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Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Nishi, Inada, Obihiro 080-8555, Japan
(Requests for offprints should be addressed to H Inoue who is now at Department of Animal and Grassland Research, National Agricultural Research Center for Kyushu Okinawa Region (KONARC), 2421 Suya, Goshi 861-1192, Japan; Email: fujino{at}obihiro.ac.jp)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, ghrelin is a peptide that is primarily produced in the oxyntic mucosa of the stomach of pigs (Govoni et al. 2005). In contrast to PYY3–36, ghrelin is known to have stimulatoryeffects on food intake in rats (Nakazato et al. 2001) and humans (Wren et al. 2001). In pigs, plasma PYY levels increase after meal intake (Adrian et al. 1987), plasma ghrelin levels rise during fasting (Salfen et al. 2003, Govoni et al. 2005). In addition, ghrelin infusion has also been shown to increase weight gain and plasma GH levels in pigs (Salfen et al. 2004). Previous studies in rats and humans suggest that more or less PYY3–36 and ghrelin may interact closely to regulate feed intake and energy expenditure. In pigs, so far, there are no reports on the anorexigenic and metabolic effects of PYY3–36. Therefore, in this study, we investigated the effects of PYY3–36 on feed intake and plasma PYY levels in pigs during ad libitum and fast-refed conditions. Furthermore, we examined whether PYY3–36 treatment could affect plasma acyl-ghrelin and growth hormone (GH) levels during fasting conditions.
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Materials and Methods |
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All experimental procedures were approved by the Institutional Animal Care and Use Committee of Obihiro University of Agriculture and Veterinary Medicine.
Crossbred (Large White x Landrace x Duroc) castrated male pigs were housed in individual pens with rubber slotted floors. Commercial diet (crude protein 16%, crude fat 2.5%, crude fiber 5%, and crude ash 7%) was available ad libitum and was supplied twice a day at 0900 and 1700 h and it showed that feed intake levels in all pigs were consistent prior to the start of experiment. Water was accessible all the time. Animals were anesthetized and indwelling catheters were inserted into the extra-jugular vein 3 days prior to treatment (Phung et al. 2000). To facilitate infusion of peptide and blood sampling at the same time, another catheter was inserted in the contralateral extra-jugular vein. Patency of the catheter was maintained with heparinized saline. All experiments were carried out after feed intake levels of the catheterized pigs returned to normal.
Peptides and method of administration
Porcine PYY3–36 (AKPEAPGEDASPEELSRYYASLR-HYLNLVTRQRY-NH2) and [Cys-0]-porcine PYY4–36 were synthesized by solid-phase peptide synthesis method using the Fmoc (9-fluorenylmethoxycarbonyl) protection strategy with Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu), Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Boc-Ala-OH, and Rink Amide MBHA resin and purified by reverse phase HPLC (TSKgel ODS-120A; TOSOH, linear gradient of 0–60% CH3CN). The purified peptides were lyophilized and stored at –30 °C. PYY3–36 was dissolved in 5 ml sterile saline to come up with a dose of 30 µg/kg body weight (BW) for single bolus i.v. injection. For infusion, PYY3–36 (0.25 µg/kg BW per min) was prepared just prior to administration via dilution in 120 ml saline and infused over a period of 120 min.
Blood sampling
The blood samples (5 ml each) were collected at – 30, 0, 15, 30, 60, 90, 120, 150, and 180 min relative to injection time, and at – 30, 0, 30, 60, 90, 120, 135, 150, and 180 min relative to the start of the infusion period. Blood samples were immediately transferred to centrifuge tubes containing heparin (10 IU/ml) and chilled on ice. Plasma samples were obtained after centrifugation at 3000 r.p.m. and 4 °C for 30 min, and stored at –30 °C until plasma assay. For acyl-ghrelin assay, 50 µl 1 M HCl/ml plasma was added to each sample, and stored at –30 °C until plasma acyl-ghrelin assay.
Feed intake measurement
In experiments 1 and 2, pre-weighed feed was given immediately after i.v. injection of PYY3–36. In experiment 3, pre-weighed feed was given after termination of infusion. Feed intake was measured at 1, 3, 6, 12, and 24 h after feed was given. The residual feed was returned to be given after measuring. Feed intake levels were calculated after monitoring the spillage of feed and expressed as feed consumed (g) per kg BW to minimize the large individual differences.
Experimental design
Experiment 1: Effect of i.v. injection of PYY3–36 on feed intake in ad libitum-fed pigs Five crossbred castrated pigs (initiation study BW ± S.E.M., 71.4 ± 2.2 kg) were continuously fed ad libitum prior to PYY3–36 treatment. Pigs were given a single bolus i.v. injection of sterile saline or PYY3–36 (30 µg/kg BW) at 0900 h. After injection, pre-weighed feed was given immediately, and intake levels were monitored until 24 h post-treatment. Blood sampling and feed intake measurements were carried out as mentioned earlier. Animals were repeatedly used in a randomized 2 x 2 crossover design and treatments were carried out at 2-day intervals.
Experiment 2: Effect of i.v. injection of PYY3–36 on feed intake during overnight fast-refed condition In this study, six overnight-fasted pigs (48.5 ± 1.3 kg BW) were initially used. Following PYY3–36 (30 µg/kg BW) injection, pre-weighed feed was immediately given. Blood sampling and feed intake measurements were carried out as mentioned earlier. Animals were repeatedly used in a randomized 2 x 2 crossover design and treatments were carried out at 2-day intervals.
Experiment 3: Effect of 2 h i.v. infusion of PYY3–36 on feed intake in fast-refed pigs To compare the effects of PYY3–36 according to the difference of administration procedure, we investigated the effects of i.v. infusion on feed intake and plasma PYY levels in fast-refed pigs. Five pigs (85.3 ± 2.9 kg BW) were studied in a randomized 2 x 2 crossover design and treatments were carried out at 2-day intervals. PYY3–36 (0.25 µg/kg BW per min) or saline were infused for 2 h (total 30 µg/kg BW per 120 min) during fasting. After infusion, pre-weighed feed was immediately given. Blood sampling and feed intake measurements were carried out as mentioned earlier.
Experiment 4: Effects of i.v. injection of PYY3–36 on plasma acyl-ghrelin and GH levels after an overnight fast To study the feeding-unrelated effect of PYY3–36 on plasma parameters, five pigs (81.2 ± 5.5 kg BW) were fasted overnight for ~6 h prior to the single bolus i.v. injections of either sterile saline or PYY3–36 (30 µg/kg BW) at 0900 h. Blood was collected as mentioned earlier. These pigs were repeatedly used in a randomized 2 x 2 crossover design and treatments were carried out at 2-day intervals.
Plasma assays
Plasma PYY, acyl-ghrelin, and GH concentrations were measured by double-antibody RIA procedures. For measurement of porcine PYY, polyclonal antibody for [Cys-0]-porcine PYY4–36 was generated in rabbit by a similar method as in the previously described study (ThidarMyint et al. 2006) and used at final dilution of 1/20 000. Synthesized porcine PYY3–36 was radioiodinated by the chloramine-T method (Tai et al. 1975) and purified by HPLC. Initially, PYY3–36 standard or plasma samples were incubated with anti-porcine PYYantiserum diluted in assay buffer (0.05 M phosphosaline containing 1% BSA, pH 7.4) and 125I-porcine PYY3–36 tracer (6000 c.p.m./100 µl assay buffer containing 1% normal rabbit serum). After 24-h incubation period, precipitating reagent containing goat anti-rabbit IgG was added to the reaction mixture and incubated for 30 min. The supernatant was discarded after centrifugation and radioactivity of the pallet was counted with a gamma counter (ARC 1000, Aloka, Japan). The antibody used in this assay system recognizes porcine PYY3–36 and PYY1–36 (422704; Phoenix Pharmaceuticals, Inc., Belmont, USA), but it does not recognize porcine NPY (491028; Peptide Institute, Inc. Japan), human PYY1–36 (422787; Phoenix), and human PP (421041; Phoenix) (Fig. 1
). Displacement curve of 125I-labeled porcine PYY3–36 with 25, 50, 75, and 100 µl porcine plasma was parallel to the standard curve. Recovery of known amounts of porcine PYY3–36 added to a pool of porcine plasma was 96.4% of the added amount. The average assay sensitivity for all studies was 0.1 ng/ml. The mean intra-assay coefficient of variance was 9.4%.
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Plasma GH concentrations were measured as previously described by Inoue et al.(2005). Porcine GH antiserum (AFP422801) and porcine GH (AFP10864B) were obtained from Dr A F Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, USA). The average recovery of the added amount was 107.7% and the mean intra-assay coefficient of variance was 12.6%.
Statistical analysis
All data are presented as means ± S.E.M. Feed intake was analyzed using one-way ANOVA. Hormonal data for individual time points between control and PYY3–36-treated groups were compared using Student’s paired t-test. All analyses were performed using SPSS for Windows, version 10.0.0 (SPSS, Chicago, IL, USA). P < 0.05 was considered significant.
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Results |
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Basal plasma PYY levels in the control group did not change throughout the experiment (2.2 ± 0.2 ng/ml). Plasma PYY levels were elevated after injection of porcine PYY3–36 (Fig. 2A) and were maintained at higher levels until 120 min compared with the values of saline-injected group.
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. Single bolus injection of PYY3–36 significantly reduced cumulative feed intake within 3-h post-treatment. Cumulative feed intake levels at 1 and 3 h after PYY3–36 treatment were reduced by 68.5 ± 18.0% (P < 0.01) and 44.2 ± 18.6% (P < 0.05) respectively, as compared with the saline-treated group. Injection of PYY3–36 also tended to reduce the feed intake until 24 h post-injection in ad libitum-fed pigs (40.9 ± 13.9, 16.1 ± 1.9, and 16.3 ± 7.8% for 6, 12, and 24 h cumulative feed intake compared with saline injection group), although no significant difference was observed. Experiment 2: Effect of i.v. injection of PYY3–36 on feed intake during overnight fast-refed condition
Averaged basal plasma PYY level was 0.7 ± 0.1 ng/ml in fast-refed pigs. Similar to the ad libitum-fed condition, elevated plasma PYY levels responsive to porcine PYY3–36 injection decreased within 60 min post-treatment (Fig. 3A).
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. Administration of PYY3–36 significantly reduced feed intake 1-h post-treatment as compared with saline group (12.9 ± 1.9 vs 20.6 ± 1.3 g/kg BW, P < 0.01). Thereafter, feed intake levels returned to similar values as the saline groups. Experiment 3: Effect of 2 h i.v. infusion of PYY3–36 on feed intake in fast-refed pigs
Salineinfusion did not modify theplasma PYY levels throughout the study (Fig. 4A). In the PYY3–36 group, plasma PYY levels were maintained at high levels compared with the values of the saline group (27.3 ± 2.1 vs 0.6 ± 0.1 ng/ml, P < 0.001) during infusion and were reduced to less than half the values at 15 min after termination of PYY3–36 infusion (135 min).
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). Experiment 4: Effects of i.v. injection of PYY3–36 on plasma acyl-ghrelin and GH levels after an overnight fast
As shown in Fig. 5A, plasma PYY levels were elevated by porcine PYY3–36 injection and decreased to values below half of the peak level at 30 min post-treatment. Plasma acyl-ghrelin levels did not change after injection of saline or PYY3–36 (Fig. 5B). Plasma GH levels significantly increased 30 min after PYY3–36 injection compared with the saline-injected group (7.8 ± 2.1 vs 3.0 ± 0.6 ng/ml, P < 0.05; Fig. 5C).
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. Plasma PYY levels were significantly higher in ad libitum-fed conditions than in the fasting condition (2.2 ± 0.2 vs 0.8 ± 0.1 ng/ml, P < 0.001).
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Discussion |
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It is well known that the post-prandial increase in plasma PYY levels is dependent on the level of calorie intake in humans; higher calorie intake induces higher concentrations of PYY for a longer duration (Adrian et al. 1985); together with evidence from that report, our data show that plasma PYY levels are elevated in the ad libitum-fed condition compared with the fasting condition in pigs. Furthermore, the proportion of PYY3–36 to total plasma PYY has been shown to increase during post-prandial periods (Grandt et al. 1994). Chelikani et al.(2005) also reported in non-food-deprived rats that the anorexigenic potency and efficacy of 15 min i.v. infusion of PYY3–36, just prior to the onset of the darkness cycle, is about three times less than a 3 h i.v. infusion at that time. These studies show that both the level and duration of increase in plasma PYY3–36 concentration may thus influence satiety and contribute to the termination of feed intake.
On the other hand, the result of our infusion study differed from those reported by Batterham et al.(2002) in humans, but are rather similar to the previous report in rats by Chelikani et al.(2005). The report in humans shows that i.v. infusion of PYY3–36 reduced calorie intake over a period of 12 h, while in the present study in pigs, PYY3–36 infusion reduced feed intake only for a period of 1 h. Although it is difficult to compare these reports with our studies because of the differences in species, experimental condition, infusion time, dose, and nutrient state, it can be considered for some of the reasons. First, in the human study, subjects could freely select the meal because of a buffet meal style. Furthermore, food intake levels were measured by calorie intake but not by weight. Secondly, the involvement of the experimental stress and/or length of acclimatization period might be the possible reason; i.p. injection of PYY3–36 to 16 h fasted and non-acclimated mice did not reduce feed intake, but significantly reduced feed intake in acclimated mice (Halatchev et al. 2004). All together, it is suggestive that experimental protocol/stress/acclimatization and other factors influence the anorexigenic potency of PYY3–36.
Recently, two conflicting studies reported on the effects of PYY3–36 on plasma acyl-ghrelin concentrations. Batterham et al.(2003) reported that i.v. infusion of PYY3–36 to obese and lean subjects suppressed caloric intake and significantly decreased plasma total ghrelin levels, while Adams et al.(2004) reported that i.p. injection of PYY3–36 to overnight-fasted mice reduced feed intake significantly, but there was no effect on plasma acyl- and total ghrelin levels. In this study, bolus injection of PYY3–36 did not affect the plasma acyl-ghrelin levels in fasted pigs. Therefore, it can be considered that PYY3–36 might not be a major regulator of ghrelin secretion in pigs.
Furthermore, we investigated whether PYY3–36 affects plasma GH levels, which are known as indicators of energy expenditure, in pigs because NPY, which belongs to the PP-hold family similar to PYY (Berglund et al. 2003), has also been demonstrated to stimulate GH secretion from anterior pituitary cells of pigs, cattle, and sheep, but not rats (McMahon et al. 2001, Barb & Barrett 2005). Moreover, the effect of NPY on GH secretion is possibly mediated by NPY Y2 receptor (Y2-R) (Suzuki et al. 1996, Korbonits et al. 1999). In this study, we showed that i.v. bolus injection of PYY3–36, a potent Y2-R agonist, significantly increased plasma GH levels, and the plasma GH peak was seen 30 min after injection. Therefore, it is suggested that this effect of PYY3–36 on plasma GH levels is a direct effect on anterior pituitary cells by excessive PYY3–36 by bolus i.v. injection.
In summary, we have shown that peripheral administration of PYY3–36 in pigs can reduce feed intake. Moreover, plasma PYY levels fluctuated with the changes in energy balance leading to the suggestion that plasma PYY3–36 levels influence satiety and contribute to the termination of feed intake in pigs. Furthermore, the effect of PYY3–36 was potent and acute, which result is reflective of the short half-life of i.v. administered PYY3–36. Moreover, i.v. injection of PYY3–36 did not affect plasma acyl-ghrelin levels in the fasted condition indicating that at least in pigs, PYY3–36 is not a major regulator of ghrelin secretion. However, since the administration of PYY3–36 increased plasma GH levels, it is suggested that PYY 3–36, apart from its influence on feed intake, may also have other metabolic functions that are crucial in the regulation of energy homeostasis in pigs.
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Acknowledgements |
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References |
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Adrian TE, Ferri GL, Bacarese-Hamilton AJ, Fuessl HS, Polak JM & Bloom SR 1985 Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 89 1070–1077.[ISI][Medline]
Adrian TE, Bacarese-Hamilton AJ, Smith HA, Chohan P, Manolas KJ & Bloom SR 1987 Distribution and postprandial release of porcine peptide YY. Journal of Endocrinology 113 11–14.[Abstract]
Barb CR & Barrett JB 2005 Neuropeptide Y modulates growth hormone but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture. Domestic Animal Endocrinology 29 548–555.[CrossRef][ISI][Medline]
Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA et al. 2002 Gut hormone PYY(3–36) physiologically inhibits food intake. Nature 418 650–654.[CrossRef][Medline]
Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA & Bloom SR 2003 Inhibition of food intake in obese subjects by peptide YY3–36. New England Journal of Medicine 349 941–948.
Berglund MM, Hipskind PA & Gehlert DR 2003 Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Experimental Biology and Medicine 228 217–244.
Chelikani PK, Haver AC & Reidelberger RD 2005 Intravenous infusion of peptide YY(3–36) potently inhibits food intake in rats. Endocrinology 146 879–888.
Govoni N, De Iasio R, Cocco C, Parmeggiani A, Galeati G, Pagotto U, Brancia C, Spinaci M, Tamanini C, Pasquali R et al. 2005 Gastric immunolocalization and plasma profiles of acyl-ghrelin in fasted and fasted-refed prepuberal gilts. Journal of Endocrinology 186 505–513.
Grandt D, Schimiczek M, Beglinger C, Layer P, Goebell H, Eysselein VE & Reeve JR Jr 1994 Two molecular forms of peptide YY (PYY) are abundant in human blood: characterization of a radioimmunoassay recognizing PYY 1–36 and PYY 3–36. Regulatory Peptides 51 151–159.[CrossRef][ISI][Medline]
Halatchev IG, Ellacott KL, Fan W & Cone RD 2004 Peptide YY3–36 inhibits food intake in mice through a melanocortin-4 receptor-independent mechanism. Endocrinology 145 2585–2590.
Inoue H, Watanuki M, Hnin TM, Ito T, Kuwayama H & Hidari H 2005 Effects of fasting and refeeding on plasma concentrations of leptin, ghrelin, insulin, growth hormone and metabolites in swine. Animal Science Journal 76 367–374.[CrossRef]
Korbonits M, Little JA, Forsling ML, Tringali G, Costa A, Navarra P, Trainer PJ & Grossman AB 1999 The effect of growth hormone secretagogues and neuropeptide Yon hypothalamic hormone release from acute rat hypothalamic explants. Journal of Neuroendocrinology 11 521–528.[CrossRef][ISI][Medline]
McMahon CD, Radcliff RP, Lookingland KJ & Tucker HA 2001 Neuroregulation of growth hormone secretion in domestic animals. Domestic Animal Endocrinology 20 65–87.[CrossRef][ISI][Medline]
Moran TH, Smedh U, Kinzig KP, Scott KA, Knipp S & Ladenheim EE 2005 Peptide YY(3–36) inhibits gastric emptying and produces acute reductions in food intake in rhesus monkeys. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 288 R384–R388.
Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K & Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature 409 194–198.[CrossRef][Medline]
Nonaka N, Shioda S, Niehoff ML & Banks WA 2003 Characterization of blood–brain barrier permeability to PYY3–36 in the mouse. Journal of Pharmacology and Experimental Therapeutics 306 948–953.
Phung LT, Inoue H, Nou V, Lee HG, Vega RA, Matsunaga N, Hidaka S, Kuwayama H & Hidari H 2000 The effects of growth hormone-releasing peptide-2 (GHRP-2) on the release of growth hormone and growth performance in swine. Domestic Animal Endocrinology 18 279–291.[CrossRef][ISI][Medline]
Salfen BE, Carroll JA & Keisler DH 2003 Endocrine responses to short-term feed deprivation in weanling pigs. Journal of Endocrinology 178 541–551.[Abstract]
Salfen BE, Carroll JA, Keisler DH & Strauch TA 2004 Effects of exogenous ghrelin on feed intake, weight gain, behavior, and endocrine responses in weanling pigs. Journal of Animal Science 82 1957–1966.
Scott V, Kimura N, Stark JA & Luckman SM 2005 Intravenous peptide YY3–36 and Y2 receptor antagonism in the rat: effects on feeding behaviour. Journal of Neuroendocrinology 17 452–457.[CrossRef][ISI][Medline]
Sileno AP, Brandt GC, Spann BM & Quay SC 2006 Lower mean weight after 14 days intravenous administration peptide YY(3–36) (PYY(3–36)) in rabbits. International Journal of Obesity 30 68–72.[CrossRef][ISI][Medline]
Suzuki N, Okada K, Minami S & Wakabayashi I 1996 Inhibitory effect of neuropeptide Y on growth hormone secretion in rats is mediated by both Y1- and Y2-receptor subtypes and abolished after anterolateral deaf-ferentation of the medial basal hypothalamus. Regulatory Peptides 65 145–151.[CrossRef][ISI][Medline]
Tai HH, Korsch B & Chey WY 1975 Preparation of 125I-labeled secretin of high specific radioactivity. Analytical Biochemistry 69 34–42.[CrossRef][ISI][Medline]
Tatemoto K & Mutt V 1980 Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 285 417–418.[CrossRef][Medline]
ThidarMyint H, Yoshida H, Ito T & Kuwayama H 2006 Dose-dependent response of plasma ghrelin and growth hormone concentrations to bovine ghrelin in Holstein heifers. Journal of Endocrinology 189 655–664.
Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA & Bloom SR 2001 Ghrelin enhances appetite and increases food intake in humans. Journal of Clinical Endocrinology and Metabolism 86 5992–5995.
Received 7 March 2006
Received in final form 12 July 2006
Accepted 21 July 2006
Made available online as an Accepted Preprint 7 August 2006
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| HOME | HELP | CONTACT US | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), porcine PYY1–36 (
), porcine NPY (
), human PYY1–36 (
), and human PP (
). Inhibition of 125I-labeled porcine PYY3–36 binding to antisera by serial dilution of pooled porcine plasma (•) parallel to the curve was obtained using control porcine PYY3–36 as standard. Each point is the mean of triplicate determinations. B/B0, bound/bound in zero standard.
P < 0.01).
