Pflfigers A r c h - Eur J Physiol (1995) 431 : 66-75 �9 Springer-Verlag 1995

X i a o m e i F u - C h e n g �9 Y o u n e s A n i n i �9 J a c q u e s C h a r i o t T h i e r r y V o i s i n �9 J e a n - P a u l G a l m i c h e �9 C l a u d e R o z 6

Peptide YY release after intraduodenal, intraileal, and intracolonic administration of nutrients in rats

Received: 13 March 1995 / Received after revision: 6 June 1995 / Accepted: 14 July 1995

A b s t r a c t Peptide YY (PYY) release was studied by measuring radioimmunoassayable PYY in the arterial plasma of anaesthetized rats receiving into the duode- num, ileum or colon either a complete semi-liquid meal (3 ml, 21 kJ) or elemental nutrients as isocaloric or isoosmolar solutions. PYY release induced by the intraduodenal meal peaked at 60 min and lasted more than 120 min. The integrated response of PYY over 120 min was larger when the meal was administered into the duodenum than into the ileum. The undigested meal induced no release of PYY over a 120-rain period when administered into the colon. When injected into the duodenum in isocaloric amounts to the meal, glu- cose and amino acids led to the release of as much PYY as did the meal, whereas oleic acid led to the release of less PYY. Part of these responses were due to osmolarity, since administration of intraduodenal hyperosmolar saline led to the release of about half as much PYY as did hyperosmolar glucose. In moderate amounts, and injected as a solution isoosmolar to plasma, oleic acid was a major PYY releaser; the amounts released were at least two times larger when oleic acid was administered into the duodenum than into the ileum and colon. Isoosmolar glucose and amino acids led to the release of no PYY when injected into the duodenum, but were nearly as active as oleic acid in the colon. Short-chain fatty acids induced the release of PYY when injected into the colon, but not into the ileum. Hexamethonium suppressed PYY release induced by the intraduodenal meal, but did not change PYY release induced by glucose or oleic acid in the colon. Urethane anaesthesia did not reduce PYY release induced by the intraduodenal meal. These

X. Fu-Cheng �9 Y. Anini �9 J. Chariot ' T.Voisin - C. Roz~ ( [ ] ) INSERM U 410, Facult6 de M~decine X Bichat, BP 416, F-75870 Paris, Cedex 18, France

J-P. Galmiche Clinique des Maladies de l 'Appareil Digestif, C H U Nord, F- 44035 Nantes, France

results suggest that two mechanisms at least contribute to PYY release in the rat. An indirect, neural mecha- nism, involving nicotinic synapses, is prominent in the proximal small intestine; the stimulation is transmitted to ileal and colonic L-cells by undetermined pathways, but contact of nutrients with L-cells is not needed. Another mechanism, probably direct and quantitatively smaller, occurs in the distal intestine when nutrients come into contact with the mucosa containing L-cells. Glucose, fatty acids and amino acids stimulate differen- tially the proximal and distal mechanisms.

K e y w o r d s Peptide YY �9 Hormone release �9 D u o d e n u m I l e u m Colon. Nutrients

Introduction

Peptide YY (PYY), a 36-amino-acid peptide first isolated from porcine duodenum [34], is produced by endocrine L-cells of the distal digestive tract: distal ileum, caecum, colon, and rectum [1, 5, 14, 20, 26]. The distribution of immunoreactive PYY in the rat, as measured by radioimmunoassay, is very similar to L- cell distribution in dogs, monkeys and humans, demon- strating low concentrations in the upper small intestine and much larger concentrations in the ileum and colon [14, 26]. In contrast, mucosal PYY receptors occur mainly in the duodenum and jejunum, while a three- fold lower density is found in the ileum, and no recep- tors are detectable in the large intestine of the rat [21].

PYY has a variety of biological effects on the diges- tive system, including inhibition of gastric secretion [2, 6, 17, 18], of exocrine pancreatic secretion [9, 13, 19, 22, 27, 30], of intestinal secretion [8], and of gastroin- testinal motility [37, 38], in laboratory animals and humans.

Plasma PYY levels increase after oral ingestion of food [2, 16, 20], and after intraintestinal or intracolonic

67

administration of nutrients [5, 15] in animals and humans. However the comparative amount of PYY release induced by constituents of food, i.e. fat, carbo- hydrate and protein, has not been extensively studied so far, especially in rats. PYY release involves at least two different mechanisms in dogs. Direct stimulation of L-cells by the luminal contents occurs in the distal digestive tract, during the late stages of digestion, whereas indirect stimulation originates in the proximal bowel in the early stages of digestion and is transmit- ted to L-cells through neural and humoral pathways [16, 25, 28, 40]. In cultured intestinal L-cells, PYY is released by sodium oleate, bombesin [4, 11], and glu- cose-dependent insulinotropic peptide GIP [11].

The aim of the present study was to compare the variations of plasma PYY in response to a standard meal and to elemental nutrients selectively adminis- tered into the duodenum, ileum and colon in rats, and to test the dependency of PYY release on nicotinic mechanisms.

blood loss, an equal volume of Haemaccel (polymerized bovine gelatin hydrolysate, Hoechst Laboratories, France) was injected through the carotid catheter after each blood sampling. After 3-min centrifugation at 10,000 rpm (Biofuge 13, Heraeus, Les Ulis, France), the plasma samples were collected and stored at - 2 0 ~ until PYY assay (up to 15 days).

Studies in conscious rats

Some rats were anaesthetized for a short time with 50 mg/kg ket- amine plus 10 mg/kg pentobarbital, i.p., and were fitted with a transpyloric duodenal catheter and a carotid catheter as described above. The carotid catheter was routed under the skin to exit at the back of the head and the rats were allowed to recover from anaesthesia in Bollman-type cages. After complete recovery, i.e. about 2 h after surgery, intraduodenal meal administration and carotid blood sampling were carried out for 2 h, as for anaesthetized rats. At the end of the experiment the rats were sacrificed by an overdose of anaesthetic.

Control of intestinal transit of the duodenal meal

Materials and methods

Studies in anaesthetized rats

In rats given the intraduodenal meal (containing charcoal as a marker) the whole small bowel was withdrawn and laid out. The position of the coloured meal front was measured and expressed as a percentage of the total small intestine length.

Male Wistar rats (300 350 g) were used. They were fasted 18 h before experiments, with free access to water, and anaesthetized with urethane (1.25 g/kg, i.m).

Meal and nutrients

Meal

Intestinal catheter for administration of nutrients

After a median laparotomy, some of the rats (n = 84) were equipped with a transpyloric duodenal catheter. A 10-cm piece of Silastic tubing (1.57 x 2.40 mm, ID x OD) was inserted through the fore- stomach, passed into the proximal duodenum through the pylorus, and secured by a ligature over the pylorus and by a purse-string suture at the forestomach. Other rats were fitted with a similar catheter, inserted either in the ileum (35 cm distal to the pylorus; n = 30) or in the colon (5 mm distal to the caecum; n = 42). The catheter was secured by a ligature surrounding the bowel and was exteriorized through the laparotomy incision before closing the abdomen.

Arterial catheter for blood sampling

A short polythene catheter (Clay Adams PE 50, 0.58 • 0.965 mm, I D x O D , length 15mm) continued by a Silastic catheter (0.64 x 1.19 mm, ID • OD, length 15 cm) was inserted into a carotid artery and secured by a double ligature around the polythene part of the catheter. The rats received 250 I U / k g heparin, injected through the carotid catheter as soon as it was secured. The catheter was then filled with saline and closed by a small "bulldog" vascu- lar clamp. Blood samples (0.6 or 1 ml during stimulation or under basal conditions, respectively) were collected in Eppendorf 1.5-ml tubes containing 2 mg ethylenediaminetetraacetate (EDTA) and 500 kIU aprotinine (50 gl Trasylol at 10,000 kIU/ml) for 1 ml whole blood, after having discarded the first drops, corresponding to the dead space of the catheter. Blood samples were collected before intestinal administration of nutrients, and then after 15, 30, 60 and 120 min (180 min in some experiments). To compensate for

The meal consisted of 3 ml of a semi-liquid diet, containing 21 kJ, provided as 57% carbohydrate (Caloreen: small glucose polymers), 13% lipid (Liprocil: medium-chain triglycerides), and 30% protein (lactoserum protein), a generous gift of Sopharga, Puteaux, France. Activated charcoal (Norit A, Aldrich Chemicals, USA, 20mg/3 ml) was added as a non-absorbable coloured marker.

Isocaloric nutrient solutions

In order to administer the same caloric amount (21 kJ) of pure nutrients as in the mixed intraduodenal meal, we used 3 ml of the following solutions: glucose 417 g/l; oleic acid 186 g/1 (pH 6.5, emulsified with 1% Tween 80), or a mixture of amino acids 410 g/1 (Vint6ne, Clintec Nutrition Clinique, Amilly, France, 3.2-fold concentrated by lyophilization). As was the mixed meal, these solutions were hyperosmolar: glucose 2300 mosmol/1, oleic acid 1350 mosmol/l and amino acids 3650 mosmol/1. To test the effect of hyperosmolarity and the specific effects of nutrients, the effect of hyperosmolar glucose was compared to that of hyperosmolar NaC1 (2300 mosmol / l = 67.3 g/t).

Isoosmolar solutions of nutrients (300 mosmol / l)

These solutions were glucose 54 g/l; oleic acid 42.3 g/1 (pH 6.5, emulsified by 1% Tween 80), amino acids 34 g/1 (a 1/3.8 dilution of Vint6ne in distilled water), short-chain fatty acids: a mixture of acetic acid 5.85 g/1 (65%), propionic acid 2.22 g/l (20%), butyric acid 1.98 g/1 (15%), pH 6.5, these proportions corresponding to those usually found in the colonic content of normal rats (C. Cherbut, personal communication).

68

In some experiments, hexamethonium (6.7mg/kg bolus + 6.7 mg/kg.h, i.v.) was administered from 60 min before to 120 min after the administration of nutrients.

All meals and solutions were given in the bowel as a 3-ml bolus over 3 rain; 0.9% saline was used as control.

All chemicals were purchased from Sigma.

PYY radioimmunoassay

Plasma PYY levels were measured by a sensitive and specific radioimmunoassay developed in our laboratory, according to Taylor [35] and Adrian et al. [2]. Antibody A4D (a generous gift of JC Cuber, INSERM U 45, Lyon, France) was raised in rabbits against synthetic porcine PYY. Antibody A4D cross-reacts 100% with porcine PYY 1 36 and PYY 3-36 and less than 0.1% with neuropeptide Y and pancreatic polypeptide. Synthetic porcine PYY (Neosystem, Strasbourg, France) was radiolabelled with Na125I using the chloramine T method [36]. Purification of radiolabelled PYY was performed by reverse-phase high performance liquid chro- matography (HPLC). The HPLC column was isocratically eluted with 31% (v/v) acetonitrile in water with 0.1% (v/v) trifluoroacetic acid. Two main tracers were obtained in these conditions: [1251- Tyrl]-monoiodo-PYY and [12SI-Tyr36]monoiodo-PYY. Both trac- ers were identically displaced by unlabelled porcine PYY. In these experiments, we used [12SI-Tyrl]-monoiodo-PYY. Fractions con- taining the radiolabelled tracer were stored at 20 o C in acetic acid buffer (0.1 M) with 0.1% bovine serum albumin and used in radioimmunoassays after proper dilution with the assay buffer. The assay buffer was 0.1 M sodium phosphate, pH 7.4, containing 0.01% sodium azide, and 0.1% bovine serum albumin. Synthetic porcine PYY was used as the standard, ranging from 3.125 to 100 pg/tube; dilution was achieved using the assay buffer. Briefly, the radioimmunoassay was performed in 500 gl incubation volume. Plasma samples were 100 gl under stimulated conditions, completed with 100 gl Haemaccel and 200 gl under basal conditions. In con- trols (zero) and standards, 200 gl Haemaccel was added. Antibody was added at a final dilution of 1 : 250,000. Assay tubes were prein- cubated for 24 h at 4 ~ C. The tracer (3000 dpm. 100 g l - t. tube 1) was added and assay tubes were incubated for 48 h at 4~ Antibody-bound tracer was separated from unbound tracer by adding 500 gl per tube ice-cold dextran-coated charcoal (0.5 % char- coal in 0.1 M phosphate buffer, containing 0.05% Dextran T-70). After centrifugation (4000 rpm, 15 min), the supernatants were aspirated and the radioactivity of the pellets was counted using a Beckman gamma counter. The samples were determined in dupli- cate and concentrations were determined from the standard curve. Non-specific binding and zero standard binding were determined to be < 5% and > 30%, respectively. The minimum detectable con- centration of PYY was 20 pg/ml, the intraassay coefficient of vari- ation 8.9%, and the interassay coefficient of variation 7.5-13.0% for values ranging between 31.25 and 1000 pg/ml. Recovery of PYY (250 pg/ml) added to plasma was 91.8%. Standard curves run in Haemaccel and in hormone-free plasma were not significantly different, indicating that rat plasma did not interfere in the assay.

Analysis of results

Results are expressed as means + SEM of actual values, or of vari- ations over basal level (A). PYY concentrations are given in pg/mI. Integrated increases of plasma PYY over basal values during the 120 min after the administration of the nutrients (area under the curve) were calculated and expressed as ng" m l - 1. 120 min 1.

Statistical analysis of the data was performed by the Student - Fisher's t-test or analysis of variance (ANOVA), followed by a multigroup comparison test (Fisher PLSD) when the global difference was significant. Differences with P < 0.05 were consid- ered significant.

Results Effect of intraduodenal, intraileal and intracolonic meal on plasma PYY

Plasma PYY concentrations increased after the intraduodenal meal (at 15 to 120 min, P < 0.001 vs basal value, Fig. 1A). Plasma PYY reached a peak level of 395 + 45 pg/ml at 60 min, i. e. about 15-fold the basal level, and still remained at sevenfold the basal level at 120 min. The same meal administered into the ileum induced a progressive and weaker increase ( P < 0 . 0 5 vs basal value) at all times studied, reaching 267 + 64 pg/ml at 120 min and 265 _+ 52 pg/ml at 180 min (not shown). At 30 and 60 min, the plasma PYY level was lower (P < 0.05)

Fig. 1A, B Effect of intraduodenal (ID), intraileal (H) and intra- colonic (IC) administration (an'ow) of a meal (3 ml of a semi-liq- uid diet containing 21 kJ provided as 57% carbohydrate, 13% lipid and 30% protein) on plasma peptide YY (PYY). Mean + SEM, n = 6 rats per group except ID group (n = 18). A Plasma PYY lev- els as a function of time; B 120-min integrated PYY increases over basal values, compared with their respective controls (saline). �9 P < 0.05, o . .P < 0.001 vs their respective controls, op < 0.05, ooop < 0.001 vs ID meal. *P < 0.05 vs ID meal at 30 and 60 rain

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69

than after intraduodenal administration. The P Y Y release integrated over 120 min was weaker after intraileal than after intraduodenal administration (17.5 + 4.6 ng'm1-1" 120 min -1 in the ileum vs 30.1 + 3.4 ng.ml 1"120 min -1 in the duodenum, P < 0.05, Fig. 1B). Administering the meal into the colon pro- duced no significant PYY release throughout the 120 min (0.49 + 1.55 ng'ml -* ' 120 rain -1.

Controls performed by injecting 3 ml saline into the duodenum, ileum or colon led to the release of very little PYY (1.41 + 0.59, 0.32 + 0.64 and -0 .17 + 0.76 ng 'ml -1 . 120 min 1, respectively, not significant)�9

Effect of intraduodenal administration of nutrients in amounts isocaloric to the meal

After isocaloric (21 k J) intraduodenal administration of glucose, plasma PYY significantly increased from a basal level of 16 + 5 pg/ml to 383 + 33 pg/ml at 60 min and 419 + 65 pg/ml at 120 min (Fig. 2A). The integrated increase over 120 min was identical to that measured after the meal (32.6+3.1 ng" ml -~-120 min -1 and 30.1 + 3.4 ng'm1-1"120 min -1 respectively, Fig. 2B).

Isocaloric amino acids significantly increased plasma PYY levels from 30 min to 120 min. The inte- grated release (30.6 + 4.6 ng" ml - 1.120 min - ~) did no t significantly differ from that produced by glucose�9

Isocaloric oleic acid significantly (P < 0.05 vs basal value) increased plasma PYY between 15 min and 120 min. Plasma PYY plateaued to about 150 pg/ml at 60 min and 120 min, a significantly lower level (P < 0.01) than the peak observed after the intraduo- denal meal: 395 + 45 pg/ml at 60 min. The integrated PYY release was 12.2 + 3.1 ng'm1-1- 120 min -1 after oleic acid versus 30.1 + 3.4 ng 'ml 1. 120 min -1 after the intraduodenal meal (P < 0.05).

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Fig. 2A, B Effect of intradnodenal administration (ID, arrow,,) of glucose (Glu), oleic acid (OA) and a mixture of amino acids (AA) in amounts isocaloric with the meal (21 kJ) on plasma PYY. Mean • SEM, n = 6 rats per group. A Plasma PYY as a function of time; B 120-min integrated PYY increases over basal values. *P < 0.05, ***P < 0.001 vs saline; o p < 0.05 vs ID meal. In order not to clutter panel A, the effect of the meal was not drawn and the significance levels of plasma PYY means vs their respective basal values (P < 0.05 for all values but amino acids at 15 min) were omitted

Effects of intraduodenal hyperosmolarity on plasma PYY

Nutrients administered instead of the meal in isocaloric amounts are strongly hyperosmolar. Their effect might thus involve a non-specific effect of hyperosmolarity, which was tested by comparing the effect of isocaloric glucose (2300 mosmol/1) to the effect of a NaC1 solu- tion of identical osmolarity. Isoosmolar glucose and NaC1 solutions were tested as controls.

While isoosmolar (300 mosmol/1) solutions of NaC1 and glucose led to the release of very little PYY (NaC1 1.41 + 0.59 ng'ml -I" 120 min -1 and glucose 1.69 + 0.40 ng-ml 1"120 min- l : Fig. 3B), hyperos- molar (2300 mosmol/1) solutions of NaC1 and glucose induced the release of large amounts of PYY (inte- grated release 32.6 + 3.1 ng 'ml -*-120 min 1 with glucose, 16.7 + 1.4 n g ' m l - l ' 1 2 0 min -1 with NaC1,

P < 0.001 vs their respective isoosmolar solution). The integrated PYY release after administration of hyper- osmolar glucose was larger than that after administra- tion of hyperosmolar NaC1 (P < 0.001), and lasted for a longer time. The level of plasma PYY 60 and 120 min after administration of hyperosmolar glucose was larger than that after administration of hyperosmolar NaC1 (P < 0.05 and P < 0.01, respectively, Fig. 3A).

Effect of intraduodenal nutrients provided as isoosmolar solutions

Isoosmolar (300mosmol/1) oleic acid (3ml), when administered into the duodenum, significantly increased plasma PYY levels from 28 + 3 pg/ml to 156 + 30 pg/ml (P < 0.05 vs basal) at 30 min and to 251 + 41 pg/ml after 60 min (P < 0.05, Fig. 4A). The

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integrated release was 19.7 + 3.4 ng 'ml 1. 120 min-1 (P < 0.01 vs saline, Fig. 4B). Administration of isoos- molar glucose and amino acids released no apprecia- ble amounts of PYY.

Effect of intraileal nutrients provided as isoosmolar solutions

Intraileal administration of 3 ml isoosmolar oleic acid significantly increased plasma PYY levels between 15min and 120min (Fig. 5A). The PYY level increased more rapidly than after intraduodenal injection of the same solution. The peak value occurred after 30 min and the PYY level then plateaued until 120 min. The integrated increase (9.5 + 2.2 ng.m1-1. 120 min -1, P < 0.01 vs saline, Fig. 5B) was smaller than that observed after intraduodenal administration (19.7 + 3.4 ng'm1-1" 120 min 1, p < 0.05).

Intraileal administration of isoosmolar glucose induced the released of approximately the same amount

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of PYY as did oleic acid between 15 min and 120 min (P < 0.01 vs basal value). The peak values at 30 min were identical and the integrated release (6.9 + 0.9 ng 'ml 1. 120 min -1) did not significantly differ from the release induced by oleic acid.

Injection of 3 ml isoosmolar amino acids into the ileum did not release significant amounts of PYY.

Isoosmolar short-chain fatty acids did not release significant amounts of PYY during the first 60 min. A weak but significant release of plasma PYY (68 + 10 pg/ml, P < 0.05 vs basal value) was observed at 120 rain and 59 + 8.6 pg/ml, not significantly different from basal value, at 180 rain (not shown). However the integrated increase (1.6 + 0.5 n g ' m l - : " 120 min-1) did not significantly differ from saline-treated controls (Fig. 5B).

Effect of intracolonic nutrients provided as isoosmolar solutions

Intracolonic administration of isoosmolar oleic acid, short-chain fatty acids, glucose and amino acids significantly increased plasma PYY (Fig. 6A). Oleic acid and short-chain fatty acids were equally active and

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Glu AA OA SCFA Fig 5A, B Effect of intraileal administration (H, arrow) of 3 ml isoosmolar solution (300 mosmol/1) of different substances on plasma PYY: saline, glucose (Glu), oleic acid (OA), short-chain fatty acids (SCFA: 65% acetic, 20% propionic and 15% butyric), and amino acids (AA). Mean + SEM, n = 6 rats per group. A Plasma PYY as a function of time; B 120 min integrated PYY increases over basal values; op < 0.05 vs basal level. In order not to clutter pannel A the significance of plasma PYY vs basal level for oleic acid and glucose was not shown (P < 0.05 at all times from 15 to 120 rain). **P < 0.01 vs saline

produced a fast PYY release with peak values occur- ring after only 15 min (72 + 5 pg/ml, P < 0.001 and P < 0.05 vs basal values for oleic acid and short-chain fatty acids respectively), followed by a plateau. PYY release after administration of glucose and amino acids occurred more slowly, and reached 82 pg/ml and 86 pg/ml at 120 min, respectively. The plasma PYY increase after administration of glucose was significant only at 60 min. The plasma PYY increase after admin- istration of amino acids was significant (P < 0.05) between 60 min and 120 min. The integrated PYY release was not significantly different (Fig. 6B) between oleic acid (7.8 + 2.1 pg.m1-1. 120 min-1), short-chain fatty acids (7.1 + 1 . 3 p g m l - l " 1 2 0 m i n - t ) , glucose (7.3 + 2.6 pg 'ml -1 - 120 rain-l) , and amino acids (4.2 + 1.5 ng'm1-1- 120 rain -1) although PYY release tended to be less after administration of amino acids.

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Fig. 6A, B Effect of intracolonic administration (IC, arrow) of 3 ml isoosmolar solutions (300 mosmol/1) of different substances on plasma PYY: saline, glucose (Glu), oleic acid (OA), short-chain fatty acids (SCFA: 65% acetic, 20% propionic and 15% butyric), and amino acids (AA). Mean + SEM, n = 6 rats per group. A Plasma PYY as a function of time; B 120-min integrated PYY increases over basal values. *P < 0.05; ***P < 0.001 vs saline. In order not to clutter panel A, the significance of plasma PYY increases was omitted

Effect of hexamethonium on the plasma PYY increase induced by intraduodenal and intracolonic administration of nutrients

Hexamethonium inhibited by 91% the PYY release induced by the intraduodenal meal (Fig. 7, P < 0.0l), and by 72% the PYY release induced by the intra- duodenal administration of isoosmolar oleic acid (P < 0.05). In contrast, the increase of plasma PYY induced by intracolonic administration of oleic acid (Fig. 7) and intracolonic glucose (not shown) was not modified by hexamethonium.

PYY release after the intraduodenal meal in conscious and in anaesthetized rats

In conscious rats given the intraduodenal meal, plasma PYY peaked at 30 min and returned to basal values at 120 min (Fig. 8A). PYY release after 60 min and

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Fig. 7 Integrated increases of plasma PYY after the intraduode- nal meal (ID meal), isoosmolar intraduodenal oleic acid (ID OA), and intracolonic oleic acid (IC OA), administered alone (full columns) or after application of hexamethonium (hatched columns). *P < 0.05 vs ID OA alone, **P < 0.01 vs ID meal alone

120min was smaller than in anaesthetized rats (P < 0.05 and P < 0.01, respectively). The integrated PYY increase (16.5 + 1.6 ng.m1-1. 120 min -1) in con- scious rats was less than in anaesthetized rats by 45 % (Fig. 8B, P < 0.05).

Intestinal transit of the intraduodenal meal

In anaesthetized rats the front of the charcoal-marked intraduodenal meal was 89.8 + 2.4 cm distal to the pylorus, i. e. in the terminal ileum (total length of the small bowel, about 100 cm). In conscious rats the front of the meal was 79.2 + 3.1 cm distal to the pylorus (P---0.02 vs anaesthetized rats). In hexamethonium- treated anaesthetized rats, the front of the meal was 67.3 + 6.8 cm distal to the pylorus (P < 0.05 vs con- trol rats). The transit of the elemental nutrients solutions was not measured.

Discussion

The peak PYY response observed after administration of the intraduodenal meal in our experiments was similar to that observed in rats by Jin et al. (A = 336 pg/ml) [20], and larger than those reported, also in rats, by Aponte et al. ( k = 120pg/ml) [5] and Rudnicki et al. (A = 50 pg/ml) [33]. The time course of PYY release was similar to that observed by some authors [20, 33], while others [5] reported a later peak (150 min). However these authors used feeding condi- tions different from ours: spontaneous ingestion by trained rats [5, 20], or oesophageal gavage [33], so that gastric emptying may have modulated the response and

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Fig. 8A, B Comparative effects of the intraduodenal meal in con- scious and in anaesthetized rats. The effect of saline in conscious rats was added as a control. A Plasma PYY as a function of time; B 120-min integrated PYY increases over basal values. *P < 0.05, **P < 0.01 between conscious and anaesthetized rats

explain the differences from our own results, which were obtained by direct duodenal administration of nutrients.

The duration of the PYY response after a meal can- not be ascertained from our study, since we did not measure PYY release for more than 2 h or 3 h. However, as far as duodenal administration is con- cerned, the peak of plasma PYY occurred most fre- quently at 60 min. In dogs, plasma PYY was more than twice the basal level for more than 2 h [25], or for more than 3 h [15, 16] after a meal, and in the human, a large meal (4500 kcal) increased circulating PYY for more than 5 h [1].

Three factors at least influence the amount of released PYY after a meal: caloric amount, osmolar- ity, and nature of nutrients. A complete analysis of these three factors is beyond the scope of this study, since it would necessitate a large number of dose response curves drawn in a systematic fashion by keeping two factors constant and varying the third one.

It was reported in the human that PYY release increased with the caloric load of the meal, and that an equal and small (530 kcal) caloric load of lipids (double cream) released more PYY than protein (steamed cod) or glucose [1]. Similarly, we found that

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low intraduodenal loads of oleic acid released more PYY than did amino acids or glucose, but this was no longer true when using high duodenal loads of the same substances. Thus, large caloric loads of glucose and amino acids released more PYY than did isocaloric amounts of oleic acid. However, under these condi- tions, solutions of glucose and amino acids were more hyperosmolar than the oleic acid solution, and osmo- larity probably made the difference, since a hyperos- molar solution of NaC1 placed in the duodenum led to the release of a large amount of PYY. This may correspond either to direct stimulation of osmorecep- tors, or to a large efflux of water into the gut lumen, inducing distension and stimulation of mechanorecep- tots. In both cases, the mechanism should have been initially neural, sensitive, and then transmitted to PYY cells.

When hyperosmolar solutions were compared, hyperosmolar glucose released more PYY (especially at 120 min) than did hyperosmolar NaC1. The PYY response to glucose thus involved some specific effect added to that of osmolarity. For this effect to occur, the amount of glucose has to be greater than a certain threshold, since 3 ml of intraduodenal isoosmolar glu- cose released no PYY. Although aimed at comparing isocaloric loads, the conditions of macronutriment experiments we carried out with hyperosmolar solu- tions were probably far from physiological. However, they allowed us to show that PYY release induced by hyperosmolar glucose was not only due to osmolarity per se.

We sought to analyse proximal and distal mecha- nisms of PYY release by injecting nutrients into the duodenum, ileum and colon. Although in physiologi- cal situations the ratio of hydrolysed versus intact nutri- ents is different in the proximal and distal gut, we chose to inject into the gut lumen pure hydrolysis products: fatty acids, amino acids, glucose. To support this posi- tion, it has been reported that triglyceride hydrolysis is required so that triglycerides can stimulate the release of PYY in dogs [23] and in rats [5]. Since PYY was released by hyperosmolar non-caloric solutions, and since it was not possible to obtain both isocaloric and isoosmolar solutions, of the same volume, we chose to inject isoosmolar (300mosmol/1) solutions of glucose, amino acids, oleic acid and short-chain fatty acids. Under these conditions, oleic acid was the most efficient PYY releaser found, but the amount of PYY released by oleic acid varied with the segment of intestine studied: the order of potency was duodenum > ileum = colon. Glucose did not induce the release of PYY when administered into the duo- denum, but was about as potent as oleic acid in the ileum and colon. Amino acids were inactive when injected into the duodenum or ileum, but they induced the release of some PYY when injected into the colon. Finally, short-chain fatty acids injected into the ileum released no PYY, while they were as potent as oleic

acid in the colon. (short-chain fatty acids were not injected into the duodenum since they do not physio- logically occur in this part of the intestine). The efficiency of short-chain fatty acids agrees with previ- ous results obtained in the isolated perfused colon of rats [29] and rabbits [24].

These data suggest that two mechanisms at least participate in PYY release in the rat. A proximal (duodenal or duodenojejunal, neural) mechanism (distant from PYY cells) is mainly activated by oleic acid, hyperosmolarity and large caloric loads of glu- cose or amino acids. A colonic mechanism (directly on PYY cells?) is activated by all nutrients present in the colonic lumen.

The intermediate results obtained when nutrients are placed into the ileum are not clear. A hypothesis is that the absorption of short-chain fatty acids and amino acids in this segment might be very fast, preventing them from reaching the colon lumen by digestive tran- sit and from affecting colonic L-cells. This hypothesis also implies that no significant direct effect of short- chain fatty acids and amino acids occurs at ileal L- cells.

A direct effect of intraluminal nutrients on L-cells can take place only when L-cells are present, i.e. in the distal segments of the digestive tract (ileum, caecum, colon, rectum). When the intraluminal progression of the intraduodenal meal was measured with charcoal as a non-absorbable marker, the most forward part of the meal never reached the ileocaecal valve after 120 min, excluding any direct caecocolic effect, but allowing a direct effect of the meal on ileal L-cells. These results agree with those of Jin et al. [20], who found that only 8% of an non-absorbable marker added to the meal reached the distal third of small intestine 120 min after the meal.

Some of our results agree with the results published by other groups, but others do not; the differences can be attributed to the type of species investigated, to the type of nutrient and to the site of intestinal administration.

In rats [5], intraduodenal administration of sodium ora te led to the progressive release of PYY, while intraileal administration of sodium o ra t e caused the release of PYY more quickly. The plasma PYY level was identical 120 min after intraduodenal admin- istration and 60 min after intraileal administration [5]. In dogs, only the administration of intraduodenal fatty acids led to the quick release of PYY with a peak at 60 min, while intracolonic amino acids, fatty acids and glucose led to a more progressive release of PYY [15]. In a primary culture of PYY cells from canine colon, a low concentration (< 10mM) of sodium oleate, which probably does not alter mem- brane stability, led to the selective release of PYY [4]. In contrast, in a study performed in humans, none of the nutrients placed into the colon (isotonic and hyper- tonic glucose, oleic acid and casein hydrolysate)

74

released P Y Y [3]. In rabbit isolated perfused colon, oleic acid, added with deoxycholic acid concentrat ions which led to the release of PYY, did not modi fy P Y Y release [7]. A PYY-releasing effect o f bile acids was also repor ted in the rat isolated perfused colon [29], in humans [3] and indirectly suggested in dogs, since in this species bile diversion decreased fat- induced P Y Y release [12].

The response o f P Y Y to the in t raduodenal meal was totally suppressed by hexamethonium. This suggests that the whole effect o f the in t raduodenal meal used neural pathways compris ing at least one nicotinic synapse. In contrast , hexamethon ium did not affect P Y Y release induced by intracolonic oleic acid or glu- cose, suggesting that intracolonic nutrients directly stimulate L-cells and do not involve nicotinic synapses. Similar differences were repor ted in dogs [39, 40], excluding ganglionic intervent ion at the colonic level. These results also agree with the fact that P Y Y release induced by pectin and bile salts in the isolated perfused rat colonic segment [29], as well as P Y Y release induced by deoxycholic acid in isolated perfused rabbit colon [7], are not changed by tetrodotoxin. The absence of a neural mechan i sm suggests a direct effect o f intra- colonic nutrients on L-cells. However the progressive release of P Y Y observed with intracolonic glucose and amino acids in our experiments as well as with all intracolonic nutrients releasing P Y Y in experiments of others [5, 15] does not favour the idea of a direct effect. The media t ion of an alternative, non-neura l interme- diary mechan ism can thus be suggested.

The same L-cells that release P Y Y also contain proglucagon and can release proglucagon-der ived pep- tides, especially glicentin, oxyn tomodul in and glucagon-like peptide 1. In fetal rat intestinal cultures, sodium oleate stimulates the release o f P Y Y and o f proglucagon-der ived peptides, p redominan t ly glicentin and oxyntomodul in [10, 11]. The p lasma level o f proglucagon-derived peptides increases after duodenal or ileal adminis t ra t ion of emulsified corn oil or after ileal glucose (1 M), whereas the lower concentra t ion o f 200 m M glucose does not modi fy the release of proglucagon-der ived peptides [31, 32]. To our knowl- edge no systematic in vivo compara t ive study o f P Y Y and proglucagon-derived peptides release has been made to date.

Finally, since (for technical and ethical reasons) these experiments were pe r fo rmed in anaesthet ized rats, we checked whether anaesthesia might reduce P Y Y release by the meal. The data show that P Y Y release in conscious rats was not greater than in anaes- thetized rats (it was in fact smaller), so that it seems likely that the releasing mechanisms have not been underes t imated by per forming the experiments in anaesthet ized rats.

Acknowledgements Partial financial support was provided by the Conseil Scientifique of Facult6 X. Bichat, and by Association Charles Debray.

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