Intravenous Infusion of Peptide YY(3–36) Potently Inhibits Food Intake in Rats
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内分泌学杂志 2005年第2期
Department of Veterans Affairs–Nebraska Western Iowa Health Care System, Omaha, Nebraska 68105; and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska 68178
Address all correspondence and requests for reprints to: Roger D. Reidelberger, Ph.D., Department of Veterans Affairs–Nebraska Western Iowa Health Care System (151), 4101 Woolworth Avenue, Omaha, Nebraska 68105. E-mail: roger.reidelberger@med.va.gov.
Abstract
Peptide YY (3–36) [PYY (3–36)] is postulated to act as a hormonal signal from the gut to the brain to inhibit food intake and gastric emptying. A mixed-nutrient meal produces a prolonged 2–3 h increase in plasma levels of both PYY (3–36) and PYY (1–36). We determined the dose-dependent effects of 3-h iv infusions of PYY (3–36) and PYY (1–36) (0.5–50 pmol·kg–1·min–1) at dark onset on food intake in non-food-deprived rats. PYY (3–36) dose-dependently inhibited food intake: the minimal effective dose was 5 pmol·kg–1·min–1; the estimated potency (mean effective dose) and efficacy (maximal percent inhibition) were 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively. PYY (1–36) was an order of magnitude less potent than PYY (3–36). Similar total doses of PYY (3–36) (0.9–30 nmol/kg) infused during the 15-min period just before dark onset also dose-dependently inhibited food intake, albeit with a lower potency and efficacy. Other experiments showed that PYY (3–36) inhibited food intake in sham-feeding rats and was more effective in reducing intake of a mixed-nutrient liquid diet than 15% aqueous sucrose. We conclude that: 1) iv infusions of PYY (3–36), which are more likely than ip injections to mimic postprandial increases in plasma PYY (3–36), potently inhibit food intake in a dose-dependent manner; 2) PYY (1–36) is an order of magnitude less potent than PYY (3–36); and 3) PYY (3–36) can inhibit food intake independently of its action to inhibit gastric emptying. It remains to be determined whether iv doses of PYY (3–36) that reproduce postprandial increases in plasma PYY (3–36) are sufficient to inhibit food intake.
Introduction
PEPTIDE YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide comprise the pancreatic polypeptide-fold family of structurally related brain-gut peptides. PYY is synthesized in the gastrointestinal tract as well as the central and peripheral nervous systems (1, 2, 3, 4, 5, 6, 7, 8, 9). The two major molecular forms of PYY found in gut and circulation are PYY (1–36) and PYY (3–36) (10, 11, 12). Endocrine cells of the ileum, colon, and rectum provide a major source of PYY (13, 14). PYY (3–36) binding sites have been identified in numerous brain regions with moderate to high densities of binding found in the olfactory bulb, hippocampus, subfornical organ, lateral posterior thalamic nucleus, piriform cortex, septal complex, the medial preoptic area and lateral and arcuate nuclei of the hypothalamus, area postrema, and the nucleus of the solitary tract (15, 16).
Food intake releases PYY (1–36) and PYY (3–36) into the circulation (11, 12, 13, 14). Systemic administration of one or both of these PYY isoforms has been reported to inhibit food intake, gastric emptying, intestinal fluid and electrolyte secretion, gallbladder contraction, and exocrine pancreatic secretion (17). It remains to be determined whether either isoform acts physiologically by endocrine, neurocrine, and/or paracrine mechanisms to produce these effects. If a PYY isoform acts as a blood-borne hormonal signal to produce an effect, then it is important to determine whether the effect is produced by iv doses of the isoform that reproduce meal-induced increases in plasma levels of the isoform.
Food intake increases plasma levels of PYY immunoreactivity in humans from about 15 to 25 pM within 30 min, a level that is sustained for at least 2 h (18, 19). PYY (3–36) accounts for 37 and 54% of total PYY immunoreactivity in basal and postprandial plasma, respectively (11). In rats, a liquid, mixed-nutrient meal increases plasma PYY immunoreactivity from a basal level of 42 pM to a peak of 160 pM within 30 min; plasma PYY then gradually declines to basal levels over a 3-h period (20). The assay used in this study did not differentiate between PYY (1–36) and PYY (3–36). Batterham et al. (21) reported that a bolus ip injection of PYY (3–36) in rats produces a transient increase in plasma PYY (3–36) that peaks at 15 min. Together these results suggest that meal-induced increases in plasma levels of PYY isoforms are likely to be better simulated by continuous iv rather than bolus ip administration of the peptides.
Batterham et al. first reported that the gut-brain peptide PYY (3–36) inhibits food intake when administered by ip injection to rats and mice (21) and iv infusion to humans (18, 21). They also reported that twice-daily ip injections of PYY (3–36) to rats for 7 d produced a sustained decrease in body weight gain. Other investigators have since confirmed that ip injections of PYY (3–36) decrease food intake in rodents (22, 23, 24, 25, 26). In contrast, numerous studies by 42 investigators from 12 laboratories failed to show that ip injections of PYY (3–36) reduce food intake or body weight in rodents (27). One of these studies did show that continuous sc administration of PYY (3–36) by osmotic pump for 7 d reduced food intake in mice during the first 3 d but had no effect on body weight. Our initial interpretation of these studies was that continuous administration of PYY (3–36) might be a more reliable method than bolus injection for reducing food intake.
Factors that promote gastric distention by inhibiting gastric emptying can reduce food intake. We recently reported that iv infusion of PYY (3–36) potently inhibits gastric emptying in rats (28). If circulating PYY (3–36) reduces food intake in part by inhibiting gastric emptying, then it would be important to determine whether iv administration of PYY (3–36) reduces gastric emptying and food intake with a similar potency and whether PYY (3–36) reduces food intake in sham-feeding animals in which ingested food rapidly drains through a gastric cannula. No such studies have been reported.
The aims of the present study were to determine the dose-dependent effects of 3-h iv infusions of PYY (3–36) and PYY (1–36) at dark onset on food intake and meal parameters in non-food-deprived rats and determine whether shorter 15-min infusions of similar total doses of PYY (3–36) are less potent or effective. We also determined the effects of iv infusions of PYY (3–36) and PYY (1–36) on ingestion of liquid food in food-deprived rats with gastric cannulas closed (real feeding) and open (sham feeding).
Materials and Methods
Animals
Male Sprague Dawley rats (Charles River, Kingston, NY) weighing 300–485 g were housed individually in hanging wire-mesh cages in a room with controlled temperature (19–21 C) and a 12-h light, 12-h dark cycle (lights off at 1700 h). The animals were provided pelleted rat chow (Labdiet, 5001 Rodent diet, PMI Nutrition International, Brentwood, MO) and water ad libitum. The Animal Studies Subcommittee of the Omaha Veterans Affairs Medical Center approved the experimental protocol.
Peptides
Rat PYY (1–36) was purchased from Phoenix Pharmaceuticals Inc. (Belmont, CA). Rat PYY (3–36) was synthesized by solid-phase methodology using the Fmoc protection strategy and purified by reverse-phase HPLC as described elsewhere (28).
Surgical procedures
The procedure for implantation of a jugular vein catheter for peptide infusions has been described previously (29). Catheters were kept patent by flushing with 0.2 ml of 50% dextrose on alternate days and plugged with stainless steel wire. Rats used for sham-feeding studies were also implanted with a gastric cannula using an established procedure (30). The animals were allowed at least 1 wk to recover from surgery before being subjected to experimental procedures.
Experiments
Effects of 3-h iv infusions of PYY (3–36) and PYY (1–36) on solid food intake in non-food-deprived rats.
For each animal used in these experiments, the jugular vein catheter was connected to a 40-cm length of tubing passed through a protective spring coil connected between a light-weight saddle (IITC, Woodland Hills, CA) worn by the rat and an infusion swivel. Rats had ad libitum access to ground chow that was provided fresh each day at 1400 h. Animals were adapted to experimental conditions for at least 1 wk before the start of experiments. Non-food-deprived rats (n = 16) received a 3-h jugular vein infusion of PYY (3–36) (0, 0.5, 1.7, 5, 17, or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min) beginning 15 min before dark onset (1700 h). Food intake cumulated hourly for 17 h after dark onset, and meal parameters [meal size, number of meals, and satiety ratio (postmeal interval per meal size)] cumulated hourly during the 3-h infusion period were determined, as described previously, from continuous computer recordings of changes in food bowl weight (29). PYY (3–36) was administered via a syringe infusion pump (PHD2000, Harvard Apparatus, South Natick, MA); pumps were turned on and off by a computer program. Each rat received each dose of PYY (3–36) in random order at approximately 48-h intervals. At the end of the experiment, the patency of jugular vein catheters was determined by iv injection of 0.2 ml of the short-acting anesthetic Propoflo (Abbott Laboratories, North Chicago, IL). A catheter was considered patent if the rat lost consciousness immediately on injection of the anesthetic; only data from such Propoflo-positive rats were included in statistical analyses. In a separate experiment of identical design, rats (n = 16) received 3-h jugular vein infusions of PYY (1–36) (0, 1.7, 5, 17, and 50 pmol·kg–1· min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min).
Effects of 15-min iv infusions of PYY (3–36) on solid food intake in non-food-deprived rats.
Experiments were identical with those described above for the 3-h infusions of PYY (3–36) except for the doses and duration of PYY (3–36) infusion. Non-food-deprived rats (n = 16) received a 15-min jugular vein infusion of PYY (3–36) (0, 60, 200, 600, or 2000 pmol·kg–1· min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min) beginning 15 min before dark onset (1700 h). Food intake cumulated hourly for 17 h after dark onset, and meal parameters [meal size, number of meals, and satiety ratio (postmeal interval per meal size)] cumulated hourly for the first 3 h of the dark period were determined as described above. The total amounts of PYY (3–36) delivered at 60, 200, and 600 pmol·kg–1·min–1 (0.9, 3, and 9 nmol/kg, respectively) were equivalent to those delivered in the preceding experiment when PYY (3–36) was infused for 3 h at 5, 17, and 50 pmol·kg–1·min–1, respectively.
Effects of iv infusions of PYY (3–36) and PYY (1–36) on ingestion of liquid food in food-deprived rats with either gastric cannula closed (real feeding) or open (sham feeding).
Rats with jugular vein and gastric cannulas were adapted to the following procedures, which took about 1 wk. Every other day after an overnight food deprivation, rats were restrained in Bollman cages for approximately 60–90 min, stomachs were flushed with warm water, and 15% aqueous sucrose solution or the mixed-nutrient liquid diet Peptamen 1.5 (Nestle, Deerfield, IL; 1.5 kcal/ml, 18% protein, 49% carbohydrate, 33% fat, 550 mOsm/kg) was provided for 30 min. After adaptation to these procedures, in separate experiments, rats (n = 15) with either an open or closed gastric cannula received iv infusion of PYY (3–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min) for either 40 or 90 min beginning either 10 or 60 min, respectively, before 30-min access to 15% sucrose or Peptamen. Each rat received each treatment in random order at intervals of at least 48 h. During intervening days, rats had free access to rat chow and a liquid diet (Ensure Plus, Abbott Laboratories, Columbus, OH; 1.5 kcal/ml). Data from a sham-feeding experiment was considered valid only if the weight of the gastric contents recovered was equal to the weight of liquid consumed (± 10%). At the end of each experiment, the patency of jugular vein catheters was determined by iv injection of 0.2 ml of the short-acting anesthetic Propoflo. In a separate experiment of identical design, rats (n = 15) with either an open or closed gastric cannula received an iv infusion of PYY (1–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for 90 min beginning 60 min before 30 min access to Peptamen.
Statistical analyses
Values are presented as group means ± SE. Data from each of the experiments determining the dose-response effects of PYY (3–36) and PYY (1–36) on food intake and meal parameters (meal size, number of meals, and satiety ratio) were analyzed separately by one-way repeated-measures ANOVA. Planned comparisons of treatment means were evaluated by paired t tests; differences were considered significant if P < 0.05. A general nonlinear, least-squares curve-fitting method was used to fit the dose-response data for the effects of PYY (1–36) and PYY (3–36) on solid food intake during the first 3 h of the dark period to the following sigmoidal dose-response equation: Y = a + (d – a)/{1 + 10[(c – X) * b]}, where Y is the response, X is the logarithm of the administered dose, a is the response for 0 dose, d is the response for infinite dose, c is the logarithm of the dose producing a response halfway between a and d (ED50), and b denotes the steepness of the dose-response curve.
Results
Effects of 3-h iv infusions of PYY (3–36) on solid food intake in non-food-deprived rats
PYY (3–36) infusion during the first 3 h of the dark period dose-dependently inhibited cumulative food intake (Figs. 1A and 2A). The minimal effective dose (5 pmol·kg–1·min–1) produced a maximal inhibition of 41% at 1 h, which decreased to 7% inhibition by 9 h. The maximal effective dose (50 pmol·kg–1·min–1) produced a sustained inhibition of cumulative intake for 11 h, with a peak inhibition of 69% at 1 h, decreasing to 14% at 11 h. Nonlinear regression fitting of the dose-response data to the sigmoidal equation gave the following relationship between food intake during the 3-h infusion period and PYY (3–36) dose: intake (grams) = 7.79 – 3.70/{1 + 10 [(1.16 – X) * –1.23]} [r2 = 0.30; F(4,76) = 285, P < 0.0001]. The estimated potency (ED50) and efficacy (maximal percent inhibition) were 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively (Fig. 3).
FIG. 1. Effects of 3-h iv infusions of PYY (3–36) (A) and PYY (1–36) (B) on food intake in 13 and 12 rats, respectively. Non-food-deprived rats received a 3-h jugular vein infusion of each peptide beginning 15 min before dark onset. Food intake was determined from continuous computer recordings of changes in food bowl weight. *, P < 0.05, , P < 0.01, , P < 0.001, compared with the vehicle-treated group.
FIG. 2. Effects of 3-h iv infusions of PYY (3–36) on 3-h food intake and meal parameters in the experiment described in Fig. 1A. A, Amount of food consumed (g). B, Number of meals consumed. C, Meal size (g). D, Satiety ratio (minutes per gram; postmeal interval per meal size). *, P < 0.05, , P < 0.01, , P < 0.001, compared with the vehicle-treated group.
FIG. 3. Dose-response effects of iv infusions of PYY (3–36) and PYY (1–36) for either 15 min or 3 h on food intake in rats. Data are from those presented in Figs. 1 and 5. The curve for the 3-h infusions of PYY (3–36) represents a nonlinear regression fitting of data to a sigmoidal equation; the best fit equation was: food intake (grams) = 7.79 – 3.70/{1 + 10[(1.16 – log(dose)) * (–1.23)]} [r2 = 0.30, F(4, 76) = 285, P < 0.0001]. The estimated potency (ED50) and efficacy (maximal percent inhibition) were 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively. Regression fitting of the data for 3-h infusions of PYY (1–36) did not converge. For the 15-min infusions of PYY (3–36) the best fit equation was: food intake (grams) = 6.27 – 3.97/{1 + 10[(2.72 – log(dose)) * (–1.39)]} [r2 = 0.15, F(4, 59) = 142, P < 0.0001]. The estimated ED50 and efficacy were 530 pmol·kg–1·min–1 (7.9 nmol/kg) and 37%, respectively. *, P < 0.05, , P < 0.01, , P < 0.001, compared with the vehicle-treated group.
PYY (3–36) reduced food intake during the 3-h infusion period by reducing meal size and increasing the satiety ratio (postmeal interval per meal size) (Fig. 2, A–D). PYY (3–36) at 5 pmol·kg–1·min–1 reduced cumulative food intake at 2 and 3 h by 15 and 19%, respectively, yet had no significant effect on meal parameters. PYY (3–36) at 17 pmol·kg–1·min–1 reduced cumulative food intake at 1, 2, and 3 h by 49, 31, and 28%; reduced mean meal size at 2 and 3 h by 24 and 21%; and increased mean satiety ratio at 3 h by 55%. PYY (3–36) at 50 pmol·kg–1·min–1 reduced cumulative intake at 1, 2, and 3 h by 64, 43, and 43%; reduced mean meal size at 1, 2, and 3 h by 38, 38, and 39%; and increased mean satiety ratio at 1, 2, and 3 h by 316, 206, and 201%, respectively.
Effects of 3-h iv infusions of PYY (1–36) on solid food intake in non-food-deprived rats
PYY (1–36) infusion during the first 3 h of the dark period dose-dependently inhibited cumulative food intake (Fig. 1B and 4A). PYY (1–36) appeared to be about 10-fold less potent than PYY (3–36) in reducing food intake (Fig. 3). The minimal effective dose (17 pmol·kg–1·min–1) inhibited cumulative intake by 13 and 11% at 3 and 5 h, respectively. The maximal effective dose (50 pmol· kg–1·min–1) produced a sustained inhibition of cumulative intake for 17 h, with a peak inhibition of 27% at 3 h, decreasing to 9% at 17 h. Nonlinear regression fitting of the dose-response data to the sigmoidal equation was not successful due to the relatively low efficacy of PYY (1–36) over the dose range administered.
FIG. 4. Effects of 3-h iv infusions of PYY (1–36) on 3-h food intake and meal parameters in the experiment described in Fig. 1B. A, Amount of food consumed (g). B, Number of meals consumed. C, Meal size (g). D, Satiety ratio (minutes per gram; postmeal interval per meal size). *, P < 0.05, , P < 0.01, compared with the vehicle-treated group.
PYY (1–36) reduced food intake during the 3-h infusion period by decreasing the number of meals consumed and increasing the satiety ratio (Fig. 4, A–D). PYY (1–36) at 17 pmol·kg–1·min–1 reduced cumulative food intake at 3 h by 14% yet had no significant effect on meal parameters. PYY (1–36) at 50 pmol·kg–1·min–1 reduced cumulative food intake at 2 and 3 h by 20 and 27%, reduced the number of meals at 2 and 3 h by 21 and 20%, and increased the mean satiety ratio at 1 and 3 h by 75 and 25%, respectively.
Effects of 15-min iv infusions of PYY (3–36) on solid food intake in non-food-deprived rats
PYY (3–36) infusions during the 15-min period just before dark onset dose-dependently inhibited cumulative food intake, albeit with a potency and efficacy that were significantly less than those for the 3-h infusions of PYY (3–36) (Figs. 3, 5, and 6A). The minimal effective dose (60 pmol·kg–1·min–1) for the 15-min infusions produced a transient inhibition of 17% at 2 h. The maximal effective dose (2000 pmol·kg–1·min–1) produced a sustained inhibition of cumulative intake for 7 h, with a peak inhibition of 50% at 2 h, decreasing to 12% at 7 h. Nonlinear regression fitting of the dose-response data to the sigmoidal equation gave the following relationship between food intake during the first 3 h of the dark period and PYY (3–36) dose: intake (grams) = 6.27 – 3.97/{1 + 10[(2.72 – X) * –1.39]} [r2 = 0.15, F(4, 59) = 142, P < 0.0001]. The ED50 of 7.9 nmol/kg (530 pmol·kg–1·min–1) was 3-fold greater than that for the 3-h infusions of PYY (3–36) (2.6 nmol/kg) (Fig. 3). The 15-min infusions of PYY (3–36) were also less effective. The 3- and 9-nmol/kg doses of PYY (3–36) suppressed cumulative intake for 2–3 h when delivered over a 15-min period (200- and 600-pmol·kg–1· min–1) and for 11 h when delivered over a 3-h period (17- and 50-pmol·kg–1·min–1)(Figs. 1A and 5).
FIG. 5. Effects of 15-min iv infusions of PYY (3–36) on food intake in 12 rats. Non-food-deprived rats received a 15-min jugular vein infusion of PYY (3–36) beginning 15 min before dark onset. Food intake was determined from continuous computer recordings of changes in food bowl weight. *, P < 0.05, , P < 0.01, compared with the vehicle-treated group.
FIG. 6. Effects of 15 min iv infusions of PYY (3–36) on 3-h food intake and meal parameters in the experiment described in Fig. 5. A, Amount of food consumed (g). B, Number of meals consumed. C, Meal size (g). D, Satiety ratio (minutes per gram; postmeal interval per meal size). *, P < 0.05, , P < 0.01, compared with the vehicle-treated group.
The 15-min infusions of PYY (3–36) at dark onset reduced food intake during the first 3 h of the dark period primarily by reducing meal size and increasing the satiety ratio (Fig. 6, A–D). PYY (3–36) at 60 pmol·kg–1·min–1 reduced cumulative food intake at 2 h by 17%, reduced number of meals at 2 h by 17%, and increased mean satiety ratio at 1 h by 58%. PYY (3–36) at 200 pmol·kg–1·min–1 reduced cumulative food intake at 2 h by 25% and increased mean satiety ratio at 1 and 2 h by 122 and 63%, respectively. PYY (3–36) at 600 pmol·kg–1·min–1 reduced cumulative intake at 2 and 3 h by 33 and 18% and reduced mean meal size at 1, 2, and 3 h by 36, 24, and 10%, respectively. PYY (3–36) at 2000 pmol·kg–1·min–1 reduced cumulative intake at 2 and 3 h by 50 and 31%; reduced mean meal size at 1, 2, and 3 h by 34, 34, and 26%; and increased mean satiety ratio at 2 and 3 h by 105 and 54%, respectively.
Effects of iv infusions of PYY (3–36) and PYY (1–36) on ingestion of liquid food in food-deprived rats with either gastric cannula closed (real feeding) or open (sham feeding)
PYY (3–36) infusion inhibited food intake similarly in real-feeding and sham-feeding rats and was more effective in reducing intake of a mixed-nutrient liquid diet than 15% aqueous sucrose. PYY (3–36) infusion at 50 pmol·kg–1·min–1 had no significant effect on real feeding or sham feeding of sucrose, whether the PYY (3–36) infusion began 10 or 60 min before 30-min access to sucrose (Fig. 7, A and B). PYY (3–36) infusion at 50 pmol·kg–1·min–1 also had no significant effect on real feeding or sham feeding of Peptamen when the PYY (3–36) infusion began 10 min before 30-min access to Peptamen (Fig. 8A). In contrast, PYY (3–36) inhibited real feeding by 25% and sham feeding by 14%, when PYY (3–36) infusion began 60 min before access to Peptamen (Fig. 8B). Under the same conditions, PYY (1–36) infusion inhibited real feeding of Peptamen by 11% but had no effect on sham feeding of Peptamen (Fig. 9).
FIG. 7. Effects of iv infusion of PYY (3–36) on ingestion of 15% aqueous sucrose in 15 rats with either gastric cannula closed (real feeding) or open (sham feeding). After an overnight fast, rats with either an open or closed gastric cannula received an iv infusion of PYY (3–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for either 40 (A) or 90 min (B) beginning either 10 or 60 min, respectively, before 30-min access to sucrose.
FIG. 8. Effects of iv infusion of PYY (3–36) on ingestion of the mixed-nutrient liquid diet Peptamen in 15 rats with either gastric cannula closed (real feeding) or open (sham feeding). After an overnight fast, rats with either an open or closed gastric cannula received an iv infusion of PYY (3–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for either 40 (A) or 90 min (B) beginning either 10 or 60 min, respectively, before 30-min access to Peptamen. *, P < 0.05, , P < 0.001, compared with the vehicle-treated group.
FIG. 9. Effects of iv infusion of PYY (1–36) on ingestion of the mixed-nutrient liquid diet Peptamen in 15 rats with either gastric cannula closed (real feeding) or open (sham feeding). After an overnight fast, rats with either an open or closed gastric cannula received an iv infusion of PYY (1–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for 90 min beginning 60 min before 30-min access to Peptamen. *, P < 0.05, compared with the vehicle-treated group.
Discussion
Our results demonstrate several important properties of the effects of the gut-brain PYY isoforms PYY (3–36) and PYY (1–36) on food intake. First, PYY (3–36) dose-dependently inhibits food intake when administered by continuous iv infusion to non-food-deprived rats during the first 3 h of the dark period. The estimated potency (ED50) and efficacy (maximal percent inhibition) are 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively. Second, PYY (1–36) is an order of magnitude less potent than PYY (3–36) in decreasing food intake. Third, similar total doses of PYY (3–36) (0.9–30 nmol/kg) infused during the 15-min period just before dark onset also dose-dependently inhibit food intake in non-food-deprived rats, albeit with a reduced potency and efficacy. Fourth, PYY (3–36) reduces food intake by decreasing meal size and increasing the satiety ratio (postmeal interval per meal size). Fifth, PYY (3–36) is more potent and/or efficacious in reducing ingestion of a mixed-nutrient liquid diet than 15% aqueous sucrose. And sixth, PYY (3–36) inhibits food intake in sham-feeding rats, indicating that PYY (3–36) can reduce food intake independently of its action to inhibit gastric emptying.
Using the same experimental model, we previously determined the effects of 3-h iv infusions of cholecystokinin (CCK)-8, amylin, calcitonin (CT), salmon CT, CT gene-related peptide, and adrenomedullin at dark onset on food intake in non-food-deprived rats (31, 32). The rank order of ED50s [pmol·kg–1·min–1] of these peptides including PYY (3–36) are salmon CT [1], amylin [6], PYY (3–36) [15], CCK8 [18], CT-gene-related peptide [26], and adrenomedullin [35]. Thus, PYY (3–36) has a potency for suppressing food intake that is similar to that for the putative satiety peptides CCK and amylin.
Batterham et al. (18, 21) first reported that exogenous PYY (3–36) inhibits food intake when administered by ip injection to rats and mice (21) and iv infusion to humans. They also reported that twice-daily ip injections of PYY (3–36) to rats for 7 d produced a sustained decrease in body weight gain. Other investigators have since confirmed that ip injections of PYY (3–36) decrease food intake in rodents (22, 23, 24, 25, 26). In contrast, numerous studies by 42 investigators from 12 laboratories have failed to demonstrate that ip injections of PYY (3–36) reduce food intake or body weight in rodents (27). One of these studies did show, however, that continuous sc administration of PYY (3–36) by osmotic pump for 7 d reduced food intake in mice during the first 3 d of administration. Our initial interpretation of these studies was that continuous administration of PYY (3–36) may be a more reliable method than bolus administration for reducing food intake. Batterham et al. (21) reported that bolus ip injection of PYY (3–36) in rats produces a transient increase in plasma PYY (3–36) that peaks at 15 min. We show here that 15-min iv infusions of PYY (3–36) at dark onset dose-dependently inhibit food intake in non-food-deprived rats, albeit with a potency and efficacy that are less than those produced by 3-h iv infusions of PYY (3–36) during the first 3 h of the dark period. Thus, PYY (3–36) dose-dependently inhibits food intake in non-food-deprived rats whether administered iv at dark onset for 15 min or 3 h.
The mechanisms through which PYY (3–36) inhibits food intake have not been clearly defined. Batterham et al. (21) proposed that PYY (3–36) acts as a hormonal signal from the gut to the brain to inhibit food intake. If this is true, then it would be important to determine whether postprandial increases in plasma PYY (3–36) are sufficient to inhibit food intake. Batterham et al. (21) reported that ip injection of an anorexic dose of PYY (3–36) increases plasma PYY (3–36) in rats to a level that is similar to that produced by food intake in rats. However, PYY (3–36) administered ip could act locally to affect feeding before being absorbed into the circulation, which would preclude the establishment of a meaningful correlation between the feeding response to PYY (3–36) and an increase in plasma PYY (3–36). In the same paper, Batterham et al. provided evidence that iv infusion of PYY (3–36) inhibits food intake in humans when administered at a dose that increases plasma immunoreactive PYY to a level that is within the normal postprandial range previously reported for humans. However, the assay methodology used in their studies did not differentiate between PYY (1–36) and PYY (3–36), and PYY (3–36) accounts for only about 50% of total PYY immunoreactivity in postprandial human blood (11). Thus, it remains to be established that PYY (3–36) acts as a blood-borne signal to decrease food intake in humans or rats.
We are not aware of any reports that have compared the effects of systemic administration of PYY (3–36) and PYY (1–36) on food intake. Our results demonstrate that PYY (3–36) is about an order of magnitude more potent than PYY (1–36) in reducing food intake in rats. Possible reasons for this difference in potency include differences in plasma-clearance rates of PYY (3–36) and PYY (1–36), their access to target tissues (e.g. brain uptake), and/or their receptor binding properties. PYY (1–36) and PYY (3–36) have been reported to have similar plasma half-lives of 8–12 min in dogs (33). Plasma half-lives of PYY (1–36) and PYY (3–36) in rats, and the relative blood-brain barrier permeabilities of each peptide, remain to be determined.
PYY (1–36) and PYY (3–36) bind with various degrees of affinity to at least six receptor subtypes, Y1 to Y6, all of which have been cloned except for the Y3 receptor (17). Ligand binding studies show that PYY (1–36) binds to Y1, Y2, Y4, and Y5 receptors, whereas PYY (3–36) selectively binds to Y2 receptors with an affinity that is similar to that exhibited by PYY (1–36) (17, 34). Differences in the tertiary structure of both forms likely explain the differences in their ligand binding affinities (34, 35). The juxtaposition of both the amino- and carboxy-terminals of the molecule is required for Y1 receptor binding, whereas only the carboxy-terminal helix confers selectivity for Y2 receptor binding (34). PYY (1–36) contains both structural features, and hence it can bind to both Y1 and Y2 receptors. Several studies described further below suggest that PYY (3–36) inhibits food intake by acting at central Y2 receptors and that PYY (1–36) potently stimulates food intake by acting at central Y1 or Y5 receptors (36). These results, together with our finding that PYY (3–36) is more potent than PYY (1–36) in suppressing food intake, suggest that PYY (3–36) and PYY (1–36) may each act at Y2 receptors to inhibit food intake, whereas PYY (1–36) might also act at Y1 receptors to stimulate food intake, which counteracts its inhibitory action through Y2 receptors.
Several lines of evidence suggest that circulating PYY (3–36) may gain access to the brain to activate Y2 receptors in the arcuate nucleus of the hypothalamus to inhibit food intake. First, high levels of Y2 mRNA expression, moderate to high densities of PYY (3–36) binding, and activation of Y2 receptors by agonist-stimulated binding of [35S]GTP have been detected in the hypothalamus of rats as well as the hippocampus, thalamus, and brain stem areas (16, 37, 38). Second, central administration of PYY (3–36), or the Y2 agonist N-acetyl [Leu28, Leu31] NPY (24–36), into the arcuate but not the paraventricular nucleus of the hypothalamus dose-dependently inhibits food intake in rats (21), whereas administration of PYY (3–36) into the cerebral ventricles either has no effect or potently increases food intake (27). Third, in rodents, ip injection of PYY (3–36) decreases NPY mRNA expression in the hypothalamus (21, 22), either has no effect (21) or increases proopiomelanocortin (POMC) mRNA expression in the hypothalamus (22), causes a nearly 2-fold increase in the number of c-Fos-positive cells in the arcuate nucleus (21), and produces a 13% (23) to 260% increase in c-Fos-positive POMC neurons (21). Thus, Batterham et al. (21) postulated that the anorexic response to circulating PYY (3–36) is mediated by a Y2 receptor-mediated mechanism in the arcuate nucleus, which inhibits NPY neurons and disinhibits POMC neurons. However, mice lacking POMC have since been found to retain sensitivity to the acute anorectic effects of ip injection of PYY (3–36) (39). Thus, it appears that melanocortin signaling is not essential for the anorexic effects of PYY (3–36) and that the peptide could act by atypical mechanisms on POMC neurons, at other sites in the central nervous system, or in the periphery.
The contribution of the brain stem in mediating the anorexic response to PYY (3–36) is less clear. Intraperitoneal injection of PYY (3–36) produces a nonsignificant increase in c-Fos in the nucleus of the solitary tract (23). Ablation of the area postrema was also found to enhance, rather than diminish, the anorexic response to PYY (3–36) in rats (26). Thus, PYY (3–36) may act in part in the area postrema to increase rather than decrease food intake.
Factors that promote gastric distention by inhibiting gastric emptying can reduce food intake. If circulating PYY (3–36) reduces food intake in part by inhibiting gastric emptying, then it would be important to determine whether iv administration of PYY (3–36) reduces gastric emptying and food intake with a similar potency. We recently demonstrated that PYY (3–36) inhibits gastric emptying of liquid in rats with a minimal effective dose of 5 pmol·kg–1·min–1 and an ED50 of 37 pmol·kg–1·min–1 (28), which is comparable with those observed in the present study for the effects of 3-h infusions of PYY (3–36) on food intake (5 and 15 pmol·kg–1·min–1, respectively). PYY (3–36) was also an order of magnitude more potent than PYY (1–36) in inhibiting gastric emptying.
In an attempt to further assess the role of gastric emptying in mediating the anorexic response to PYY (3–36), in the present study, we determined the effects of iv infusion of PYY (3–36) on real feeding and sham feeding of liquid food in food-deprived rats. In the sham-feeding model, in which ingested liquid rapidly drains through a gastric cannula, the negative feedback effect of gastric distention on food intake that can result from peptide-induced inhibition of gastric emptying, is eliminated. We found that PYY (3–36) infusion at 50 pmol·kg–1·min–1 similarly inhibited real feeding and sham feeding of the mixed-nutrient liquid diet Peptamen but had no effect on ingestion of 15% aqueous sucrose. These results suggest that PYY (3–36) can inhibit food intake independently of its action to inhibit gastric emptying. It is not clear why PYY (3–36) reduced ingestion of rat chow and Peptamen but not 15% sucrose. A plausible explanation is that the mixed-nutrient diets evoke mechanism(s) that interact with PYY (3–36) to inhibit food intake. What these mechanism(s) might be is not clear.
In summary, the present study demonstrates that: 1) iv infusions of PYY (3–36), which are more likely than ip injections to mimic postprandial increases in plasma PYY (3–36), potently inhibit food intake in a dose-dependent manner; 2) PYY (1–36) is an order of magnitude less potent than PYY (3–36); 3) PYY (3–36) reduces food intake by decreasing meal size and increasing the satiety ratio; and 4) PYY (3–36) can inhibit food intake independently of its action to inhibit gastric emptying. These results support the hypothesis that PYY (3–36) acts as a hormonal signal from the gut to the brain to inhibit food intake. It remains to be determined whether iv doses of PYY (3–36) that reproduce postprandial increases in plasma PYY (3–36) are sufficient to inhibit food intake.
Acknowledgments
We thank Linda Kelsey and Dean Heimann for their expert technical assistance.
References
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Address all correspondence and requests for reprints to: Roger D. Reidelberger, Ph.D., Department of Veterans Affairs–Nebraska Western Iowa Health Care System (151), 4101 Woolworth Avenue, Omaha, Nebraska 68105. E-mail: roger.reidelberger@med.va.gov.
Abstract
Peptide YY (3–36) [PYY (3–36)] is postulated to act as a hormonal signal from the gut to the brain to inhibit food intake and gastric emptying. A mixed-nutrient meal produces a prolonged 2–3 h increase in plasma levels of both PYY (3–36) and PYY (1–36). We determined the dose-dependent effects of 3-h iv infusions of PYY (3–36) and PYY (1–36) (0.5–50 pmol·kg–1·min–1) at dark onset on food intake in non-food-deprived rats. PYY (3–36) dose-dependently inhibited food intake: the minimal effective dose was 5 pmol·kg–1·min–1; the estimated potency (mean effective dose) and efficacy (maximal percent inhibition) were 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively. PYY (1–36) was an order of magnitude less potent than PYY (3–36). Similar total doses of PYY (3–36) (0.9–30 nmol/kg) infused during the 15-min period just before dark onset also dose-dependently inhibited food intake, albeit with a lower potency and efficacy. Other experiments showed that PYY (3–36) inhibited food intake in sham-feeding rats and was more effective in reducing intake of a mixed-nutrient liquid diet than 15% aqueous sucrose. We conclude that: 1) iv infusions of PYY (3–36), which are more likely than ip injections to mimic postprandial increases in plasma PYY (3–36), potently inhibit food intake in a dose-dependent manner; 2) PYY (1–36) is an order of magnitude less potent than PYY (3–36); and 3) PYY (3–36) can inhibit food intake independently of its action to inhibit gastric emptying. It remains to be determined whether iv doses of PYY (3–36) that reproduce postprandial increases in plasma PYY (3–36) are sufficient to inhibit food intake.
Introduction
PEPTIDE YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide comprise the pancreatic polypeptide-fold family of structurally related brain-gut peptides. PYY is synthesized in the gastrointestinal tract as well as the central and peripheral nervous systems (1, 2, 3, 4, 5, 6, 7, 8, 9). The two major molecular forms of PYY found in gut and circulation are PYY (1–36) and PYY (3–36) (10, 11, 12). Endocrine cells of the ileum, colon, and rectum provide a major source of PYY (13, 14). PYY (3–36) binding sites have been identified in numerous brain regions with moderate to high densities of binding found in the olfactory bulb, hippocampus, subfornical organ, lateral posterior thalamic nucleus, piriform cortex, septal complex, the medial preoptic area and lateral and arcuate nuclei of the hypothalamus, area postrema, and the nucleus of the solitary tract (15, 16).
Food intake releases PYY (1–36) and PYY (3–36) into the circulation (11, 12, 13, 14). Systemic administration of one or both of these PYY isoforms has been reported to inhibit food intake, gastric emptying, intestinal fluid and electrolyte secretion, gallbladder contraction, and exocrine pancreatic secretion (17). It remains to be determined whether either isoform acts physiologically by endocrine, neurocrine, and/or paracrine mechanisms to produce these effects. If a PYY isoform acts as a blood-borne hormonal signal to produce an effect, then it is important to determine whether the effect is produced by iv doses of the isoform that reproduce meal-induced increases in plasma levels of the isoform.
Food intake increases plasma levels of PYY immunoreactivity in humans from about 15 to 25 pM within 30 min, a level that is sustained for at least 2 h (18, 19). PYY (3–36) accounts for 37 and 54% of total PYY immunoreactivity in basal and postprandial plasma, respectively (11). In rats, a liquid, mixed-nutrient meal increases plasma PYY immunoreactivity from a basal level of 42 pM to a peak of 160 pM within 30 min; plasma PYY then gradually declines to basal levels over a 3-h period (20). The assay used in this study did not differentiate between PYY (1–36) and PYY (3–36). Batterham et al. (21) reported that a bolus ip injection of PYY (3–36) in rats produces a transient increase in plasma PYY (3–36) that peaks at 15 min. Together these results suggest that meal-induced increases in plasma levels of PYY isoforms are likely to be better simulated by continuous iv rather than bolus ip administration of the peptides.
Batterham et al. first reported that the gut-brain peptide PYY (3–36) inhibits food intake when administered by ip injection to rats and mice (21) and iv infusion to humans (18, 21). They also reported that twice-daily ip injections of PYY (3–36) to rats for 7 d produced a sustained decrease in body weight gain. Other investigators have since confirmed that ip injections of PYY (3–36) decrease food intake in rodents (22, 23, 24, 25, 26). In contrast, numerous studies by 42 investigators from 12 laboratories failed to show that ip injections of PYY (3–36) reduce food intake or body weight in rodents (27). One of these studies did show that continuous sc administration of PYY (3–36) by osmotic pump for 7 d reduced food intake in mice during the first 3 d but had no effect on body weight. Our initial interpretation of these studies was that continuous administration of PYY (3–36) might be a more reliable method than bolus injection for reducing food intake.
Factors that promote gastric distention by inhibiting gastric emptying can reduce food intake. We recently reported that iv infusion of PYY (3–36) potently inhibits gastric emptying in rats (28). If circulating PYY (3–36) reduces food intake in part by inhibiting gastric emptying, then it would be important to determine whether iv administration of PYY (3–36) reduces gastric emptying and food intake with a similar potency and whether PYY (3–36) reduces food intake in sham-feeding animals in which ingested food rapidly drains through a gastric cannula. No such studies have been reported.
The aims of the present study were to determine the dose-dependent effects of 3-h iv infusions of PYY (3–36) and PYY (1–36) at dark onset on food intake and meal parameters in non-food-deprived rats and determine whether shorter 15-min infusions of similar total doses of PYY (3–36) are less potent or effective. We also determined the effects of iv infusions of PYY (3–36) and PYY (1–36) on ingestion of liquid food in food-deprived rats with gastric cannulas closed (real feeding) and open (sham feeding).
Materials and Methods
Animals
Male Sprague Dawley rats (Charles River, Kingston, NY) weighing 300–485 g were housed individually in hanging wire-mesh cages in a room with controlled temperature (19–21 C) and a 12-h light, 12-h dark cycle (lights off at 1700 h). The animals were provided pelleted rat chow (Labdiet, 5001 Rodent diet, PMI Nutrition International, Brentwood, MO) and water ad libitum. The Animal Studies Subcommittee of the Omaha Veterans Affairs Medical Center approved the experimental protocol.
Peptides
Rat PYY (1–36) was purchased from Phoenix Pharmaceuticals Inc. (Belmont, CA). Rat PYY (3–36) was synthesized by solid-phase methodology using the Fmoc protection strategy and purified by reverse-phase HPLC as described elsewhere (28).
Surgical procedures
The procedure for implantation of a jugular vein catheter for peptide infusions has been described previously (29). Catheters were kept patent by flushing with 0.2 ml of 50% dextrose on alternate days and plugged with stainless steel wire. Rats used for sham-feeding studies were also implanted with a gastric cannula using an established procedure (30). The animals were allowed at least 1 wk to recover from surgery before being subjected to experimental procedures.
Experiments
Effects of 3-h iv infusions of PYY (3–36) and PYY (1–36) on solid food intake in non-food-deprived rats.
For each animal used in these experiments, the jugular vein catheter was connected to a 40-cm length of tubing passed through a protective spring coil connected between a light-weight saddle (IITC, Woodland Hills, CA) worn by the rat and an infusion swivel. Rats had ad libitum access to ground chow that was provided fresh each day at 1400 h. Animals were adapted to experimental conditions for at least 1 wk before the start of experiments. Non-food-deprived rats (n = 16) received a 3-h jugular vein infusion of PYY (3–36) (0, 0.5, 1.7, 5, 17, or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min) beginning 15 min before dark onset (1700 h). Food intake cumulated hourly for 17 h after dark onset, and meal parameters [meal size, number of meals, and satiety ratio (postmeal interval per meal size)] cumulated hourly during the 3-h infusion period were determined, as described previously, from continuous computer recordings of changes in food bowl weight (29). PYY (3–36) was administered via a syringe infusion pump (PHD2000, Harvard Apparatus, South Natick, MA); pumps were turned on and off by a computer program. Each rat received each dose of PYY (3–36) in random order at approximately 48-h intervals. At the end of the experiment, the patency of jugular vein catheters was determined by iv injection of 0.2 ml of the short-acting anesthetic Propoflo (Abbott Laboratories, North Chicago, IL). A catheter was considered patent if the rat lost consciousness immediately on injection of the anesthetic; only data from such Propoflo-positive rats were included in statistical analyses. In a separate experiment of identical design, rats (n = 16) received 3-h jugular vein infusions of PYY (1–36) (0, 1.7, 5, 17, and 50 pmol·kg–1· min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min).
Effects of 15-min iv infusions of PYY (3–36) on solid food intake in non-food-deprived rats.
Experiments were identical with those described above for the 3-h infusions of PYY (3–36) except for the doses and duration of PYY (3–36) infusion. Non-food-deprived rats (n = 16) received a 15-min jugular vein infusion of PYY (3–36) (0, 60, 200, 600, or 2000 pmol·kg–1· min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min) beginning 15 min before dark onset (1700 h). Food intake cumulated hourly for 17 h after dark onset, and meal parameters [meal size, number of meals, and satiety ratio (postmeal interval per meal size)] cumulated hourly for the first 3 h of the dark period were determined as described above. The total amounts of PYY (3–36) delivered at 60, 200, and 600 pmol·kg–1·min–1 (0.9, 3, and 9 nmol/kg, respectively) were equivalent to those delivered in the preceding experiment when PYY (3–36) was infused for 3 h at 5, 17, and 50 pmol·kg–1·min–1, respectively.
Effects of iv infusions of PYY (3–36) and PYY (1–36) on ingestion of liquid food in food-deprived rats with either gastric cannula closed (real feeding) or open (sham feeding).
Rats with jugular vein and gastric cannulas were adapted to the following procedures, which took about 1 wk. Every other day after an overnight food deprivation, rats were restrained in Bollman cages for approximately 60–90 min, stomachs were flushed with warm water, and 15% aqueous sucrose solution or the mixed-nutrient liquid diet Peptamen 1.5 (Nestle, Deerfield, IL; 1.5 kcal/ml, 18% protein, 49% carbohydrate, 33% fat, 550 mOsm/kg) was provided for 30 min. After adaptation to these procedures, in separate experiments, rats (n = 15) with either an open or closed gastric cannula received iv infusion of PYY (3–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA; 50 μl/min) for either 40 or 90 min beginning either 10 or 60 min, respectively, before 30-min access to 15% sucrose or Peptamen. Each rat received each treatment in random order at intervals of at least 48 h. During intervening days, rats had free access to rat chow and a liquid diet (Ensure Plus, Abbott Laboratories, Columbus, OH; 1.5 kcal/ml). Data from a sham-feeding experiment was considered valid only if the weight of the gastric contents recovered was equal to the weight of liquid consumed (± 10%). At the end of each experiment, the patency of jugular vein catheters was determined by iv injection of 0.2 ml of the short-acting anesthetic Propoflo. In a separate experiment of identical design, rats (n = 15) with either an open or closed gastric cannula received an iv infusion of PYY (1–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for 90 min beginning 60 min before 30 min access to Peptamen.
Statistical analyses
Values are presented as group means ± SE. Data from each of the experiments determining the dose-response effects of PYY (3–36) and PYY (1–36) on food intake and meal parameters (meal size, number of meals, and satiety ratio) were analyzed separately by one-way repeated-measures ANOVA. Planned comparisons of treatment means were evaluated by paired t tests; differences were considered significant if P < 0.05. A general nonlinear, least-squares curve-fitting method was used to fit the dose-response data for the effects of PYY (1–36) and PYY (3–36) on solid food intake during the first 3 h of the dark period to the following sigmoidal dose-response equation: Y = a + (d – a)/{1 + 10[(c – X) * b]}, where Y is the response, X is the logarithm of the administered dose, a is the response for 0 dose, d is the response for infinite dose, c is the logarithm of the dose producing a response halfway between a and d (ED50), and b denotes the steepness of the dose-response curve.
Results
Effects of 3-h iv infusions of PYY (3–36) on solid food intake in non-food-deprived rats
PYY (3–36) infusion during the first 3 h of the dark period dose-dependently inhibited cumulative food intake (Figs. 1A and 2A). The minimal effective dose (5 pmol·kg–1·min–1) produced a maximal inhibition of 41% at 1 h, which decreased to 7% inhibition by 9 h. The maximal effective dose (50 pmol·kg–1·min–1) produced a sustained inhibition of cumulative intake for 11 h, with a peak inhibition of 69% at 1 h, decreasing to 14% at 11 h. Nonlinear regression fitting of the dose-response data to the sigmoidal equation gave the following relationship between food intake during the 3-h infusion period and PYY (3–36) dose: intake (grams) = 7.79 – 3.70/{1 + 10 [(1.16 – X) * –1.23]} [r2 = 0.30; F(4,76) = 285, P < 0.0001]. The estimated potency (ED50) and efficacy (maximal percent inhibition) were 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively (Fig. 3).
FIG. 1. Effects of 3-h iv infusions of PYY (3–36) (A) and PYY (1–36) (B) on food intake in 13 and 12 rats, respectively. Non-food-deprived rats received a 3-h jugular vein infusion of each peptide beginning 15 min before dark onset. Food intake was determined from continuous computer recordings of changes in food bowl weight. *, P < 0.05, , P < 0.01, , P < 0.001, compared with the vehicle-treated group.
FIG. 2. Effects of 3-h iv infusions of PYY (3–36) on 3-h food intake and meal parameters in the experiment described in Fig. 1A. A, Amount of food consumed (g). B, Number of meals consumed. C, Meal size (g). D, Satiety ratio (minutes per gram; postmeal interval per meal size). *, P < 0.05, , P < 0.01, , P < 0.001, compared with the vehicle-treated group.
FIG. 3. Dose-response effects of iv infusions of PYY (3–36) and PYY (1–36) for either 15 min or 3 h on food intake in rats. Data are from those presented in Figs. 1 and 5. The curve for the 3-h infusions of PYY (3–36) represents a nonlinear regression fitting of data to a sigmoidal equation; the best fit equation was: food intake (grams) = 7.79 – 3.70/{1 + 10[(1.16 – log(dose)) * (–1.23)]} [r2 = 0.30, F(4, 76) = 285, P < 0.0001]. The estimated potency (ED50) and efficacy (maximal percent inhibition) were 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively. Regression fitting of the data for 3-h infusions of PYY (1–36) did not converge. For the 15-min infusions of PYY (3–36) the best fit equation was: food intake (grams) = 6.27 – 3.97/{1 + 10[(2.72 – log(dose)) * (–1.39)]} [r2 = 0.15, F(4, 59) = 142, P < 0.0001]. The estimated ED50 and efficacy were 530 pmol·kg–1·min–1 (7.9 nmol/kg) and 37%, respectively. *, P < 0.05, , P < 0.01, , P < 0.001, compared with the vehicle-treated group.
PYY (3–36) reduced food intake during the 3-h infusion period by reducing meal size and increasing the satiety ratio (postmeal interval per meal size) (Fig. 2, A–D). PYY (3–36) at 5 pmol·kg–1·min–1 reduced cumulative food intake at 2 and 3 h by 15 and 19%, respectively, yet had no significant effect on meal parameters. PYY (3–36) at 17 pmol·kg–1·min–1 reduced cumulative food intake at 1, 2, and 3 h by 49, 31, and 28%; reduced mean meal size at 2 and 3 h by 24 and 21%; and increased mean satiety ratio at 3 h by 55%. PYY (3–36) at 50 pmol·kg–1·min–1 reduced cumulative intake at 1, 2, and 3 h by 64, 43, and 43%; reduced mean meal size at 1, 2, and 3 h by 38, 38, and 39%; and increased mean satiety ratio at 1, 2, and 3 h by 316, 206, and 201%, respectively.
Effects of 3-h iv infusions of PYY (1–36) on solid food intake in non-food-deprived rats
PYY (1–36) infusion during the first 3 h of the dark period dose-dependently inhibited cumulative food intake (Fig. 1B and 4A). PYY (1–36) appeared to be about 10-fold less potent than PYY (3–36) in reducing food intake (Fig. 3). The minimal effective dose (17 pmol·kg–1·min–1) inhibited cumulative intake by 13 and 11% at 3 and 5 h, respectively. The maximal effective dose (50 pmol· kg–1·min–1) produced a sustained inhibition of cumulative intake for 17 h, with a peak inhibition of 27% at 3 h, decreasing to 9% at 17 h. Nonlinear regression fitting of the dose-response data to the sigmoidal equation was not successful due to the relatively low efficacy of PYY (1–36) over the dose range administered.
FIG. 4. Effects of 3-h iv infusions of PYY (1–36) on 3-h food intake and meal parameters in the experiment described in Fig. 1B. A, Amount of food consumed (g). B, Number of meals consumed. C, Meal size (g). D, Satiety ratio (minutes per gram; postmeal interval per meal size). *, P < 0.05, , P < 0.01, compared with the vehicle-treated group.
PYY (1–36) reduced food intake during the 3-h infusion period by decreasing the number of meals consumed and increasing the satiety ratio (Fig. 4, A–D). PYY (1–36) at 17 pmol·kg–1·min–1 reduced cumulative food intake at 3 h by 14% yet had no significant effect on meal parameters. PYY (1–36) at 50 pmol·kg–1·min–1 reduced cumulative food intake at 2 and 3 h by 20 and 27%, reduced the number of meals at 2 and 3 h by 21 and 20%, and increased the mean satiety ratio at 1 and 3 h by 75 and 25%, respectively.
Effects of 15-min iv infusions of PYY (3–36) on solid food intake in non-food-deprived rats
PYY (3–36) infusions during the 15-min period just before dark onset dose-dependently inhibited cumulative food intake, albeit with a potency and efficacy that were significantly less than those for the 3-h infusions of PYY (3–36) (Figs. 3, 5, and 6A). The minimal effective dose (60 pmol·kg–1·min–1) for the 15-min infusions produced a transient inhibition of 17% at 2 h. The maximal effective dose (2000 pmol·kg–1·min–1) produced a sustained inhibition of cumulative intake for 7 h, with a peak inhibition of 50% at 2 h, decreasing to 12% at 7 h. Nonlinear regression fitting of the dose-response data to the sigmoidal equation gave the following relationship between food intake during the first 3 h of the dark period and PYY (3–36) dose: intake (grams) = 6.27 – 3.97/{1 + 10[(2.72 – X) * –1.39]} [r2 = 0.15, F(4, 59) = 142, P < 0.0001]. The ED50 of 7.9 nmol/kg (530 pmol·kg–1·min–1) was 3-fold greater than that for the 3-h infusions of PYY (3–36) (2.6 nmol/kg) (Fig. 3). The 15-min infusions of PYY (3–36) were also less effective. The 3- and 9-nmol/kg doses of PYY (3–36) suppressed cumulative intake for 2–3 h when delivered over a 15-min period (200- and 600-pmol·kg–1· min–1) and for 11 h when delivered over a 3-h period (17- and 50-pmol·kg–1·min–1)(Figs. 1A and 5).
FIG. 5. Effects of 15-min iv infusions of PYY (3–36) on food intake in 12 rats. Non-food-deprived rats received a 15-min jugular vein infusion of PYY (3–36) beginning 15 min before dark onset. Food intake was determined from continuous computer recordings of changes in food bowl weight. *, P < 0.05, , P < 0.01, compared with the vehicle-treated group.
FIG. 6. Effects of 15 min iv infusions of PYY (3–36) on 3-h food intake and meal parameters in the experiment described in Fig. 5. A, Amount of food consumed (g). B, Number of meals consumed. C, Meal size (g). D, Satiety ratio (minutes per gram; postmeal interval per meal size). *, P < 0.05, , P < 0.01, compared with the vehicle-treated group.
The 15-min infusions of PYY (3–36) at dark onset reduced food intake during the first 3 h of the dark period primarily by reducing meal size and increasing the satiety ratio (Fig. 6, A–D). PYY (3–36) at 60 pmol·kg–1·min–1 reduced cumulative food intake at 2 h by 17%, reduced number of meals at 2 h by 17%, and increased mean satiety ratio at 1 h by 58%. PYY (3–36) at 200 pmol·kg–1·min–1 reduced cumulative food intake at 2 h by 25% and increased mean satiety ratio at 1 and 2 h by 122 and 63%, respectively. PYY (3–36) at 600 pmol·kg–1·min–1 reduced cumulative intake at 2 and 3 h by 33 and 18% and reduced mean meal size at 1, 2, and 3 h by 36, 24, and 10%, respectively. PYY (3–36) at 2000 pmol·kg–1·min–1 reduced cumulative intake at 2 and 3 h by 50 and 31%; reduced mean meal size at 1, 2, and 3 h by 34, 34, and 26%; and increased mean satiety ratio at 2 and 3 h by 105 and 54%, respectively.
Effects of iv infusions of PYY (3–36) and PYY (1–36) on ingestion of liquid food in food-deprived rats with either gastric cannula closed (real feeding) or open (sham feeding)
PYY (3–36) infusion inhibited food intake similarly in real-feeding and sham-feeding rats and was more effective in reducing intake of a mixed-nutrient liquid diet than 15% aqueous sucrose. PYY (3–36) infusion at 50 pmol·kg–1·min–1 had no significant effect on real feeding or sham feeding of sucrose, whether the PYY (3–36) infusion began 10 or 60 min before 30-min access to sucrose (Fig. 7, A and B). PYY (3–36) infusion at 50 pmol·kg–1·min–1 also had no significant effect on real feeding or sham feeding of Peptamen when the PYY (3–36) infusion began 10 min before 30-min access to Peptamen (Fig. 8A). In contrast, PYY (3–36) inhibited real feeding by 25% and sham feeding by 14%, when PYY (3–36) infusion began 60 min before access to Peptamen (Fig. 8B). Under the same conditions, PYY (1–36) infusion inhibited real feeding of Peptamen by 11% but had no effect on sham feeding of Peptamen (Fig. 9).
FIG. 7. Effects of iv infusion of PYY (3–36) on ingestion of 15% aqueous sucrose in 15 rats with either gastric cannula closed (real feeding) or open (sham feeding). After an overnight fast, rats with either an open or closed gastric cannula received an iv infusion of PYY (3–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for either 40 (A) or 90 min (B) beginning either 10 or 60 min, respectively, before 30-min access to sucrose.
FIG. 8. Effects of iv infusion of PYY (3–36) on ingestion of the mixed-nutrient liquid diet Peptamen in 15 rats with either gastric cannula closed (real feeding) or open (sham feeding). After an overnight fast, rats with either an open or closed gastric cannula received an iv infusion of PYY (3–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for either 40 (A) or 90 min (B) beginning either 10 or 60 min, respectively, before 30-min access to Peptamen. *, P < 0.05, , P < 0.001, compared with the vehicle-treated group.
FIG. 9. Effects of iv infusion of PYY (1–36) on ingestion of the mixed-nutrient liquid diet Peptamen in 15 rats with either gastric cannula closed (real feeding) or open (sham feeding). After an overnight fast, rats with either an open or closed gastric cannula received an iv infusion of PYY (1–36) (0 or 50 pmol·kg–1·min–1 in 0.15 M NaCl, 0.1% BSA) for 90 min beginning 60 min before 30-min access to Peptamen. *, P < 0.05, compared with the vehicle-treated group.
Discussion
Our results demonstrate several important properties of the effects of the gut-brain PYY isoforms PYY (3–36) and PYY (1–36) on food intake. First, PYY (3–36) dose-dependently inhibits food intake when administered by continuous iv infusion to non-food-deprived rats during the first 3 h of the dark period. The estimated potency (ED50) and efficacy (maximal percent inhibition) are 15 pmol·kg–1·min–1 (2.6 nmol/kg) and 47%, respectively. Second, PYY (1–36) is an order of magnitude less potent than PYY (3–36) in decreasing food intake. Third, similar total doses of PYY (3–36) (0.9–30 nmol/kg) infused during the 15-min period just before dark onset also dose-dependently inhibit food intake in non-food-deprived rats, albeit with a reduced potency and efficacy. Fourth, PYY (3–36) reduces food intake by decreasing meal size and increasing the satiety ratio (postmeal interval per meal size). Fifth, PYY (3–36) is more potent and/or efficacious in reducing ingestion of a mixed-nutrient liquid diet than 15% aqueous sucrose. And sixth, PYY (3–36) inhibits food intake in sham-feeding rats, indicating that PYY (3–36) can reduce food intake independently of its action to inhibit gastric emptying.
Using the same experimental model, we previously determined the effects of 3-h iv infusions of cholecystokinin (CCK)-8, amylin, calcitonin (CT), salmon CT, CT gene-related peptide, and adrenomedullin at dark onset on food intake in non-food-deprived rats (31, 32). The rank order of ED50s [pmol·kg–1·min–1] of these peptides including PYY (3–36) are salmon CT [1], amylin [6], PYY (3–36) [15], CCK8 [18], CT-gene-related peptide [26], and adrenomedullin [35]. Thus, PYY (3–36) has a potency for suppressing food intake that is similar to that for the putative satiety peptides CCK and amylin.
Batterham et al. (18, 21) first reported that exogenous PYY (3–36) inhibits food intake when administered by ip injection to rats and mice (21) and iv infusion to humans. They also reported that twice-daily ip injections of PYY (3–36) to rats for 7 d produced a sustained decrease in body weight gain. Other investigators have since confirmed that ip injections of PYY (3–36) decrease food intake in rodents (22, 23, 24, 25, 26). In contrast, numerous studies by 42 investigators from 12 laboratories have failed to demonstrate that ip injections of PYY (3–36) reduce food intake or body weight in rodents (27). One of these studies did show, however, that continuous sc administration of PYY (3–36) by osmotic pump for 7 d reduced food intake in mice during the first 3 d of administration. Our initial interpretation of these studies was that continuous administration of PYY (3–36) may be a more reliable method than bolus administration for reducing food intake. Batterham et al. (21) reported that bolus ip injection of PYY (3–36) in rats produces a transient increase in plasma PYY (3–36) that peaks at 15 min. We show here that 15-min iv infusions of PYY (3–36) at dark onset dose-dependently inhibit food intake in non-food-deprived rats, albeit with a potency and efficacy that are less than those produced by 3-h iv infusions of PYY (3–36) during the first 3 h of the dark period. Thus, PYY (3–36) dose-dependently inhibits food intake in non-food-deprived rats whether administered iv at dark onset for 15 min or 3 h.
The mechanisms through which PYY (3–36) inhibits food intake have not been clearly defined. Batterham et al. (21) proposed that PYY (3–36) acts as a hormonal signal from the gut to the brain to inhibit food intake. If this is true, then it would be important to determine whether postprandial increases in plasma PYY (3–36) are sufficient to inhibit food intake. Batterham et al. (21) reported that ip injection of an anorexic dose of PYY (3–36) increases plasma PYY (3–36) in rats to a level that is similar to that produced by food intake in rats. However, PYY (3–36) administered ip could act locally to affect feeding before being absorbed into the circulation, which would preclude the establishment of a meaningful correlation between the feeding response to PYY (3–36) and an increase in plasma PYY (3–36). In the same paper, Batterham et al. provided evidence that iv infusion of PYY (3–36) inhibits food intake in humans when administered at a dose that increases plasma immunoreactive PYY to a level that is within the normal postprandial range previously reported for humans. However, the assay methodology used in their studies did not differentiate between PYY (1–36) and PYY (3–36), and PYY (3–36) accounts for only about 50% of total PYY immunoreactivity in postprandial human blood (11). Thus, it remains to be established that PYY (3–36) acts as a blood-borne signal to decrease food intake in humans or rats.
We are not aware of any reports that have compared the effects of systemic administration of PYY (3–36) and PYY (1–36) on food intake. Our results demonstrate that PYY (3–36) is about an order of magnitude more potent than PYY (1–36) in reducing food intake in rats. Possible reasons for this difference in potency include differences in plasma-clearance rates of PYY (3–36) and PYY (1–36), their access to target tissues (e.g. brain uptake), and/or their receptor binding properties. PYY (1–36) and PYY (3–36) have been reported to have similar plasma half-lives of 8–12 min in dogs (33). Plasma half-lives of PYY (1–36) and PYY (3–36) in rats, and the relative blood-brain barrier permeabilities of each peptide, remain to be determined.
PYY (1–36) and PYY (3–36) bind with various degrees of affinity to at least six receptor subtypes, Y1 to Y6, all of which have been cloned except for the Y3 receptor (17). Ligand binding studies show that PYY (1–36) binds to Y1, Y2, Y4, and Y5 receptors, whereas PYY (3–36) selectively binds to Y2 receptors with an affinity that is similar to that exhibited by PYY (1–36) (17, 34). Differences in the tertiary structure of both forms likely explain the differences in their ligand binding affinities (34, 35). The juxtaposition of both the amino- and carboxy-terminals of the molecule is required for Y1 receptor binding, whereas only the carboxy-terminal helix confers selectivity for Y2 receptor binding (34). PYY (1–36) contains both structural features, and hence it can bind to both Y1 and Y2 receptors. Several studies described further below suggest that PYY (3–36) inhibits food intake by acting at central Y2 receptors and that PYY (1–36) potently stimulates food intake by acting at central Y1 or Y5 receptors (36). These results, together with our finding that PYY (3–36) is more potent than PYY (1–36) in suppressing food intake, suggest that PYY (3–36) and PYY (1–36) may each act at Y2 receptors to inhibit food intake, whereas PYY (1–36) might also act at Y1 receptors to stimulate food intake, which counteracts its inhibitory action through Y2 receptors.
Several lines of evidence suggest that circulating PYY (3–36) may gain access to the brain to activate Y2 receptors in the arcuate nucleus of the hypothalamus to inhibit food intake. First, high levels of Y2 mRNA expression, moderate to high densities of PYY (3–36) binding, and activation of Y2 receptors by agonist-stimulated binding of [35S]GTP have been detected in the hypothalamus of rats as well as the hippocampus, thalamus, and brain stem areas (16, 37, 38). Second, central administration of PYY (3–36), or the Y2 agonist N-acetyl [Leu28, Leu31] NPY (24–36), into the arcuate but not the paraventricular nucleus of the hypothalamus dose-dependently inhibits food intake in rats (21), whereas administration of PYY (3–36) into the cerebral ventricles either has no effect or potently increases food intake (27). Third, in rodents, ip injection of PYY (3–36) decreases NPY mRNA expression in the hypothalamus (21, 22), either has no effect (21) or increases proopiomelanocortin (POMC) mRNA expression in the hypothalamus (22), causes a nearly 2-fold increase in the number of c-Fos-positive cells in the arcuate nucleus (21), and produces a 13% (23) to 260% increase in c-Fos-positive POMC neurons (21). Thus, Batterham et al. (21) postulated that the anorexic response to circulating PYY (3–36) is mediated by a Y2 receptor-mediated mechanism in the arcuate nucleus, which inhibits NPY neurons and disinhibits POMC neurons. However, mice lacking POMC have since been found to retain sensitivity to the acute anorectic effects of ip injection of PYY (3–36) (39). Thus, it appears that melanocortin signaling is not essential for the anorexic effects of PYY (3–36) and that the peptide could act by atypical mechanisms on POMC neurons, at other sites in the central nervous system, or in the periphery.
The contribution of the brain stem in mediating the anorexic response to PYY (3–36) is less clear. Intraperitoneal injection of PYY (3–36) produces a nonsignificant increase in c-Fos in the nucleus of the solitary tract (23). Ablation of the area postrema was also found to enhance, rather than diminish, the anorexic response to PYY (3–36) in rats (26). Thus, PYY (3–36) may act in part in the area postrema to increase rather than decrease food intake.
Factors that promote gastric distention by inhibiting gastric emptying can reduce food intake. If circulating PYY (3–36) reduces food intake in part by inhibiting gastric emptying, then it would be important to determine whether iv administration of PYY (3–36) reduces gastric emptying and food intake with a similar potency. We recently demonstrated that PYY (3–36) inhibits gastric emptying of liquid in rats with a minimal effective dose of 5 pmol·kg–1·min–1 and an ED50 of 37 pmol·kg–1·min–1 (28), which is comparable with those observed in the present study for the effects of 3-h infusions of PYY (3–36) on food intake (5 and 15 pmol·kg–1·min–1, respectively). PYY (3–36) was also an order of magnitude more potent than PYY (1–36) in inhibiting gastric emptying.
In an attempt to further assess the role of gastric emptying in mediating the anorexic response to PYY (3–36), in the present study, we determined the effects of iv infusion of PYY (3–36) on real feeding and sham feeding of liquid food in food-deprived rats. In the sham-feeding model, in which ingested liquid rapidly drains through a gastric cannula, the negative feedback effect of gastric distention on food intake that can result from peptide-induced inhibition of gastric emptying, is eliminated. We found that PYY (3–36) infusion at 50 pmol·kg–1·min–1 similarly inhibited real feeding and sham feeding of the mixed-nutrient liquid diet Peptamen but had no effect on ingestion of 15% aqueous sucrose. These results suggest that PYY (3–36) can inhibit food intake independently of its action to inhibit gastric emptying. It is not clear why PYY (3–36) reduced ingestion of rat chow and Peptamen but not 15% sucrose. A plausible explanation is that the mixed-nutrient diets evoke mechanism(s) that interact with PYY (3–36) to inhibit food intake. What these mechanism(s) might be is not clear.
In summary, the present study demonstrates that: 1) iv infusions of PYY (3–36), which are more likely than ip injections to mimic postprandial increases in plasma PYY (3–36), potently inhibit food intake in a dose-dependent manner; 2) PYY (1–36) is an order of magnitude less potent than PYY (3–36); 3) PYY (3–36) reduces food intake by decreasing meal size and increasing the satiety ratio; and 4) PYY (3–36) can inhibit food intake independently of its action to inhibit gastric emptying. These results support the hypothesis that PYY (3–36) acts as a hormonal signal from the gut to the brain to inhibit food intake. It remains to be determined whether iv doses of PYY (3–36) that reproduce postprandial increases in plasma PYY (3–36) are sufficient to inhibit food intake.
Acknowledgments
We thank Linda Kelsey and Dean Heimann for their expert technical assistance.
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