating ghrelin due to disinhibition of pathways regulating this parameter was tested in PYY knockout mice. In addition, we asked whether the anorexigenic effects of PYY[3–36] are compromised under conditions of high physiological levels of circulating ghrelin or by an orexigenic dose of ghrelin. In our model, PYY[3–36] reduced short-term food intake regardless of the prevailing endogenous plasma ghrelin levels and without lowering the circulating ghrelin concentration. Furthermore, diminished food intake in response to PYY[3–36] remained robust despite coadministration of an orexigenic dose of ghrelin. Because peripherally derived ghrelin would be expected to influence NPY/AgRP and POMC circuits in the hypothalamus (4, 16, 17, 37, 38), the data support the hypothesis that anorexigenic properties of PYY[3–36] arise through alternative pathways. This hypothesis is bolstered by our observation that PYY[3–36] decreased acute food intake in agouti mice, which display down-regulated hypothalamic MSH signaling.
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Materials and Methods |
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Peptides
Synthetic human PYY[3–36] and acyl-ghrelin were synthesized in-house or purchased from Anaspec, Inc. (San Jose, CA), respectively. The purity and identity of the peptides were confirmed by mass spectroscopy. Peptide stock solutions in deionized water were aliquoted into polypropylene microcentrifuge tubes, lyophilized, and frozen (–20 C) until freshly redissolved in physiological saline on each experimental day. Injectates were prepared in borosilicate glass vials by dissolving an aliquot of stock solution in saline.
Ghrelin injectates were formulated to deliver doses of 84, 281, 845, and 2536 µg/kg. However, a post hoc study using an acyl-ghrelin-specific RIA (see below) indicated that injectates prepared in borosilicate glass delivered 10%, 43%, 45%, and 58% of the calculated doses noted above, respectively; therefore, the doses reported herein account for this fact. Preliminary results using spectrophotometric methods indicated negligible loss of PYY[3–36] in glass at all but the lowest injectate concentrations used (i.e. 30% loss; taken into account when reporting the two lowest doses herein).
Food intake measurements in response to PYY[3–36] or ghrelin administration
Depending on the study, animals were fasted overnight (
16 h) or were postabsorptive (food withdrawn 2–3 h before injection). Injections and subsequent food intake measurements commenced at 0900 h (early light cycle), except in some studies in which injections occurred at 1730 h (late light cycle). Mice were treated i.p. with 100 µl injectate, a preweighed food container was introduced, and, unless otherwise noted, food weights were determined at 30, 60, 120, 180, and 360 min postinjection. Consistent with the literature (4, 5, 15, 32, 39), the food intake responses to these peptides was most robust at the earliest time points after injection (see Results), so unless otherwise stated, graphs depict values derived from 30 min postinjection. Doses for PYY[3–36] were based on those reported to successfully lower food intake in this model (33).
Blood sampling and plasma hormone assays
Blood was collected into EDTA-flushed syringes via terminal cardiac puncture from mice lightly anesthetized with isoflurane (except in PYY knockouts, where conscious animals were bled via tail nick into sodium-heparin-coated tubes). Samples were placed on ice and promptly centrifuged (7200 x g, 3 min), and plasma was frozen at –80 C until analysis. Plasma ghrelin was measured in two separate RIAs to determine total (desacyl- plus acyl-ghrelin) and acyl-ghrelin; the latter employed an antibody specific to the octanoylated form of the peptide (Linco Research, Inc., St. Louis, MO). Both assays display 100% cross-reactivity among mouse, rat, and human ghrelin. Only acyl-ghrelin was analyzed in PYY knockout studies due to limited sample availability. Plasma GH was determined using a rat GH RIA (Linco Research, Inc.), whereas plasma total PYY (PYY[1–36] and PYY[3–36]) were determined by RIA using an antibody specific to human PYY (Linco Research, Inc.).
Statistics
Values are presented as the mean ± SEM, and sample sizes are noted in the figure legends. The effects of peptide dose or treatment on the parameters tested within a given time period were evaluated by one-way ANOVA, followed by Fisher’s protected least significant difference test (Systat 10.2, Systat, Richmond, CA). Statistical analyses of hormone concentrations did not include those samples yielding unacceptably high replicate variability or out of range values. Values were considered statistically significant at P < 0.05.
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Results |
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). As expected, an elevated ghrelin concentration was observed in overnight-fasted mice compared with postabsorptive animals sampled in the early light cycle. Interestingly, animals sampled during the late light cycle displayed significantly lower plasma ghrelin levels compared with other groups. Acyl-ghrelin represented 21%, 17%, and 14% of total plasma ghrelin in overnight-fasted, early light cycle, and late light cycle mice, respectively.
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). The effects of PYY[3–36] were not influenced by the prevailing circulating ghrelin levels at the time of injection, considering the wide range of plasma ghrelin concentrations observed under these conditions (Fig. 1
). At the highest dose, cumulative food intake remained significantly reduced at least through 180 min postinjection (Fig. 3A), an effect that was independent of the time of day (data not shown). PYY[3–36] injection in a separate cohort of mice increased the levels of total plasma PYY immunoreactivity significantly (P < 0.01–0.05) compared with saline-treated controls; at 15 min postinjection values were 140 ± 7, 12,404 ± 1,717, 23,464 ± 3,331, 28,208 ± 5,087, and 36,698 ± 3,317 pg/ml at doses of 0, 19, 92, 275, and 918 µg/kg, respectively.
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). Plasma acyl-ghrelin concentrations in the same samples were also unchanged relative to controls over 60 min postinjection, but were reduced at 120 min postinjection (Table 1).
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Orexigenic action of peripherally administered ghrelin is altered by fasting and time of day
Because one goal of our studies was to explore the food intake effects of coinjection of PYY[3–36] and ghrelin, it was imperative to establish whether ghrelin is an effective orexigen under conditions used in the PYY[3–36] food intake studies. In mice treated with the highest dose of ghrelin during the early light cycle, 60, 120, and 180 min food intakes were, respectively, 1.5-, 1.4-, and 1.3-fold higher compared with controls, but these differences did not achieve statistical significance (by ANOVA, P 
0.1; Fig. 3B). At 30 min postinjection, food intake increased significantly (up to 1.8-fold) in ghrelin-treated postabsorptive mice during the early light cycle (Fig. 4B), but this treatment was ineffective in overnight-fasted mice and animals injected during the late light cycle (Fig. 4, A and C).
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) approximately 3- to 10-fold greater than the high physiological concentration observed with overnight fasting (see Fig. 1). Considering that only a single postinjection time point was sampled (15 min), it is not known whether higher plasma ghrelin levels would have been observed at earlier or later times after injection. At the three highest doses of ghrelin, plasma GH increased significantly by approximately 5- to 15-fold compared with control levels (Fig. 5B), indicating peptide bioactivity and communication between injected ghrelin and CNS sites of action.
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). For instance, at the lowest dose of PYY[3–36] coinjected with ghrelin, cumulative food intake was normalized to control levels, and increasing doses of PYY[3–36] further decreased food intake (Fig. 6).
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MSH signaling), consistent with the hypothesis that peripherally administered PYY[3–36] engages different sites of action than circulating ghrelin. Because ghrelin is believed to inhibit hypothalamic POMC neuronal circuits (17, 37), we evaluated whether PYY[3–36] is anorexigenic when this pathway is compromised. PYY[3–36] administration significantly reduced short-term food intake in agouti (Ay) mice and their nonobese counterparts to the same extent (Fig. 7), indicating that the peptide has activities that do not require fully functional MSH signaling.
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Discussion |
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MSH cascade required for the anorexigenic effect of PYY[3–36]? Under each condition tested, PYY[3–36] administration at pharmacological doses diminished short-term food intake significantly in mice, consistent with observations in rats administered the peptide peripherally or via direct ARC injections (5). The data also concur with reports that examined PYY[3–36] effects on short-term food intake in mice (31, 32, 33). During the preparation of this manuscript, Halatchev et al. (32) reported that PYY[3–36] lowered intake in male C57BL/6J mice only after acclimation to daily handling and injection. However, PYY[3–36] reduced food intake in our naive mouse model, suggesting that strain, gender, or other factors influence its anorexigenic properties in rodents. The anorexigenic properties of PYY[3–36] in these rodent models coupled to evidence of diminished appetite and food intake in human subjects infused with the peptide (5, 34) support the idea that appetitive behavior is influenced by changes in circulating PYY[3–36]. Despite such strong associations, the degree to which normal physiological fluctuations in circulating PYY[3–36] impact meal patterns and energy balance remains to be clarified further.
PYY[3–36] administration lowered acute food intake regardless of prevailing plasma ghrelin levels, which differed significantly depending on time of day and nutritional state. Even in fasted animals where ghrelin levels are in the high physiological range, inhibition of short-term food intake after PYY[3–36] injection remained robust. These results demonstrate that pharmacological administration of PYY[3–36] may override putative counteracting orexigenic effects of endogenous ghrelin acting on shared CNS sites of action. Alternatively, the anorexigenic circuits engaged by PYY[3–36] may be distinct from the orexigenic pathways activated by ghrelin (see additional discussion below).
Over the time frame in which the anorexigenic effects of PYY[3–36] are apparent, injection of the peptide did not decrease circulating ghrelin levels. These results contrast with a human study in which 90-min peptide infusion significantly lowered the plasma total ghrelin concentration (34). Although this correlated with decreased appetite and food intake in humans, our results in mice indicate that diminution of circulating ghrelin levels is not universally associated with the anorexigenic effects of PYY[3–36]. Importantly, no difference in postabsorptive plasma acyl-ghrelin was observed in male PYY knockout mice (see Results), providing additional evidence of a dissociation between PYY activity and the regulation of ghrelin levels. However, a reduction in plasma acyl-ghrelin was observed at 2 h post-PYY[3–36] injection (Table 1). Thus, additional studies are required to elucidate whether genetic or pharmacological modulation of PYY[3–36] activity influences the circulating ghrelin concentration and to evaluate whether important differences exist between rodent and human responses.
Using postabsorptive mice in the early light cycle (where ghrelin increased food intake and PYY[3–36] had the opposite effect), we found that the anorexigenic properties of PYY[3–36] were not compromised by coinjection of an orexigenic dose of ghrelin. Coupled with the reduced food intake in PYY[3–36]-treated mice in the face of high physiological circulating ghrelin levels (see above), at least two possibilities emerge. First, the anorexigenic signals resulting from pharmacological administration of PYY[3–36] may be dominant to the orexigenic impact of ghrelin within shared pathways in the CNS. Second, PYY[3–36] may engage circuits whose activities are minimally impacted by ghrelin signaling. The latter concept is supported by the finding that PYY[3–36] decreased food intake in Ay mice to the same degree as in their nonobese counterparts (Fig. 7; also see Ref. 37); a similar result was reported in POMC (40) and melanocortin-4 receptor (MC4R) (32) knockout mice. Because signaling via the hypothalamic melanocortin system is inhibited in these animal models, these data are consistent with a site of PYY[3–36] action different from the hypothalamic NPY/AgRP-POMC neuronal circuit. Importantly, peripherally administered ghrelin loses its orexigenic activity in Ay (37) or in MC3R/MC4R knockout mice (17), and intracerebroventricular ghrelin is ineffective under conditions of diminished NPY activity (4), supporting the view that ghrelin activates hypothalamic NPY/AgRP neurons with subsequent inactivation of POMC (MSH-secreting) neurons. Also supporting the hypothesis that PYY[3–36] and ghrelin operate through different CNS circuits, Andersson et al. (38) recently reported that peripherally administered ghrelin and leptin regulate (in opposite directions) hypothalamic AMP kinase activity and acetyl-coenzyme A carboxylase phosphorylation, whereas PYY[3–36] did not influence these parameters. The brainstem has been implicated as an important hypothalamic partner in regulating energy balance (41), and this structure expresses the Y2 receptor and displays PYY[3–36] binding (42, 43). Therefore, it will be important to establish whether PYY[3–36] engages neurons in the brainstem that, in turn, modulate energy balance. In addition, the possibility that PYY[3–36] acts downstream of hypothalamic NPY/AgRP-POMC neurons, vis-à-vis the anorexigenic peptide brain-derived neurotrophic factor (44), or engages uncharacterized pathways remains an open question.
Early reports that ghrelin administration increases food intake, body weight, and adiposity in rodents (2, 3, 4) in combination with the preprandial rise in the concentration of the peptide in human plasma (21) sparked a great deal of attention to ghrelin’s potential metabolic role. More recently, elegant studies by Pinto et al. (25) demonstrated that ghrelin injections lead to remarkable shifts in hypothalamic neuronal wiring in mice, resulting in down-regulation of excitatory inputs and up-regulation of inhibitory inputs to POMC neurons (effects opposite those of leptin). These studies and others support the view that circulating ghrelin represents an important physiological regulator of energy balance, acting to increase food intake and partition calories toward adipose stores via activation of hypothalamic GHSR1a-positive NPY/AgRP neurons (1, 26). With this paradigm in mind, we were surprised to encounter marginal orexigenic effects of ghrelin in mice treated ip after fasting or in the late light cycle. Furthermore, the orexigenic activity of ghrelin in mice was only evident at plasma acyl-ghrelin concentrations at least 3-fold higher than the high physiological levels observed with fasting. In a separate study we found that cumulative food intake and retroperitoneal fat pad weights (controls, n = 16; ghrelin, n = 14) or body weight (controls, n = 32; ghrelin, n = 28) in nonobese NIH/Swiss female mice were unaffected by administration of ghrelin over approximately 2 wk (109 µg/kg/d, ip, twice daily, early and late light cycle injection) (Adams, S. H., and W. B. Won, unpublished observations). This occurred despite postinjection plasma acyl-ghrelin levels at least 3-fold higher than average fasting values (to 3239 ± 318 pg/ml), with a 4-fold increase in plasma GH compared with controls (to 13 ± 3 ng/ml; P < 0.01) at 15 min postinjection. Thus, results from our models underscore the need to clarify how physiological fluctuations in circulating ghrelin impact energy balance.
Several observations from the literature further highlight the necessity to delineate the metabolic properties of peripherally derived ghrelin. For instance, in human and rodent studies that demonstrate the peptide’s actions to promote positive energy balance (15, 18, 45), postadministration acyl-ghrelin levels appear to have been about 4-fold to more than 100-fold higher than upper fasted values (considering that acyl-ghrelin represents approximately 10–20% of the plasma total ghrelin pool) (data herein; also see Refs. 9 , 10 , and 13). Although the aforementioned rodent studies indicated that significant food intake increases may occur at doses as low as approximately 10 µg/kg (15), several reports have employed high doses (e.g. 8–15 mg/kg) to characterize ghrelin’s properties to increase food intake and adiposity (2, 17, 19). Interestingly, although diurnal patterns of circulating ghrelin appear to track feeding patterns in humans (21) and in meal-entrained sheep (23), this association is not apparent in studies with rats upon close examination of the data (20, 45). Finally, an overt metabolic phenotype is lacking in ghrelin or GHSR-1a knockout mice (46, 47), and often paradoxical patterns of circulating ghrelin are observed in obese subjects (48) or after gastric surgery-induced weight loss (49).
In summary, our results indicate that peripherally administered PYY[3–36] reduces short-term food intake in mice regardless of prevailing endogenous circulating ghrelin levels. A dissociation between the anorexigenic effects of PYY[3–36] and ghrelin secretion/action was evident in our model; food intake effects of PYY[3–36] were not associated with reduced plasma ghrelin levels, and PYY[3–36] lowered food intake despite coinjection of an orexigenic dose of ghrelin. We propose that PYY[3–36] has CNS sites of action distinct from the classic hypothalamic NPY/AgRP-POMC axis, distinguishing it from ghrelin, which probably engages this pathway as a primary event. The data also point to the need for additional evaluation of the physiological importance of gut-derived ghrelin in regulating normal diurnal patterns of food intake in rodents and humans.
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Acknowledgments |
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Footnotes |
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Received April 22, 2004.
Accepted for publication July 13, 2004.
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