Peptide YY: Metabolism and effect on pancreatic secretion in dogs

GASTROENTEROLO(;Y

1985;89:1387-92

Peptide YY: Metabolism and Effect on Pancreatic Secretion in Dogs T. N. PAPPAS, Sepulveda Medicine,

H. T. DEBAS,

and Wadsworth Veterans Los Angeles. California

and I. L. TAYLOR

Administration

Peptide YY is an ileocolonic peptide that inhibits meal-stimulated pancreatic secretion when infused in a dose of 400 pmollkg . h. In this study pancreatic secretion was monitored in response to increasing doses of secretin or cholecystokinin-octapeptide (62.5,125, 250, and 500 nglkg . h) during the simultaneous infusion of either saline or peptide YY (400 pmollkg . h). Peptide YY significantly (p < 0.05) inhibited the secretory response to the three lowest doses of each pancreatic secretogogue, reducing the bicarbonate response to the 62.5-nglkg . h dose of secretin by 86% + 6% and the protein response to the same dose of cholecystokinin by 57% ? 16%. In the second limb of the study, the half-life of peptide YY (11.7 ? 21 min) and the metabolic clearance rate (13.8 ? 1.6 ml/kg +min) were found to be similar to those of other gastrointestinal hormones. We conclude that inhibition of meal-stimulated puncreatic secretion by peptide YY can be explained by its ability to decrease the responsiveness of the pancreas to endogenous secretogogues.

Peptide YY (PYY) was initially isolated from porcine duodenal mucosa using a novel chemical assay that identifies peptides with amidated carboxyl termini (l-3). As many biologically active gastrointestinal hormones share this structural feature, this method has proved to be a productive tool for isolating physiologically important peptides. Peptide YY has been found in high concentrations in the mucosa of the ileum and colon of a variety of species including

Medical

Center

of

rats (4),dogs (5), and humans (6). Structure analysis of PYY reveals marked sequence homology with both pancreatic polypeptide (PP) and a newly isolated neurotransmitter-neuropeptide Y (2,3,7). In view of their close structural homology, it is not surprising to find that members of this family share biologic actions (2.8-11).Although PYY (2,8), like its cousin PP (g-11), is a potent inhibitor of mealstimulated pancreatic secretion, the mechanism(s) by which it exerts its inhibitory effect remains unknown. It is possible that PYY acts indirectly by inhibiting the release of pancreatic secretogogues rather than by a direct action on the pancreatic acinus itself. The present study examines this question by determining the effects of synthetic PYY on both secretin- and cholecystokinin (CCK)-stimulated pancreatic exocrine secretion in conscious dogs. A dose of PYY was selected that both inhibited mealstimulated pancreatic secretion and reproduced blood levels observed after perfusion of the small intestine with fat (8). This choice was based on the previous demonstration by others (12-14)that intestinally perfused fat releases an inhibitor of pancreatic secretion. In the second limb of the study we examined the metabolic clearance of synthetic PYY in the same animals to determine if PYY is cleared from the circulation at a rate similar to that of other established gastrointestinal hormones.

Methods Peptide Five

Received February 27. 1985. Accepted June 11. 1985. Address requests for reprints to: Ian L. Taylor, M.D.. Ph.D., Division of Gastroenterology (lllG), Sepulveda Veterans Administration Medical Center, 16111 Plummer Street. Sepulveda. California 91343. This work was supported by the Veterans Administration and a grant from the National Institutes of Health (NIADDK AM 35247). The authors thank Joan Semasinghe for manuscript preparation and Ray Melendez for skilled technical assistance. I 1985 by the American Gastroenterological Association 0016.5085/85/$3.30

and UCLA School

pared

with

pancreatic allowed maintained Purina,

YY und Pancreatic

mongrel gastric fistula

St. Louis,

weighing (Thomas

(Herrera

to recover on

dogs fistula

for several

Purina MO.)

19-25 cannula)

cannula). weeks

Caniue after

Secretion

The before

Laboratory the

immediate

kg were and animals study

pre-

chronic were

and

Chow

were

(Ralston

postoperative

Abbreviations used in this paper: CCK. cholecystokinin; cholecystokinill-octapeptide; PP. pancreatic polypeptide; peptide YY.

CCK-8, PYY,

GASTROENTEROLOGYVol.8Y,No.G

i388 PAPPAS ET AL.

period. Eighteen hours before each study, animals were fasted but ,allowed free access to water. Basal pancreatic secretion was monitored in 4 dogs with pancreatic fistula during the infusion of normal saline or PYY (460 pmolikg h). Four dogs were also studied to determine the effects of PYY on secretinand, CCK-stimulated setretion Dogs received four doses of secretin or cholecystokininoctapeptide (CCK-8) (62.5, 125, 250, 500 ngikg . h). Each dose was infused for 45 inin and the dose was increased in a step-dose fashion. During the secretin and,CCK-8 infusions each dog received either a background infusion of saline as a control or an infusion of PYY 1400 pmolikg 1h). Previous studies (8) have demonstrated that the infusion of PYY in a dose of 400 pmolikg . h inhibits meal-stimulated pancreatic secretion. In addition, this dose of PYY reproduces blood levels observed after the small intestine is perfused with fat (8), a stimulus known (13) to inhibit the pancreatic secretory response to exogenous secretogogues. During each experiment pancreatic secretion was collected continuously and divided into 15-min samples. The volume and bicarbonate and protein outputs in each sample were determined using published methods (11). A two-tailed Student’s paired t-test was used to determine differences between basal and hormonally stimulated pancreatic secretion during the saline and PYY infusions. Raw data were converted to square roots to normalize the data before statistical analysis, and probability values of ~0.05 were designated significant. Values are reported in the text as mean + standard error.

Infusates Synthetic porcine PYY (Penninsula, Belmont, Calif.) was used for the infusion studies and was dissolved in a phosphate buffer at a stock concentration of 100 kg/ml. Each dose of PYY was infused in normal saline containing 0.1% bovine serum aibumin. Synthetic CCK-8 (Penninsula) was prepared from a stock solution stored in O.l%, acetic acid containing 0.5% bovine serum albumin and infused in saline containing 0.1% bovine serum albumin. Synthetic secretin (Squibb, New York, N.Y.) was also dissolved and infused in the 0.1% bovine serum albuminsaline buffer.

plasma PYY concentration observed during infusion of PYY minus the mean basal concentration. The disappearance rate constant k was defined as the negative slope,of the least-squares linear regression of the natural logarithm of PYY concentration versus time in minutes (15). It was computed (16)using a nonlinear least-squares computer program (BMDP3R). The same computer program was used to determine if a two-compartment model fitted the disappearance curve for each animal better than a singlecompartment model. The disappearance half-time (tIrE) was then calculated from the equation tl;2 = 0.693/k. The metabolic clearance rate of PYY was calculated from the following equation: C = DIP, where C is the clearance in milliliters per kilogram per minute, D is the dose of PYY in picomoles per kilogram per minute, and P is the plateau plasma PYY concentration given in nanomolar concentrations. The volume of distribution was calculated from the plateau principle using the. following equation: V = D/PI<, where V is the volume of distribution as a fraction of body weight (milliliters per kilogram) and the other symbols are as described above. Radioimmunoassay Plasma PYY was determined by a specific radioimmunoassay using published methods (5). In brief, the antiserum [AbSlS] was raised against a porcine YY-bovine serum albumin conjugate and was used at a working titer of l:lOO,OOO. A porcine PYY label and standard were used for all assays. Pancreatic polypeptide and neuropeptide Y are, respectively, lO,OOO-fold and 500-fold less immunoreactive than PYY in this assay. Structurally unrelated synthetic peptides such as secretin and CCK-8 exhibit no cross-reactivity (
Results Basal

of Peptide

YY

infusion.

Peptide

kg . h) significantly bonate

To determine the plasma half-life of PYY, 5 animals were infused intravenously with a dose of 400 were pmolikg . h for 45 min. Two basal blood samples collected before commencing the infusion and further samples were taken every 15 min during the infusion. The infusion catheter was removed at the end of the 45-&n infusion period and blood samples were taken at 2.5,5, 7.5,10,15,3b,45,and 60 min to monitor disappearance of PYY. All blood samples were collected on ice in ethylenediaminetetraacetic acid tubes with Trasylol (500 U/ml blood). Each sample was centrifuged and the plasma was separated and stored at -20°C. For determination of metabolic clearance rate, plateau plasma PYY concentrations were taken as the 45-min

Secretion

Basal pancreatic bicarbonate was 144 * 26 pmoli60 min

4 dogs control

Metabolism

Pancreatic

YY

(p < 0.05)

to 51 ? 11 pmo]/60

inhibition

of 59%

*

infusion

(400

inhibited

basal

min,

13%.

secretion in the during the saline

Basal

representing pancreatic

pmoli bicara mean protein

secretion during the I-h saline control period was 463 i 46 mgi60 min. Peptide YY infusion decreased basal protein secretion to 185 ‘-’ 44 mgi60 min, a change

that

represented

a 59%

? 11%

inhibition

(p

< 0.05).

Secretin-Stimulated

secretin

Pancreatic

Secretion

During the saline control study, infusion of resulted in a prompt increase in pancreatic

bicarbonate nificantly

secretion. Secretory rates increased sigfrom a basal rate of 51.9 t 8.3 pmolil5

December

1985

AND BIOL,OGIC ACTIONS

METABOLISM

BICARBONATE

OF PYY

1389

PROTEIN

SECRETIN

PLUS

PYY

60

7

I

0

62.6

I

126

260

0

600

r

126

250 *p
SECRETIN w/kg/h Figure

1. Effects

of peptide

YY (PYY) (400 pmolikg

min to 588.7 I? 116.5 ~molil5 min after infusion of the lowest dose of secretin (62.5 ng/kg . h). With increasing doses, bicarbonate output increased in a dose-dependent manner until a maximal secretory rate of 3066 +- 199 pmo1/15 min was observed with the 500-ng/kg . h dose of secretin (Figure 1). When PYY was infused with secretin, no significant increase in bicarbonate secretiqn was observed with the 62.5-ngikg . h dose. In addition, the bicarbonate * h doses of responses to the 125- and 250-ngikg secretin were significantly inhibited (Figure 1). However, PYY failed to inhibit maximal bicarbonate output observed in response to the 500-ngikg . h dose of secretin. Protein secretion also increased significantly in response to secretin infusion alone, although the response was much less drtimatic than that observed with CCK-8. A maximal secretory rate of 232.5 -t 38.5 mgil5 min was observed with the highest dose of secretin (500 ngikg . h) during the saline control study. In contrast, no increase in protein output was observed when PY’Y was infused’with secretin (p ‘< 0.05).

Cholecystokinin-Stimulated Secretion

Pancreatic

During the saline control study, a prompt increase in protein output was observed in response to CCK-8; this increase reached a numerical peak with the 250-ngikg . h dose (394.7 2 121.7 mgA5 min). All four doses of CCK-8 significantly increased

h) on the pancreatic

secretory

I

I

I

62.5

response

500 n=4

to secretin.

pancreatic protein output during the saline control study. In contrast, no significant increase in protein output was observed when PYY was infused with the 62.5-ngikg . h dose of CCK, and significant inhibition of the pancreatic protein response to the 125and 250-ngikg . h doses of CCK-8 was observed. Peptide YY, however, did not inhibit the protein response to the highest dose of CCK-8, demonstrating once again that the inhibitory effect was surmountable (Figure 2). During the CCK-8 infusion, bicarbonate secretion increased fo a maximal rate of 158.5 ? 22.7 pEqll5 min during the 250-nglkg ’h dose, a response that was completely abolished by PYY. Metabolic

Clearance

Studies

The mean basal PYY concentration in these animals was 88 2 8.5 pM. During the infusion of PYY (400 pmolikg . h), plasma PYY peaked at 45 min when an increment over basal of 513 * 64 pM was observed. Peptide YY concentrations fell rapidly after removal of the infusion catheter (Figure 3). The metabolic half-life of PYY was calculated as 11.7 ? 2.1 min, with 95% confidence intervals that extended from 5.8 to 17.8 min. The metabolic clearance rate of PYY was 13.8 ? 1.6 ml/kg. min and the volume of distribution was 223 -t 33 ml/kg.

Discussion The present study demonstrates that PYY in a dose of 400 pmolikg . h inhibits basal bicarbonate

1390

GASTKO~~TEKOLOGY

PAPPAS ET AL.

BICARBONATE

Vol. 89, No. 6

PROTEIN

CCK-0

ALONE

I

160

/ T /

CCK-6

ALONE

/

0” x

40

I

0

I

62.5

I

125

I

250

I

I

I

I

0

500

I

125

62.5

* CCK-8 Figure

2. Effects of peptide

YY (PYY] (400 pmol/kg

500

P
n=4

ng/kg/h

II) on the pancreatic

output and shifts the pancreatic bicarbonate doseresponse curve to secretin to the right (Figure 1). However, the bicarbonate response to the highest dose of secretin was not decreased significantly, indicating that PYY does not inhibit the response to maximal doses of secretogogue and that the inhibition caused by PYY was surmountable. These effects on secretin-stimulated pancreatic secretion are similar to those previously reported for PP (g-11,1 7). Although the dose-response data do not fit classical models of inhibitory kinetics, they more closely fit a competitive rather than noncompetitive pattern of inhibition. An almost identical pattern of inhibition was observed when CCK-stimulated protein secretion was examined. Thus, PYY inhibited the protein response to low doses of CCK-8 but did not inhibit the maximal response. Although we did observe a bicarbonate response to CCK-8 alone, bicarbonate outputs were Z&fold less than those seen with secretin. Both the protein response to secretin and the bicarbonate response to CCK were totally abolished by PYY. We conclude from these studies that PYY inhibits both secretin- and CCK-stimulated pancreatic exocrine secretion. As such, our findings extend those of Tatemoto (Z), who reported inhibition of secretinand CCK-stimulated pancreatic secretion in a single anesthetized cat. The results suggest that our previous demonstration (8) of PYY-induced inhibition of meal-stimulated pancreatic exocrine secretion does not reflect an indirect effect of the peptide through inhibition of secretin and cholecystokinin release.

I

250

secretory

response

to cholecystokinill-octapeptide

(CCK-R).

One, however, cannot necessarily conclude from our data that PYY acts directly on the pancreatic acinus. Again we can draw a corollary from the available data on PP-induced inhibition of pancreatic exocrine secretion (18). Thus, although PP inhibits pancreatic secretion in response to secretin and CCK in vivo (g-11), it fails to inhibit hormonally stimulated secretion in vitro (18). There are several possible explanations for these observations. First, the hormonal receptors present on the acinus may be damaged or removed during preparation of the isolated acini. Second, PYY has been demonstrated to cause a dose-dependent vasoconstriction of the splanchnic ‘001

*

‘;,

60

1, =11.7min /2

c n

1

0

10

1

‘.‘.

I

I

20

30

1

1

1

40

50

60

TIME (mid Figure

3. Metabolic half-life of peptide YY (PYY) in the circulation. The mean half-life (tlr2) was 11.7 +- 2.1 min.

December

1985

circulation when injected into the superior mesenteric artery (4). It is possible that the effects of PYY on the pancreas, and that of the other PP family members, could be mediated through decreased pancreatic blood flow. In support of this hypothesis, PYY has been demonstrated to induce vasoconstriction in the cat submandibular gland (4). Finally, the recent demonstration (19) of CCK receptors on the vagal nerve fibers suggests that gastrointestinal hormones may be able to modulate parasympathetic activity. The metabolic clearance studies demonstrate that PYY has a metabolic half-life (11.7 ? 2.1 min) comparable to that of other established gastrointestinal hormones of similar size. The metabolic halflife is somewhat prolonged compared with its cousin PP. Thus bovine PP has a half-life of 6.8 min in humans (20) and canine PP has a half-life of 5.5 min in dogs (11). Comparable differences were observed in metabolic clearance rates (11) despite similar distribution volumes. We cannot exclude the possibility that the use of heterologous PYY, i.e., porcine PYY in dogs might have had effects on the metabolic parameters we were monitoring. In the case of another family member (PP), however, the porcine and canine peptides are known to be identical in structure (7). In summary, our study demonstrates that PYY has a circulatory half-life comparable to that of other gastrointestinal hormones of similar size (21). A hormonal role for this peptide is further supported by the demonstration that the majority of PYY cells have endocrine characteristics (6). In addition, we have demonstrated that PYY is released into the circulation in response to food, a response that is delayed when compared with that of its sister peptide, PP (5,22). Although these studies would support a role for PYY as a true hormone, its physiologic role remains to be determined. It is apparent that many exocrine and endocrine glands are controlled by the complex interplay of multiple stimulants and inhibitors. As the PYY response to food is both delayed and prolonged, it may serve to inhibit pancreatic secretion after intraluminal contents have passed beyond the absorptive surface of the gut. Peptide YY would be ideally suited for such an inhibitory role based on the present demonstration that it will only inhibit pancreatic secretion when the concentrations of hormonal stimulants have fallen to submaximal levels. The existence of an inhibitor of pancreatic secretion in extracts of the ileum and colon was established several years before the discovery of PYY. Harper and his coworkers (12) called this factor pancreotone and Sarles et al. (13) called it the “anti-CM hormone.” The present study coupled

AND BIOLOGIC ACTIONS OF PYY

METABOLISM

1391

with the findings of these other studies (12-14) demonstrates that PYY, pancreotone, and the “antiCCK hormone” share the ability to inhibit secretinand CCK-stimulated pancreatic secretion, Perfusion of the ileocolonic mucosa with oleic acid was also shown to inhibit pancreatic secretion (13,14). This effect was shown to be mediated by release of a hormonal inhibitor when inhibition was still shown to be demonstrable, even after denervation of the pancreas and intestine (13,14). Parabiotic studies performed in rats (13) confirmed the hormonal nature of this inhibitor. The present study demonstrates that a dose of PYY previously shown to reproduce the blood levels of PYY observed after intestinal perfusion with oleic acid (8) is capable of inhibiting both secretin- and CCK-stimulated pancreatic secretion. As such, PYY must be considered a likely candidate for the title of “pancreotone” or “anti-CCK hormone.” However, as pancreotone is reported to inhibit gallbladder contractility (l2), whereas PYY is reported to have no effect on gallbladder tone (21, the intestinal extract that constitutes pancreotone may contain more than one hormonal factor.

References 1. Tatemoto K, Mutt V. Isolation of two novel candidate hormones using a chemical method for finding natural occurring polypeptides. Nature 1980;285:417-8. 2. Tatemoto K. Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc Nat1 Acad Sci USA 1982;79:2514-8. K, Carlquist M, Mutt V. Neuropeptide Y-a novel 3. Tatemoto brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 1982;296:659-60, 4. Lundberg JM, Tatemoto K, Terenius L, et al. Localization of peptide YY (PYY) in gastrointestinal endocrine cells and effects in intestinal blood flow and motility. Proc Nat1 Acad Sci USA 1982;79:4471-5. 5. Taylor

IL. Distribution

by specific

and

radioimmunoassay

release

of peptide

YY measured

in dog. Gastroenterology

1985;

88:731-7. 6. El-Salhy chemical human 7. Chance

M, Grimelius

L, Wilander

identification

of polypeptide

gastrointestinal RE,

Moon

tract. NE,

E, et al. ImmunocytoYY (PYY) cells in the

Histochemistry

Johnson

MG.

polypeptide (HPP) and bovine pancreatic In: Jaffe BM, Behrman HR, eds. Methods immunoassay.

New York: Academic,

1983;77:15-23. Human

pancreatic-

polypeptide of hormone

(BPP). radio-

1979:657-72.

8. Papas TN, Debas HT, Goto Y, Taylor IL. Peptide meal-stimulated pancreatic and gastric secretion,

YY inhibits Am J Phys-

iol 1985;248:G118-23. 9. Greenberg GR, McCloy RF, Adrian JE, Chadwick VS, Baron JH, Bloom SR. Inhibition of pancreas and gallbladder by pancreatic polypeptide. Lancet 1978;ii:1280-2. 10. Lin T-M, Evans KC, Chance RE, Spray CF. Bovine pancreatic polypeptide: actions on gastric and dogs. Am J Physiol 1977;232:E311-5,

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in

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11. Taylor IL, Solomon TE, Walsh J, Grossman MI. Pancreatic polypeptide: metabolism and effect on pancreatic secretion in dogs. Gastroenterology 1979;76:524-8. 12. Harper AA, Hood AJC, Mushens J, Smy JR. Pancreotone, an inhibitor of pancreatic secretion in extracts of ileal and colonic mucosa. J Physiol 1979;292:455-67. 13. Sarles H, Hage G, Laugier R, Demo1 P, Bataille D. Present status of the anticholecystokinin hormone. Digestion 1979;19: 73-6. 14. Harper AA, Hood AJC, Mushens J, Smy JR. Inhibition of external pancreatic secretion by intracolonic and intraileal infusions in the cat. J Physiol 1979;292:445-54. 15. Goldstein AL, Aranon L, Kalman SM. In: Principles of drug action. New York: Harper & Row, 1969:292-317. 16. Carter DC, Taylor IL, Elashoff J, Grossman MI. Re-appraisal of the secretory potency and disappearance of pure human mini-gastrin. Gut 1979;20:705-8.

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17. Beglinger C, Taylor IL, Grossman

MI, Solomon TE. Pancreatic polypeptide inhibits exocrine pancreatic responses to six stimulants. Am J Physiol 1984;246:G286-91. 18. Kim KH, Case RM. Effects of pancreatic polypeptide on the secretion of enzymes and electrolytes by in vitro preparations of rat and cat pancreas. Yansei Med J 1980;21:99-105. 19. Zarbin MA, Wormsley JK, Innis RB, Kuhar MK. Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve. Life Sci 1981;29:697-705. 20. Adrian TE, Greenberg GR, Besterman HS, Bloom SR. Pharmacokinetics of pancreatic polypeptide in man. Gut 1978;19:907-9. 21. Walsh JH. Endocrine cells of the digestive system. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven, 1981:59-144. polypeptide: a hormone under 22. Schwartz TW. Pancreatic vagal control. Gastroenterology 1983;85:1411-25.