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Fat-induced ileal brake in the dog depends on peptide YY
GASTROENTEROLOGY 1996;110:1491–1495
Fat-Induced Ileal Brake in the Dog Depends on Peptide YY HENRY C. LIN,* XIAO–TUAN ZHAO,* LIJIE WANG,* and HELEN WONG‡ *Department of Medicine, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, Los Angeles; and ‡CURE/Digestive Diseases Research Center and School of Medicine, University of California, Los Angeles, California
Background & Aims: Fat in the distal gut inhibits transit through the proximal small intestine as the ileal brake. Although the mediator of this response is not established, peptide YY (PYY) has been considered the most likely peptide candidate because inhibition of intestinal motility by fat in the distal gut correlated with the release of PYY but not other distal gut peptides such as enteroglucagon or neurotensin. Although intravenous administration of PYY inhibits intestinal transit, the role of this peptide remains to be confirmed because systemic PYY may not exert its effect by the same regulatory pathway as fat-induced ileal brake. The aim of this study was to definitively test the hypothesis that PYY mediates fat-induced ileal brake using the technique of peptide immunoneutralization. Methods: In a fistulated dog model, intestinal transit during perfusion of the distal gut with 60 mmol/L oleate (ileal brake) was examined after intravenous administration of 0.5 mg/kg of PYY antibody (anti-PYY), nonspecific immunoglobulin G (control), or 0.15 mol/L NaCl. Intestinal transit result (cumulative percent recovery of 99mTc) was normalized within each animal against the transit result of the 0.15 mol/L NaCl experiment. Results: Intestinal transit accelerated with PYY immunoneutralization, increasing cumulative percent recovery from 25.9 { 6.2 (control) to 81.2 { 6.3 (anti-PYY). Conclusions: Fat-induced ileal brake depends on PYY.
at slows the movement of luminal contents.1 – 4 Read et al.3 and Spiller et al.4 independently described the slowing of intestinal transit through a jejunal segment during fat perfusion of the distal small intestine. This effect was termed the ileal brake.4 Because of their preferential localization to the ileum and the colon, peptide YY (PYY),1,3,5 – 7 enteroglucagon,5 and neurotensin6,8 – 10 have been hypothesized as possible peptide mediators of fat-induced ileal brake.3,4,11,12 Of this group, PYY has been considered the most likely candidate13 – 16 because the release of this peptide correlated with inhibition of intestinal motility17 and transit3 by fat in the ileum, even in the absence of an increase in the plasma level of neurotensin or enteroglucagon.3 However, the role of this peptide as the mediator of fat-induced ileal brake remains to be established because systemically administered PYY
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may act through different regulatory pathways than the endogenous PYY released by fat. To further support this role for PYY, we used the technique of peptide immunoneutralization in dogs to test the hypothesis that PYY mediates fat-induced ileal brake.
Materials and Methods General Experimental Design The procedures used in this study were approved by the Institutional Animal Care and Use Committee at CedarsSinai Medical Center in Los Angeles. In dogs equipped with duodenal and midintestinal fistulas, we compared intestinal transit during perfusion of the gut distal to the midintestinal fistula with 60 mmol/L oleate after intravenous administration of 0.5 mg/kg of PYY antibody (anti-PYY), nonspecific immunoglobulin (Ig) G (control), or 0.15 mol/L NaCl. The order of testing followed a randomization schedule.
Preparation of PYY Antibody Antibody was provided by the Antibody Core Laboratory of CURE/Digestive Diseases Research Center in Los Angeles, using the technique of multiple intradermal injections of the full-length synthetic peptide conjugated to keyhole-limpet hemocyanin in rabbits.18 For control experiments, normal rabbit IgG was used. PYY antibody and nonspecific IgG were both purified using a protein-A Sepharose column (Pharmacia Biosystems, Piscataway, NJ). The PYY antibody had no cross-reactivity with rat or human pancreatic polypeptide or neuropeptide Y at the dilution of 1:30,000.
Animal Preparation Six mongrel dogs were each surgically prepared with two chronic intestinal fistulas. Modified Thomas cannulas were placed into fistulas located approximately 10 cm (duodenal fistula, distal to the bile duct) and approximately 160 cm (midintestinal fistula) from the pylorus.19 With the flanges of the cannula resting against the inner surface of the intestinal wall, the cannulas were fixed against rotation. Just distal to the fistula, a length of Tygon tubing with a diameter of 2 mm was looped around the intestine and fixed by suture through Abbreviation used in this paper: sqrt AUC, square root area under the curve. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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the visceral peritoneum to the intestinal wall. The length of tubing used was individualized to be as short as possible without a tightening effect on the lumen. This provided a stent against which an inflated Foley balloon could be pulled to provide a water-tight seal. All dogs were given a recovery period of 4 weeks and underwent testing only after normal feeding behaviors were re-established postoperatively. This preparation had good survival, and the 6 dogs remained healthy with stable body weights and unaffected demeanor for more than 12 months of observation.
Experimental Preparations Dogs were deprived of food but not water for an 18hour period before each experiment. Thirty minutes before the start of each experiment, the intestinal cannulas were uncorked so a Foley catheter could be placed into the distal limb of each fistula. By inflating its balloon with approximately 10 mL of water and cinching the balloon up against the Tygon tubing ring, a water-tight seal was achieved at each fistula with a Foley catheter.2 The output of each fistula was then allowed to drain freely by gravity. Using this method, a 150-cm test segment was isolated between the fistulas. Five minutes before the start of intestinal perfusions, 0.5 mg/kg of anti-PYY, nonspecific IgG (control), or 0.15 mol/L NaCl was administered as an intravenous bolus at an access site placed into a limb vein. At the start of each experiment, the distal half of the gut (beyond the midintestinal fistula) was perfused with 60 mmol/L oleate delivered as a solution of mixed micelles with monolein and 10 mmol/L taurocholate in pH 7.0 phosphate buffer through the Foley catheter in the midintestinal fistula. Concurrently, the proximal half of the small intestine was perfused with pH 7.0 phosphate buffer through the Foley catheter in the duodenal fistula. Both perfusates were delivered at 2 mL/min through the occluding catheters for a total of 90 minutes. Sixty minutes after the start of the perfusion, approximately 20 mCi 99mTc chelated to diethyltriaminepentaacetic acid20 was delivered as a bolus into the test segment through the Foley catheter placed in the duodenal fistula to begin measurement of intestinal transit.
Calculation of Intestinal Transit Intestinal transit was measured by counting the radioactivity of 1-mL samples collected every 5 minutes from the diverted output of the midintestinal fistula.15,21 Using a matched dose of 99m-Tc to represent the delivered bolus, the radioactivity in the bolus and the radioactivity of the recovered fistulous output were all measured in a gamma well-counter (Packard Instruments, Meriden, CT). After correcting all counts to time zero, intestinal transit was calculated as the cumulative percentage of the delivered 99mTc dose that was recovered from the output of the midintestinal fistula over the 30-minute collection period.
Analysis of Data Intestinal transit results after intravenous anti-PYY or nonspecific IgG (control) were first normalized against the
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Table 1. The Effect of Fat-Induced Ileal Brake on Intestinal Transit After Intravenous Anti-PYY or Nonspecific IgG Intravenous agent Intestinal transit
Nonspecific IgG
Anti-PYY
Cumulative % recovered Sqrt AUC
25.9 { 6.2 15.0 { 3.5
81.2 { 6.3 34.6 { 2.3
NOTE. Values are means { SE; 60 mol/L oleate was perfused into the distal half of the gut while buffer was perfused into the proximal half of the gut. Values are normalized within each animal against the transit results of the 0.15 mol/L NaCl experiments. Sqrt AUC, square root of the area under the curve for the cumulative percent recovery of 99mTc, where 0 equals no recovery by 0 minutes and 47.4 equals theoretical, complete recovery by time 0.
result after intravenous NaCl, 0.15 mol/L. Data were normalized within each animal by subtracting the transit result of the 0.15 mol/L NaCl experiment from the result of the antiPYY or nonspecific IgG (control) experiment. This normalization allowed each animal to serve as its own control. The normalized data were then compared by determining the area under the curve (AUC) for the cumulative percent recovery of the radioactive marker. To stabilize the variance, we compared the square root values of the AUC (sqrt AUC)19 where 0 equals no recovery by 30 minutes and 47.4 equals theoretical, complete recovery by time 0 (rapid transit) using a paired t test. The computer program used was BMDP.22
Validation of Immunoneutralization of Circulating PYY To confirm adequate anti-PYY activity in the PYY antibody–treated animals, anti-PYY activities in serum collected from all experiments were measured by radioimmunoassay as previously described18,23 – 27 using 125I-labeled PYY tracers (125I-PYY1 – 36 and 125I-PYY22 – 36 ). Antibody-bound PYY was measured by serially diluting the serum obtained at 030, 0, 30, 60, and 90 minutes using the buffer for radioimmunoassay. The samples were then incubated in the presence of 125Ilabeled PYY tracers. The bound/free ratio was tested at different dilutions after incubation for 24 hours at 4⬚C.
Results Effect of PYY Immunoneutralization on FatInduced Ileal Brake Intestinal transit represented by cumulative percent recovered and sqrt AUC values are shown as Table 1 and Figure 1. For the control experiment in which nonspecific IgG was administered intravenously while 60 mmol/L oleate was perfused into the distal half of the gut, the cumulative percent recovered was 25.9 { 6.2. This result represented the slowing of intestinal transit by fat-induced ileal brake. In contrast, intestinal transit was significantly accelerated when anti-PYY was adminWBS-Gastro
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ILEAL BRAKE DEPENDS ON PYY 1493
Figure 1. Intestinal transit across a 150-cm test segment during the last 30 minutes of a 90-minute perfusion experiment. Oleate (60 mmol/L) was delivered into the distal half of the gut (beyond the midintestinal fistula) while buffer was delivered into the proximal half of the gut (between fistulas). This perfusion was used to trigger a fatinduced ileal brake. To test the effect of PYY immunoneutralization, 5 minutes before the start of the perfusions, nonspecific IgG (●) or anti-PYY (䊏) was administered intravenously. Data represented the mean values of normalized data from 6 animals.
istered before the fat perfusion (P Å 0.001). In this setting of PYY immunoneutralization, the cumulative percent recovered increased to 81.2 { 6.3 despite the fat perfusion of the distal gut. Correspondingly, sqrt AUC values increased from 15.0 { 3.5 for the control experiment to 34.6 { 2.3 for the anti-PYY experiment. The dogs did not show any visible reaction to the administration of the PYY antibody. Confirmation of Anti-PYY Activity In the anti-PYY–treated dogs, PYY immunoreactivity was detected at 1:500 serum dilution. However, the 1:10 diluted serum collected during the 0.15 mol/L NaCl or control (nonspecific IgG) experiments did not have PYY immunoreactivity. Thus, adequate anti-PYY activity was achieved after intravenous PYY antibody.
Discussion In this study, using the technique of peptide immunoneutralization, we confirmed definitively and for the first time that fat-induced ileal brake depended on PYY. In the control experiments after intravenous nonspecific IgG, intestinal transit was slowed by oleate perfused into the distal gut as the ileal brake response. In contrast, intestinal transit was significantly accelerated by intravenous anti-PYY, suggesting that fat-induced / 5e0d$$0042
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ileal brake depended on PYY. Although other mediators may also play a role, this study proved conclusively that PYY is a key mediator of the ileal brake. The term ‘‘ileal brake’’ was given by Spiller et al.4 to describe the slowing of transit through a 30-cm jejunal segment when 100 mL of 10% Lipofundin was perfused into the human small intestinal approximately 170 cm from the teeth. This slowing effect on transit was associated with inhibition of jejunal motility. Using a different technique, Read et al.3 described a similar response when orocecal transit was slowed after 100 mL of 10% Intralipid was perfused into the gut approximately 205 cm from the teeth. In that study, delayed gastric emptying was also found to be part of the ileal brake response.3 Although the effect of fat as the trigger of the ileal brake has been most extensively studied,3,4,17 proteins3 and carbohydrates3 are also effective in this response. The triggering of the ileal brake by fat may depend on the end products of fat digestion because the inhibition by triolein was significantly less than that generated by oleic acid.4 Because glycerol alone did not alter jejunal motility,4 fatty acids may be the active hydrolytic product that triggers a fat-induced ileal brake response. Because of their preferential concentrations on the distal small intestine and colon, enteroglucagon,5 neurotensin,9 – 11,28,29 and PYY14,29 – 31 have been considered possible mediators of the fat-induced ileal brake response.3,7,17 Of this group of peptides, several lines of evidence suggest that PYY is the most likely mediator: PYY is released by fat in the ileocolon11 – 13,31; fat delivered into the distal small intestine delayed intestinal transit without increasing the plasma level of either neurotensin or enteroglucagon3; inhibition of jejunal motility correlated with the release of PYY but not enteroglucagon or neurotensin17; intravenous infusion of PYY given to mimic postprandial levels of the peptide inhibited jejunal motility32,33 and small intestinal transit28,32; and whereas ileal lipids or carbohydrates both released enteroglucagon and neurotensin, only lipids released PYY.17 Previously, investigators have sought to confirm the mediator role of PYY in the ileal brake response by administering the peptide intravenously28,32,33 to show that intravenous PYY inhibited jejunal motility32,33 and transit.28,32 However, the effects of systemic PYY may not be the same as luminal fat because exogenous vs. endogenous peptides may work through different pathways. Thus, inhibition of intestinal transit28,32 by intravenous PYY suggested but did not prove the mediator role for PYY. Because an effective antagonist to PYY is not currently available, we have taken the approach of PYY immunoneutralization to show that PYY mediated the slowing of intestinal transit by fat in the distal gut. WBS-Gastro
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PYY is a 36–amino acid peptide secreted by L cells of the distal small intestine and colon.30 Along with inhibition of intestinal motility and transit,28,32,33 other reported effects of PYY have included inhibition of gastric emptying,16 stimulation of small intestinal absorption,18 and suppression of gastric,7 biliary,12 and pancreatic7 secretions. Although most of these reports were based on an associated increase in plasma levels of PYY or the effect of intravenous PYY, PYY appears to be a key peptide for the regulation of the gastrointestinal response to a meal. PYY receptors have been found on neurons in the myenteric plexus.34 This finding suggests that PYY may exert its effect not only as a hormone or a peptide that participates in a paracrine regulatory pathway but also as a neuropeptide that stimulates neural pathways.35 In summary, using the technique of PYY immunoneutralization, we found that the effect of fat-induced ileal brake on intestinal transit was greatly diminished after the administration of intravenous PYY antibody. We also found that adequate PYY antibody was available during the experiment for the immunoneutralization of this peptide. Based on these findings, we conclude that fat-induced ileal brake depended on PYY.
References 1. Borgstrom S, Arborelius J. Influence of a fatty acid on duodenal motility. Scand J Gastroenterol 1975;10:599–601. 2. Lin HC, Zhao XT, Reddy SN, Wang LJ, Gu YG. Inhibition of intestinal transit by fat depends on length of exposure to nutrient (abstr). Gastroenterology 1994;106:A531. 3. Read NW, MacFarlane A, Kinsman R. Effect of infusion of nutrient solutions into the ileum on gastrointestinal transit and plasma levels of neurotensin and enteroglucagon in man. Gastroenterology 1984;86:274–280. 4. Spiller RC, Trotman IF, Higgins BE. The ileal brake-inhibition of jejunal motility after ileal fat perfusion in man. Gut 1984;25: 365–374. 5. Baldissera FGA, Holst JJ. Glucagon-related peptides in the human Gastrointestinal mucosa. Diabetologia 1984;26:223–228. 6. Hellstrom PM, Nylander G, Rosell S. Effects of neurotensin on the transit of gastrointestinal contents in the rat. Acta Physiol Scand 1982;115:239–243. 7. Pappas TN, Debas HT, Goto Y, Taylor IL. Peptide YY inhibits mealstimulated pancreatic and gastric secretion. Am J Physiol 1985; 248:G118–G123. 8. Buhner S, Ehrlein H-J. Effects of neurotensin on motor patterns of canine duodenum and proximal jejunum. Can J Physiol Pharmacol 1989;67:1534–1539. 9. Sundler F, Hakanson R, Hammer RA, Alumets J, Carraway R, Leeman SE, Zimmerman EA. Immunohistochemcal localization of neurotensin in endocrine cells of the gut. Cell Tissue Res 1977;178:313–321. 10. Schemann M, Siegle M-L, Sahyoun H, Ehrlein H-J. Computer analysis of intestinal motility: effects of cholecystokinin and neurotensin on jejunal contraction patterns. Z Gastroenterologie 1986; 24:262–268. 11. Konturek SJ, Bilski J, Pawlik W, Tasler J, Domschke W. Adrenergic pathway in the inhibition of pancreatic secretion by peptide YY in dogs. Gastroenterology 1988;94:266–273.
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12. Lluis F, Fujimura M, Lonovics J, Guo Y-S, Gomez G, Greeley GH, Townsend CM, Thompson JC. Peptide YY and gallbladder contraction. Gastroenterology 1988;94:1441–1446. 13. Greeley GH, Himoto T, Izukura M, Gomez G, Jeng J, Hill FLC. A comparison of intraduodenally and intra-colonically administered nutrients on the release of peptide YY in the dog. Endocrinology 1989;125:1761–1765. 14. Taylor IL. Distribution and release of Peptide YY in dog measured by specific radioimmunoassay. Gastroenterology 1985;88:731– 737. 15. Zierler KL. A simplified explanation of the theory of indicator dilution for measurement of fluid flow and volume and other distributive phenomena. Bull Johns Hopkins 1958;103:199–217. 16. Pappas TN, Chang AM, Debas HT, Taylor IL. Peptide YY release by fatty acids is sufficient to inhibit gastric emptying in dog. Gastroenterology 1986;91:1386–1389. 17. Spiller RC, Trotman IF, Adrian TE, Bloom SR, Misiewicz JJ, Silk DBA. Further characterisation of the ‘‘ileal brake’’ reflex in man— effect of ileal infusion of partial digests of fat, protein, and starch on jejunal motility and release of neurotensin, enteroglucagon, and peptide YY. Gut 1988;29:1042–1051. 18. Bilchik AJ, Hines DJ, Ashley SW, Adrian TE, Liu CD, Wong H, Zinner MJ, McFadden DW. Peptide YY immunoneutralization inhibits meal–induced absorption in-vivo. Surgery 1994;116: 1153–1157. 19. Lin HC, Kim BK, Elashoff JD, Doty JE, Gu YG, Meyer JH. Gastric emptying of solid food is most potently inhibited by carbohydrates in the canine distal ileum. Gastroenterology 1992;102:793– 801. 20. Delin NA, Axelsson B, Johansson C, Popper B. Comparison of gamma camera and withdrawal methods for the measurement of gastric emptying. Scand J Gastroenterol 1978;13:867–872. 21. Johansson C. Studies of gastrointestinal interactions. Scand J Gastroenterol 1974;9(Suppl 28):1–60. 22. Dixon WJ. BMDP statistical software manual. Berkeley, CA: University of California, 1990. 23. Wong H, Andersen D, Sternini C, Ruiz C, Hull E, Walsh J, Brunicardi F. Production of a rat pancreatic polypeptide-specific monoclonal antibody and its influence on glucose homeostasis by in vivo immunoneutralization. Hybridoma 1995;14:369–376. 24. Wong H, Tache Y, Lloyd K, Yang H, Sternini C, Holzer P, Walsh J. Monoclonal antibody to rat alpha-CGRP: production, characterization, and in vivo immunoneutralization activity. Hybridoma 1993;12:93–106. 25. Wong H, Walsh J, Yang H, Tache Y, Buchan A. A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity. Peptides 1990;11:707–712. 26. Ebert R, Illmer K, Creutzfeldt W. Release of gastric inhibitor polypeptide (GIP) by intraduodenal acidification in rats and human and abolishment of the incretin effect of acid by GIP-antiserum in rats. Gastroenterology 1979;76:515–523. 27. Almdal T, Holst J, Heindorff H, Vilstrup H. Glucagon immunoneutralization in diabetic rats normalized urea synthesis and decreases nitrogen wasting. Diabetes 1992;41:12–16. 28. Savage AP, Adrian TE, Carolan G, Chatterjee VK, Bloom SR. Effects of peptide YY (PYY) on mouth to caecum intestinal transit time and on the rate of gastric emptying in healthy volunteers. Gut 1987;28:166–170. 29. Eysselein V, Eberlein G, Grandt D. Structural characterization of canine PYY. Peptides 1990;11:111–116. 30. Adrian T, Ferri G-L, Bacarese-Hamilton A, Fuessl H, Polak J, Bloom S. Human distribution and release of a putative new gut hormone, Peptide YY. Gastroenterology 1985;89:1070–1077. 31. Aponte GW, Fink AS, Meyer JH, Tatemoto K, Taylor IL. Regional distribution and release of peptide YY with fatty acids of different chain length. Am J Physiol 1985;249:G745–G750. 32. Al-Saffar A, Hellstrom PM, Nylander G. Correlation between pep-
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tide YY–induced myoelectric activity and transit of small-intestinal contents in rats. Scand J Gastroenterol 1985;20:577–582. 33. Lundberg JM, Tatemoto K, Terenius L, Hellstrom PM, Mutt V, Hokfelt T, Hamberger B. Localization of peptide YY (PYY) in Gastrointestinal endocrine cells and effects on intestinal blood flow and motility. Proc Natl Acad Sci USA 1982;79:4471–4475. 34. Martin SP, Puccio AM, Schraut WH, Lee Jr KW, Reeve J Jr, Whitcomb DC. Predominance of PYY-Y1 receptors on myenteric ganglia and PYY-Y2 receptors on enterocytes of human small intestine: use of 125I-PYY(1-36) and 125I-PYY(3-36) in receptor autoradiography (abstr). Gastroenterology 1994;106:A536.
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35. Allen J, Fitzpatrick J, Yeats J, Darcy K, Adrian T, Bloom S. Effects of peptide YY and neuropeptide Y on gastric emptying in man. Digestion 1984;30:255–262.
Received September 25, 1995. Accepted January 22, 1996. Address requests for reprints to: Henry C. Lin, M.D., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, no. 7511, Los Angeles, California 90048-1869. Supported in part by the American Heart Association, Greater Los Angeles Affiliate.
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Fat-Induced Ileal Brake in the Dog Depends on Peptide YY HENRY C. LIN,* XIAO–TUAN ZHAO,* LIJIE WANG,* and HELEN WONG‡ *Department of Medicine, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, Los Angeles; and ‡CURE/Digestive Diseases Research Center and School of Medicine, University of California, Los Angeles, California
Background & Aims: Fat in the distal gut inhibits transit through the proximal small intestine as the ileal brake. Although the mediator of this response is not established, peptide YY (PYY) has been considered the most likely peptide candidate because inhibition of intestinal motility by fat in the distal gut correlated with the release of PYY but not other distal gut peptides such as enteroglucagon or neurotensin. Although intravenous administration of PYY inhibits intestinal transit, the role of this peptide remains to be confirmed because systemic PYY may not exert its effect by the same regulatory pathway as fat-induced ileal brake. The aim of this study was to definitively test the hypothesis that PYY mediates fat-induced ileal brake using the technique of peptide immunoneutralization. Methods: In a fistulated dog model, intestinal transit during perfusion of the distal gut with 60 mmol/L oleate (ileal brake) was examined after intravenous administration of 0.5 mg/kg of PYY antibody (anti-PYY), nonspecific immunoglobulin G (control), or 0.15 mol/L NaCl. Intestinal transit result (cumulative percent recovery of 99mTc) was normalized within each animal against the transit result of the 0.15 mol/L NaCl experiment. Results: Intestinal transit accelerated with PYY immunoneutralization, increasing cumulative percent recovery from 25.9 { 6.2 (control) to 81.2 { 6.3 (anti-PYY). Conclusions: Fat-induced ileal brake depends on PYY.
at slows the movement of luminal contents.1 – 4 Read et al.3 and Spiller et al.4 independently described the slowing of intestinal transit through a jejunal segment during fat perfusion of the distal small intestine. This effect was termed the ileal brake.4 Because of their preferential localization to the ileum and the colon, peptide YY (PYY),1,3,5 – 7 enteroglucagon,5 and neurotensin6,8 – 10 have been hypothesized as possible peptide mediators of fat-induced ileal brake.3,4,11,12 Of this group, PYY has been considered the most likely candidate13 – 16 because the release of this peptide correlated with inhibition of intestinal motility17 and transit3 by fat in the ileum, even in the absence of an increase in the plasma level of neurotensin or enteroglucagon.3 However, the role of this peptide as the mediator of fat-induced ileal brake remains to be established because systemically administered PYY
F
/ 5e0d$$0042
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gasa
may act through different regulatory pathways than the endogenous PYY released by fat. To further support this role for PYY, we used the technique of peptide immunoneutralization in dogs to test the hypothesis that PYY mediates fat-induced ileal brake.
Materials and Methods General Experimental Design The procedures used in this study were approved by the Institutional Animal Care and Use Committee at CedarsSinai Medical Center in Los Angeles. In dogs equipped with duodenal and midintestinal fistulas, we compared intestinal transit during perfusion of the gut distal to the midintestinal fistula with 60 mmol/L oleate after intravenous administration of 0.5 mg/kg of PYY antibody (anti-PYY), nonspecific immunoglobulin (Ig) G (control), or 0.15 mol/L NaCl. The order of testing followed a randomization schedule.
Preparation of PYY Antibody Antibody was provided by the Antibody Core Laboratory of CURE/Digestive Diseases Research Center in Los Angeles, using the technique of multiple intradermal injections of the full-length synthetic peptide conjugated to keyhole-limpet hemocyanin in rabbits.18 For control experiments, normal rabbit IgG was used. PYY antibody and nonspecific IgG were both purified using a protein-A Sepharose column (Pharmacia Biosystems, Piscataway, NJ). The PYY antibody had no cross-reactivity with rat or human pancreatic polypeptide or neuropeptide Y at the dilution of 1:30,000.
Animal Preparation Six mongrel dogs were each surgically prepared with two chronic intestinal fistulas. Modified Thomas cannulas were placed into fistulas located approximately 10 cm (duodenal fistula, distal to the bile duct) and approximately 160 cm (midintestinal fistula) from the pylorus.19 With the flanges of the cannula resting against the inner surface of the intestinal wall, the cannulas were fixed against rotation. Just distal to the fistula, a length of Tygon tubing with a diameter of 2 mm was looped around the intestine and fixed by suture through Abbreviation used in this paper: sqrt AUC, square root area under the curve. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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the visceral peritoneum to the intestinal wall. The length of tubing used was individualized to be as short as possible without a tightening effect on the lumen. This provided a stent against which an inflated Foley balloon could be pulled to provide a water-tight seal. All dogs were given a recovery period of 4 weeks and underwent testing only after normal feeding behaviors were re-established postoperatively. This preparation had good survival, and the 6 dogs remained healthy with stable body weights and unaffected demeanor for more than 12 months of observation.
Experimental Preparations Dogs were deprived of food but not water for an 18hour period before each experiment. Thirty minutes before the start of each experiment, the intestinal cannulas were uncorked so a Foley catheter could be placed into the distal limb of each fistula. By inflating its balloon with approximately 10 mL of water and cinching the balloon up against the Tygon tubing ring, a water-tight seal was achieved at each fistula with a Foley catheter.2 The output of each fistula was then allowed to drain freely by gravity. Using this method, a 150-cm test segment was isolated between the fistulas. Five minutes before the start of intestinal perfusions, 0.5 mg/kg of anti-PYY, nonspecific IgG (control), or 0.15 mol/L NaCl was administered as an intravenous bolus at an access site placed into a limb vein. At the start of each experiment, the distal half of the gut (beyond the midintestinal fistula) was perfused with 60 mmol/L oleate delivered as a solution of mixed micelles with monolein and 10 mmol/L taurocholate in pH 7.0 phosphate buffer through the Foley catheter in the midintestinal fistula. Concurrently, the proximal half of the small intestine was perfused with pH 7.0 phosphate buffer through the Foley catheter in the duodenal fistula. Both perfusates were delivered at 2 mL/min through the occluding catheters for a total of 90 minutes. Sixty minutes after the start of the perfusion, approximately 20 mCi 99mTc chelated to diethyltriaminepentaacetic acid20 was delivered as a bolus into the test segment through the Foley catheter placed in the duodenal fistula to begin measurement of intestinal transit.
Calculation of Intestinal Transit Intestinal transit was measured by counting the radioactivity of 1-mL samples collected every 5 minutes from the diverted output of the midintestinal fistula.15,21 Using a matched dose of 99m-Tc to represent the delivered bolus, the radioactivity in the bolus and the radioactivity of the recovered fistulous output were all measured in a gamma well-counter (Packard Instruments, Meriden, CT). After correcting all counts to time zero, intestinal transit was calculated as the cumulative percentage of the delivered 99mTc dose that was recovered from the output of the midintestinal fistula over the 30-minute collection period.
Analysis of Data Intestinal transit results after intravenous anti-PYY or nonspecific IgG (control) were first normalized against the
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Table 1. The Effect of Fat-Induced Ileal Brake on Intestinal Transit After Intravenous Anti-PYY or Nonspecific IgG Intravenous agent Intestinal transit
Nonspecific IgG
Anti-PYY
Cumulative % recovered Sqrt AUC
25.9 { 6.2 15.0 { 3.5
81.2 { 6.3 34.6 { 2.3
NOTE. Values are means { SE; 60 mol/L oleate was perfused into the distal half of the gut while buffer was perfused into the proximal half of the gut. Values are normalized within each animal against the transit results of the 0.15 mol/L NaCl experiments. Sqrt AUC, square root of the area under the curve for the cumulative percent recovery of 99mTc, where 0 equals no recovery by 0 minutes and 47.4 equals theoretical, complete recovery by time 0.
result after intravenous NaCl, 0.15 mol/L. Data were normalized within each animal by subtracting the transit result of the 0.15 mol/L NaCl experiment from the result of the antiPYY or nonspecific IgG (control) experiment. This normalization allowed each animal to serve as its own control. The normalized data were then compared by determining the area under the curve (AUC) for the cumulative percent recovery of the radioactive marker. To stabilize the variance, we compared the square root values of the AUC (sqrt AUC)19 where 0 equals no recovery by 30 minutes and 47.4 equals theoretical, complete recovery by time 0 (rapid transit) using a paired t test. The computer program used was BMDP.22
Validation of Immunoneutralization of Circulating PYY To confirm adequate anti-PYY activity in the PYY antibody–treated animals, anti-PYY activities in serum collected from all experiments were measured by radioimmunoassay as previously described18,23 – 27 using 125I-labeled PYY tracers (125I-PYY1 – 36 and 125I-PYY22 – 36 ). Antibody-bound PYY was measured by serially diluting the serum obtained at 030, 0, 30, 60, and 90 minutes using the buffer for radioimmunoassay. The samples were then incubated in the presence of 125Ilabeled PYY tracers. The bound/free ratio was tested at different dilutions after incubation for 24 hours at 4⬚C.
Results Effect of PYY Immunoneutralization on FatInduced Ileal Brake Intestinal transit represented by cumulative percent recovered and sqrt AUC values are shown as Table 1 and Figure 1. For the control experiment in which nonspecific IgG was administered intravenously while 60 mmol/L oleate was perfused into the distal half of the gut, the cumulative percent recovered was 25.9 { 6.2. This result represented the slowing of intestinal transit by fat-induced ileal brake. In contrast, intestinal transit was significantly accelerated when anti-PYY was adminWBS-Gastro
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Figure 1. Intestinal transit across a 150-cm test segment during the last 30 minutes of a 90-minute perfusion experiment. Oleate (60 mmol/L) was delivered into the distal half of the gut (beyond the midintestinal fistula) while buffer was delivered into the proximal half of the gut (between fistulas). This perfusion was used to trigger a fatinduced ileal brake. To test the effect of PYY immunoneutralization, 5 minutes before the start of the perfusions, nonspecific IgG (●) or anti-PYY (䊏) was administered intravenously. Data represented the mean values of normalized data from 6 animals.
istered before the fat perfusion (P Å 0.001). In this setting of PYY immunoneutralization, the cumulative percent recovered increased to 81.2 { 6.3 despite the fat perfusion of the distal gut. Correspondingly, sqrt AUC values increased from 15.0 { 3.5 for the control experiment to 34.6 { 2.3 for the anti-PYY experiment. The dogs did not show any visible reaction to the administration of the PYY antibody. Confirmation of Anti-PYY Activity In the anti-PYY–treated dogs, PYY immunoreactivity was detected at 1:500 serum dilution. However, the 1:10 diluted serum collected during the 0.15 mol/L NaCl or control (nonspecific IgG) experiments did not have PYY immunoreactivity. Thus, adequate anti-PYY activity was achieved after intravenous PYY antibody.
Discussion In this study, using the technique of peptide immunoneutralization, we confirmed definitively and for the first time that fat-induced ileal brake depended on PYY. In the control experiments after intravenous nonspecific IgG, intestinal transit was slowed by oleate perfused into the distal gut as the ileal brake response. In contrast, intestinal transit was significantly accelerated by intravenous anti-PYY, suggesting that fat-induced / 5e0d$$0042
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ileal brake depended on PYY. Although other mediators may also play a role, this study proved conclusively that PYY is a key mediator of the ileal brake. The term ‘‘ileal brake’’ was given by Spiller et al.4 to describe the slowing of transit through a 30-cm jejunal segment when 100 mL of 10% Lipofundin was perfused into the human small intestinal approximately 170 cm from the teeth. This slowing effect on transit was associated with inhibition of jejunal motility. Using a different technique, Read et al.3 described a similar response when orocecal transit was slowed after 100 mL of 10% Intralipid was perfused into the gut approximately 205 cm from the teeth. In that study, delayed gastric emptying was also found to be part of the ileal brake response.3 Although the effect of fat as the trigger of the ileal brake has been most extensively studied,3,4,17 proteins3 and carbohydrates3 are also effective in this response. The triggering of the ileal brake by fat may depend on the end products of fat digestion because the inhibition by triolein was significantly less than that generated by oleic acid.4 Because glycerol alone did not alter jejunal motility,4 fatty acids may be the active hydrolytic product that triggers a fat-induced ileal brake response. Because of their preferential concentrations on the distal small intestine and colon, enteroglucagon,5 neurotensin,9 – 11,28,29 and PYY14,29 – 31 have been considered possible mediators of the fat-induced ileal brake response.3,7,17 Of this group of peptides, several lines of evidence suggest that PYY is the most likely mediator: PYY is released by fat in the ileocolon11 – 13,31; fat delivered into the distal small intestine delayed intestinal transit without increasing the plasma level of either neurotensin or enteroglucagon3; inhibition of jejunal motility correlated with the release of PYY but not enteroglucagon or neurotensin17; intravenous infusion of PYY given to mimic postprandial levels of the peptide inhibited jejunal motility32,33 and small intestinal transit28,32; and whereas ileal lipids or carbohydrates both released enteroglucagon and neurotensin, only lipids released PYY.17 Previously, investigators have sought to confirm the mediator role of PYY in the ileal brake response by administering the peptide intravenously28,32,33 to show that intravenous PYY inhibited jejunal motility32,33 and transit.28,32 However, the effects of systemic PYY may not be the same as luminal fat because exogenous vs. endogenous peptides may work through different pathways. Thus, inhibition of intestinal transit28,32 by intravenous PYY suggested but did not prove the mediator role for PYY. Because an effective antagonist to PYY is not currently available, we have taken the approach of PYY immunoneutralization to show that PYY mediated the slowing of intestinal transit by fat in the distal gut. WBS-Gastro
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PYY is a 36–amino acid peptide secreted by L cells of the distal small intestine and colon.30 Along with inhibition of intestinal motility and transit,28,32,33 other reported effects of PYY have included inhibition of gastric emptying,16 stimulation of small intestinal absorption,18 and suppression of gastric,7 biliary,12 and pancreatic7 secretions. Although most of these reports were based on an associated increase in plasma levels of PYY or the effect of intravenous PYY, PYY appears to be a key peptide for the regulation of the gastrointestinal response to a meal. PYY receptors have been found on neurons in the myenteric plexus.34 This finding suggests that PYY may exert its effect not only as a hormone or a peptide that participates in a paracrine regulatory pathway but also as a neuropeptide that stimulates neural pathways.35 In summary, using the technique of PYY immunoneutralization, we found that the effect of fat-induced ileal brake on intestinal transit was greatly diminished after the administration of intravenous PYY antibody. We also found that adequate PYY antibody was available during the experiment for the immunoneutralization of this peptide. Based on these findings, we conclude that fat-induced ileal brake depended on PYY.
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12. Lluis F, Fujimura M, Lonovics J, Guo Y-S, Gomez G, Greeley GH, Townsend CM, Thompson JC. Peptide YY and gallbladder contraction. Gastroenterology 1988;94:1441–1446. 13. Greeley GH, Himoto T, Izukura M, Gomez G, Jeng J, Hill FLC. A comparison of intraduodenally and intra-colonically administered nutrients on the release of peptide YY in the dog. Endocrinology 1989;125:1761–1765. 14. Taylor IL. Distribution and release of Peptide YY in dog measured by specific radioimmunoassay. Gastroenterology 1985;88:731– 737. 15. Zierler KL. A simplified explanation of the theory of indicator dilution for measurement of fluid flow and volume and other distributive phenomena. Bull Johns Hopkins 1958;103:199–217. 16. Pappas TN, Chang AM, Debas HT, Taylor IL. Peptide YY release by fatty acids is sufficient to inhibit gastric emptying in dog. Gastroenterology 1986;91:1386–1389. 17. Spiller RC, Trotman IF, Adrian TE, Bloom SR, Misiewicz JJ, Silk DBA. Further characterisation of the ‘‘ileal brake’’ reflex in man— effect of ileal infusion of partial digests of fat, protein, and starch on jejunal motility and release of neurotensin, enteroglucagon, and peptide YY. Gut 1988;29:1042–1051. 18. Bilchik AJ, Hines DJ, Ashley SW, Adrian TE, Liu CD, Wong H, Zinner MJ, McFadden DW. Peptide YY immunoneutralization inhibits meal–induced absorption in-vivo. Surgery 1994;116: 1153–1157. 19. Lin HC, Kim BK, Elashoff JD, Doty JE, Gu YG, Meyer JH. Gastric emptying of solid food is most potently inhibited by carbohydrates in the canine distal ileum. Gastroenterology 1992;102:793– 801. 20. Delin NA, Axelsson B, Johansson C, Popper B. Comparison of gamma camera and withdrawal methods for the measurement of gastric emptying. Scand J Gastroenterol 1978;13:867–872. 21. Johansson C. Studies of gastrointestinal interactions. Scand J Gastroenterol 1974;9(Suppl 28):1–60. 22. Dixon WJ. BMDP statistical software manual. Berkeley, CA: University of California, 1990. 23. Wong H, Andersen D, Sternini C, Ruiz C, Hull E, Walsh J, Brunicardi F. Production of a rat pancreatic polypeptide-specific monoclonal antibody and its influence on glucose homeostasis by in vivo immunoneutralization. Hybridoma 1995;14:369–376. 24. Wong H, Tache Y, Lloyd K, Yang H, Sternini C, Holzer P, Walsh J. Monoclonal antibody to rat alpha-CGRP: production, characterization, and in vivo immunoneutralization activity. Hybridoma 1993;12:93–106. 25. Wong H, Walsh J, Yang H, Tache Y, Buchan A. A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity. Peptides 1990;11:707–712. 26. Ebert R, Illmer K, Creutzfeldt W. Release of gastric inhibitor polypeptide (GIP) by intraduodenal acidification in rats and human and abolishment of the incretin effect of acid by GIP-antiserum in rats. Gastroenterology 1979;76:515–523. 27. Almdal T, Holst J, Heindorff H, Vilstrup H. Glucagon immunoneutralization in diabetic rats normalized urea synthesis and decreases nitrogen wasting. Diabetes 1992;41:12–16. 28. Savage AP, Adrian TE, Carolan G, Chatterjee VK, Bloom SR. Effects of peptide YY (PYY) on mouth to caecum intestinal transit time and on the rate of gastric emptying in healthy volunteers. Gut 1987;28:166–170. 29. Eysselein V, Eberlein G, Grandt D. Structural characterization of canine PYY. Peptides 1990;11:111–116. 30. Adrian T, Ferri G-L, Bacarese-Hamilton A, Fuessl H, Polak J, Bloom S. Human distribution and release of a putative new gut hormone, Peptide YY. Gastroenterology 1985;89:1070–1077. 31. Aponte GW, Fink AS, Meyer JH, Tatemoto K, Taylor IL. Regional distribution and release of peptide YY with fatty acids of different chain length. Am J Physiol 1985;249:G745–G750. 32. Al-Saffar A, Hellstrom PM, Nylander G. Correlation between pep-
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tide YY–induced myoelectric activity and transit of small-intestinal contents in rats. Scand J Gastroenterol 1985;20:577–582. 33. Lundberg JM, Tatemoto K, Terenius L, Hellstrom PM, Mutt V, Hokfelt T, Hamberger B. Localization of peptide YY (PYY) in Gastrointestinal endocrine cells and effects on intestinal blood flow and motility. Proc Natl Acad Sci USA 1982;79:4471–4475. 34. Martin SP, Puccio AM, Schraut WH, Lee Jr KW, Reeve J Jr, Whitcomb DC. Predominance of PYY-Y1 receptors on myenteric ganglia and PYY-Y2 receptors on enterocytes of human small intestine: use of 125I-PYY(1-36) and 125I-PYY(3-36) in receptor autoradiography (abstr). Gastroenterology 1994;106:A536.
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Received September 25, 1995. Accepted January 22, 1996. Address requests for reprints to: Henry C. Lin, M.D., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, no. 7511, Los Angeles, California 90048-1869. Supported in part by the American Heart Association, Greater Los Angeles Affiliate.
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