Adaptation to low-protein diet increases inhibition of gastric emptying by CCK

Peptides 24 (2003) 1929–1934

Adaptation to low-protein diet increases inhibition of gastric emptying by CCK Véronique Leray a , Jean-Pierre Segain a,b,∗ , Christine Cherbut b , Jean-Paul Galmiche a a

Centre de Recherche en Nutrition Humaine, INSERM U539, Hˆotel Dieu, 44093 Nantes, France b INRA UFDNH, Rue de la Géraudière, BP 71627, 44316 Nantes Cedex 03, France Received 23 October 2002; accepted 29 October 2003

Abstract Chronic nutritional disorders such as protein malnutrition are associated with delayed gastric emptying and increased postprandial cholecystokinin (CCK) levels. This study investigated the mechanisms involved in gastric emptying adaptation to low-protein diet. Two groups of 12 rats were adapted to a low-protein (LPD) or standard diet (SD) for 3 weeks. As compared to rats fed a SD, in rats adapted to a LPD gastric emptying was delayed, whereas postprandial CCK levels were increased. LPD enhanced antral muscle contractile response to CCK and cerulein without altering response to acetylcholine. This increased contractility was associated with up-regulation of CCK-A receptor mRNA levels in antral muscle. Our data suggest that modulation of gastric emptying after adaptation to a low-protein diet involves up-regulation of both CCK-A receptors and CCK-induced contraction of antral smooth muscle. © 2003 Elsevier Inc. All rights reserved. Keywords: Gastric emptying; Malnutrition; Cholecystokinin receptors; Gastric muscle contractility

1. Introduction Gastric emptying plays a key role in the regulation of food intake by controlling the flow of nutrients to their absorption site in the small intestine. Among the many hormones and neuropeptides involved in the regulation of gastric emptying, cholecystokinin (CCK) seems to play a prominent role in the early postprandial period. This gastrointestinal hormone is secreted by endocrine mucosal “I” cells of the upper small intestinal mucosa in response to food reaching the duodenum. The mechanism of action of this peptide hormone in decreasing the rate of gastric emptying is unclear, but it has been shown to modulate gastric motility [30]. In addition to its immediate postprandial effect, CCK can also induce satiety by stimulating neurons of the nucleus tractus solitarus [24]. The presence of nutrients in the duodenum controls the rate of gastric emptying. Most studies investigating the effect of nutrients on gastric emptying and CCK secretion have focused on short-term effects, whereas chronic nutritional adaptation has rarely been studied [11,15,17]. Several studies have suggested that CCK could play an important role in chronic nutritional disorders such as bulimia, protein ∗

Corresponding author. Tel.: +33-240-675008; fax: +33-240-675005. E-mail address: [email protected] (J.-P. Segain).

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.10.015

malnutrition or anorexia. In both humans and rats, protein malnutrition and anorexia are associated with an increase in postprandial CCK release [1,5,10] and a delay in gastric emptying [28,35]. These two events could prolong illness by enhancing satiety response. Indeed, CCK induces satiety and thus regulates body weight [21], and gastric emptying can also influence satiety by controlling gastric volume [5,26]. In a previous study, our group showed that the rate of gastric emptying in rats was altered by chronic adaptation to diets differing in protein content [32]. The present study was designed to determine the mechanisms involved in changes in gastric emptying induced by chronic adaptation to a low-protein diet. Our results indicate that restriction of chronic dietary protein induces regulation of gastric emptying through modulations of CCK release and gastric muscle sensitivity to CCK.

2. Materials and methods 2.1. Animals and chronic diets Male Wistar rats (150–190 g body weight at the beginning of the experiment) were maintained on a 12 h light–dark cycle (lights ‘on’ at 8 a.m. and ‘off’ at 8 p.m.). Two groups of 12 rats were fed a low-protein or standard diet for 21 days

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Table 1 Composition of the diets Nutrient

Low-protein diet (g/100 g diet)

Standard diet (g/100 g diet)

Proteins Carbohydrates Lipids Fibers Water Minerals Vitamin mix

9.2 75.4 4.3 1.4 4.0 5.0 0.7

17.0 58.7 3.0 4.0 12.0 5.0 0.3

Carbohydrate/fat ratioa

89/11

90/10

Total energy content (kcal/100 g)

377

330

Proteins include animal (fish) protein and vegetable (soya bean meal, yeast) protein at a ratio of 2:7. An appropriate quantity of lipids, minerals and vitamin mix was added. The standard diet was rat and mouse maintenance diet A-04 from U.A.R., France. a As fraction (kcal/kcal) of energy delivered as carbohydrate or fat.

(Table 1), with free access to water. Carbohydrate/fat ratios (kcal/kcal) were similar in both diets. During experiments, food consumption and body weights were recorded daily. 2.2. Measurement of gastric emptying Gastric emptying was measured by a previously described method [32], using a 40% peptone test-meal (3 ml) supplemented with phenol red (1 mg/ml). The pre-warmed meal (37 ◦ C) was given orally through a stainless steel tube after an 18-h fast. Forty minutes later, rats were killed by cervical dislocation. The phenol red concentration in the remaining gastric content was determined spectrophotometrically at 523 nm, and gastric emptying was calculated according to the following formula:
 Cstandard − Csample gastric emptying (%) = × 100 Cstandard where Cstandard is the initial concentration of phenol red in the test meal and Csample is the phenol red concentration in the stomach. 2.3. CCK bioassay Basal or postprandial plasma CCK levels were measured by a specific and sensitive bioassay [18]. This method is based on the ability of CCK to stimulate amylase release from isolated rat pancreatic acini. Amylase release into the medium was measured using procion yellow starch as substrate. Total acinar amylase content was measured after lysis of cells by Triton X-100. Amylase release, expressed as a percentage of total amylase content, was quantified using a standard curve constructed with CCK-8. 2.4. Measurement of gastric muscle contractility Rats were killed by cervical dislocation, and fundic and antral longitudinal muscle segments (1–1.5 cm long) were

quickly removed after midline incision along the greater curvature. Luminal content was cleaned with Krebs-bicarbonate buffer (NaCl 128 mM, KCl 4.5 mM, CaCl2 2.5 mM, MgSO4 1.18 mM, KH2 PO4 1.18 mM, NaHCO3 125 mM, d-glucose 5.55 mM, pH 7.4), and antral mucosa was gently scraped off. The segments were mounted in organ baths containing continuously oxygenated Krebs-bicarbonate solution (CO2 5%, O2 95%), suspended under a tension of 1.5 g for fundic segments and 1 g for antral segments, and allowed to equilibrate for 1 h. Isometric longitudinal mechanical activity was then recorded using a force transducer (Ugo Basile No. 7005, Comerio, VA, Italy), as previously described [29]. At the beginning of each experiment, acetylcholine (ACh, 10−6 M) was applied as the muscle contractility control. A dose–response curve to ACh, CCK-8 (Sigma, L’Isle d’Abeau Chesnes, St. Quentin Fallavier Cedex, France) and cerulein (Cerulex® , Pharmacia, Saclay, Orsay Cedex, France) was established by applying graded doses of these peptides in a non-cumulative manner, with repeated 20–30 min washes between each tested concentration. At the end of the experiment, the viability of each segment was checked by measuring response to ACh 10−6 M. Contractile response to drugs was calculated by determining the difference between the mean basal tension recorded 5 min before injection and the mean tension recorded 2 min after injection. Results were expressed as g/mm2 by normalizing tension (g) for the cross-sectional area (CSA), which was determined by the formula of Vermillion et al. [37]: CSA =

fresh tissue weight (mg)/tissue length (mm) , density (mg/mm2 ) with density = 1.05.

2.5. Semi-quantification of CCK receptor mRNA Reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described [31] in order to semi-quantify CCK receptor mRNA levels in antral longitudinal muscle-myenteric plexus preparations. Antral muscles were removed from each rat and snap-frozen separately in liquid nitrogen. Frozen muscles were then homogenized, and total RNA was isolated using the acid guanidinium thiocyanate–phenol–chloroform method [2]. Total RNA extracted from the AR42J rat pancreatic acinar cell line and rat brain was used in parallel RT-PCR reactions as a positive control for CCK-A and CCK-B receptors. RNA were treated with RQ1-Rnase-free DNase (Promega, Charbonnieres, France) at 37 ◦ C for 30 min, followed by phenol extraction and ethanol precipitation. After centrifugation, RNA pellets were dissolved in water and then quantified spectrophotometrically. Two micrograms of total RNA were reverse-transcribed in a reaction volume of 20 ␮l using random primers (Pharmacia, Saclay, Orsay Cedex, France) and Superscript II murine Moloney leukemia virus reverse transcriptase, according to the manufacturer’s instructions (Life Technologies, Cergy

V. Leray et al. / Peptides 24 (2003) 1929–1934

CCK-A R: 5 -GATTGTGATGGTGGTGGC-3 and 5 -ACAGGAAGAAGAGGACCACG-3 CCK-B R: 5 -TCATCATCGTGGTCCTGG-3 and 5 -CACCGCAATAACCACACC-3 GAPDH: 5 -ATCACCATCTTCCAGGAGCG-3 and 5 -ATCACCATCTTCCAGGAGCG-3 . Ten microlitters of the PCR reaction were electrophoresed in 1.5% agarose gel, and amplified cDNA fragments were stained with ethidium bromide. Signals were then quantified by a laser densitometer. Densitometrical values obtained with CCK-A and CCK-B receptors were normalized to GAPDH values. 2.6. Statistical analysis Values are expressed as the mean ± S.E.M. Comparison among means was performed by analysis of variance (ANOVA) followed by a post hoc test (Student’s t-test for unpaired values). P-values less than 0.05 were considered significant. All calculations were performed using Statview software (Abacus Concepts Inc., Berkeley, CA).

3. Results 3.1. Body weight of rats adapted for 3 weeks to standard or low-protein diet Body weight curves of rats fed standard or low-protein diet showed that weight gain over a 21-day period was not different between the two groups (Fig. 1). 3.2. Effects of 3-week adaptation to low-protein diet on gastric emptying and postprandial CCK release Table 2 shows that gastric emptying of a peptone test meal was significantly delayed in rats adapted to a low-protein diet as compared to rats fed a standard diet. Concomitantly, postprandial plasma CCK levels in response to the peptone test-meal were higher in rats adapted to the low-protein diet, which suggests that CCK release increased in response to the test-meal. However, basal plasma CCK levels were lower in

350 LPD SD

Body weight (g)

Pontoise, France). PCR was performed in the linear range of amplification (determined for each primer pair–cDNA combination). Thirty-five and 28 PCR cycles amplified cDNA coding for CCK-A/CCK-B receptors and GAPDH, respectively. Standard PCR reactions were performed in a 50-␮l volume containing 1 ␮l cDNA solution, 1 ␮l of each primer solution (50 mM), 1 ␮l dNTP solutions (10 mM each), 3 ␮l MgCl2 (25 mM), 5 ␮l 10× Goldstar DNA polymerase reaction buffer, and 0.1 ␮l (0.5 units) Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). Each PCR cycle consisted of 1 min at 92 ◦ C, 1 min at 58 ◦ C and 1 min at 72 ◦ C. The following sense/antisense primers (Genosys, Pampisford, UK) were designed to amplify cDNA fragments:

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300 250 200 150

7 14 Time course (day)

21

Fig. 1. Body weight gain of rats maintained on standard (SD) and low-protein (LPD) diet over a 21-day adaptation period. No difference was observed for the weight gain between the two diets.

rats fed a low-protein diet (0.61 ± 0.01 versus 1.50 ± 0.35 for the standard diet; P < 0.05). 3.3. Effects of 3-week dietary protein restriction on gastric muscle contractility to cerulein The CCK analogue cerulein was used to assess gastric muscle contractility in response to this peptide. No significant change in longitudinal fundic muscle contractility was noted after adaptation to a standard or low-protein diet (Fig. 2A), whereas longitudinal antral muscle contractility was modified after 21-day adaptation to the low-protein diet (Fig. 2B). In comparison with control rats, protein restriction was associated with a significant increase in contractile response to cerulein (P < 0.02). 3.4. Effects of 3-week dietary protein restriction on antral muscle contractility to CCK-8 As adaptation to the low-protein diet modified the contractile response of antral muscle to cerulein, experiments were performed to assess the response of longitudinal antral muscle to CCK. The dose–response curve showed that the contractility of longitudinal antral muscle strips to CCK (from 10−7 to 10−5 M) was higher in rats adapted to low-protein diet than in rats fed a standard diet (Fig. 3A). In contrast, the contractility of these strips to Ach was similar in rats adapted to low protein diet and in control rats (Fig. 3B). Table 2 Gastric emptying and postprandial CCK release in rats adapted for 21 days to standard and low-protein diets

Gastric emptying (%) Postprandial CCK release (pM)

Standard protein diet (n = 7)

Low-protein diet (n = 7)

P-value

36.96 ± 1.70 2.03 ± 0.40

28.10 ± 2.30 3.81 ± 0.45

<0.02 <0.05

Results are expressed as means ± S.E.M. The gastric emptying rate of a peptone meal (over 40 min) was measured as described in Section 2. Postprandial CCK release represents the difference between postprandial and basal plasma CCK levels.

V. Leray et al. / Peptides 24 (2003) 1929–1934

Contraction (gx10-3 /mm2)

1932

150

LPD SD

100 50

0

-11

-10 -9 -8 log [Cerulein](M)

Contraction (gx10-3 /mm 2)

(A)

30

*

LPD SD

20

-7

* *

10 0 -11

-10 -9 -8 log [Cerulein](M)

(B)

-7

Fig. 2. Comparison of gastric muscle contraction induced by cerulein in rats fed for 21 days with standard (SD) and low-protein (LPD) diets. Dose–response curves of the effect of cerulein on fundic longitudinal smooth muscle (A) and antral longitudinal smooth muscle (B). Values are expressed as means ± S.E.M. (n = 5). ∗ P < 0.02.

Fig. 4. RT-PCR semi-quantification of CCK-A receptor mRNA levels in antral muscle strips. Total RNA was isolated from antral longitudinal muscle preparations of rats fed for 21 days with standard and low-protein diets. RT-PCR was then performed to amplify CCK receptors and GAPDH mRNAs. (A) Bars represent the mean (±S.E.M.) ratio of CCK-A receptor (CCK-A R) to GAPDH densitometric values. ∗ P < 0.05. (B) Control signals and representative experimental signals for CCK-A R and GAPDH mRNAs.

3.5. Effects of 3-week adaptation to low-protein diet on CCK receptor mRNA levels in antral muscle strips

Contraction (gx10-3 /mm2)

The increase in antral muscle sensitivity to CCK-8 in rats adapted to the low-protein diet prompted us to ana15

LPD SD

***

10

** 5

*

Contraction (gx10-3 /mm2)

0 -12 -11 -10 -9 -8 -7 log [CCK8](M) (A)

(B)

300

-6 -5

LPD SD

200 100

0

lyze CCK receptor mRNA expression in antral longitudinal muscle-myenteric plexus. Although CCK-B receptor mRNA expression was detected in positive controls including the AR42J cell line and in rat brain (data not shown), no expression was detected in antral longitudinal muscle-myenteric plexus preparations. Interestingly, an increase in CCK-A receptor mRNA levels relative to GAPDH mRNA levels was observed in rats adapted to the low-protein diet as compared to rats fed a standard diet (P < 0.05) (Fig. 4).

-9

-8

-7 -6 -5 log [ACh](M)

-4

-3

Fig. 3. Comparison of antral smooth muscle contraction induced by cholecystokinin (CCK8) and acetycholine (ACh) in rats fed for 21 days with standard (SD) and low-protein (LPD) diets. Values are expressed as means ± S.E.M. (n = 11). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.002.

4. Discussion Gastric emptying is an important digestive function that can be considered as a major determinant for meal size and food intake. It controls the rate and thus the intestinal absorption of nutrients reaching the duodenum. However, adaptation to a specific diet can regulate the gastric emptying rate. For example, it was reported that adaptation of rats to high-fat diet diminishes the ability of CCK to inhibit gastric emptying [3]. In a previous study, our group showed that the rate of gastric emptying in rats was altered by chronic adaptation to diets differing in protein content [32]. Furthermore, in chronic nutritional disorders such as protein malnutrition and anorexia, gastric emptying is in fact delayed [28,35]. The molecular mechanisms involved in nutritional regulation of gastric emptying are poorly understood. The present study showed that the situation after chronic adaptation to

V. Leray et al. / Peptides 24 (2003) 1929–1934

a low-protein diet in rats is comparable to protein malnutrition, i.e. gastric emptying of the peptone test meal was delayed. At least two types of regulation were observed, which could act synergistically: (i) enhanced response of postprandial CCK release to the peptone test-meal, and (ii) increased antral muscle sensitivity to CCK due to up-regulation of CCK receptor expression. With respect to the first type of regulation, the enhanced postprandial CCK release observed in previous studies during protein malnutrition or anorexia [1,13,25] could have been mediated through increased CCK content of duodenal I cells or up-regulation of CCK gene transcription. However, no alteration of CCK mRNA levels in duodenal cells was observed in our previous experiments [32]. Alternatively, increased postprandial CCK release in response to a peptone test meal could have been due to the greater sensitivity of CCK-positive cells after chronic adaptation to low protein intake. The mechanisms by which nutrients stimulate CCK release by duodenal I cells are not well established. Nutrients could interact with I cells through chemoreceptors, and the number of these receptors could be modulated after adaptation to a particular diet [16]. It is possible that in rats adapted to low-protein diet, the sensitivity of I cells to a peptone meal is increased by a similar mechanism. Alternatively, some studies have suggested that pancreatic enzymes, e.g. trypsin or chymotrypsin, can control CCK release by degrading endogenous luminal stimulatory peptides such as pancreatic monitor peptide and CCK-releasing factor [20,23]. Interestingly, Dubick et al., reported that a low-protein diet decreased the level of pancreatic enzymes and was also associated with reduced inhibition of CCK release stimulation by these peptides [6]. With respect to the second regulatory mechanism of gastric emptying, our experiments show for the first time that chronic adaptation to a low-protein diet induces a modification of gastric muscle sensitivity to CCK through up-regulation of CCK-A receptor expression. Thus, in addition to an increase in postprandial CCK release, the contractility of antral muscle to CCK or its analogue cerulein was enhanced. This does not seem to be due to a general increase in muscle contractility/hypertrophy since the contraction of antral muscle in response to ACh was not modified and no difference in fundic muscle contractility to cerulein was observed. Notably, our results indicate that the increase in antral muscle contractility to CCK in rats adapted to a low-protein diet involves an up-regulation of CCK-A receptor expression. Although this up-regulation was observed at the mRNA level, the increased response of antral muscle to CCK suggests that CCK-A protein expression is also increased. Our RT-PCR experiments failed to detect CCK-B receptors in antral myenteric plexus-longitudinal muscle preparations, but indicated the presence of CCK-A receptors. The tissue location of CCK receptors is not well established in rats. Using immunohistochemistry, Sternini et al. found CCK-A receptor expression in neurons innervating the rat gastrointestinal tract [36]. However, these

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authors failed to detect CCK-A receptors in some cells (e.g. smooth muscle cells, D cells and chief cells) that have been described as CCK-cell targets in functional studies in dogs and pigs [19,22] and pharmacological studies in guinea pigs [12]. According to Sternini et al., this discrepancy could be due to the sensitivity of the method used to detect CCK receptors. Concerning the cellular location of CCK-A receptors in our antral myenteric plexus-longitudinal muscle preparations, it was not possible to determine which type of cell (nerve or muscle) bears CCK-A receptors. CCK can induce smooth muscle cell contraction directly or indirectly through the release of ACh. Hutchison and Dockray reported that the contractile effect of CCK-8 on innervated strips of guinea pig ileum longitudinal muscle was abolished by denervation and mediated by the release of ACh and substance P from the myenteric plexus [14]. However, in humans, D’Amato et al. showed that CCK could act both indirectly and directly depending on the gastrointestinal area and the muscle layer [4]. Up- or down-regulation of receptors often occurs in the central nervous system or peripheral organs in conjunction with a respective decrease or increase of ligand concentration [27,33]. The modulation of receptor density on target tissues may restore transmission of the hormonal message to a normal level. This kind of regulation was apparent in our study. As a result of poor stimulation of CCK secretion by the low-protein diet (lower plasma CCK levels as compared to the standard diet), up-regulation of CCK-A receptor in antral muscle compensated for low levels of circulating CCK. Similar nutritional regulation was reported in obese subjects whose high CCK levels were associated with an adaptive desensitization of CCK receptors following overeating [9]. More generally, our results suggest that nutritional mechanisms regulating gene expression adapt digestive functions such as gastric emptying to a particular diet or to chronic nutritional disorders. In fact, the regulation of gastric emptying in response to a chronic diet is probably a complex process involving the interaction of satiety signals such as CCK with a distinctly different set of pathways controlling food intake and energy homeostasis. For example, both insulin [8] and leptin [21] can enhance the satiety effect of CCK. The fact that activation of the nucleus tractus solitarius by CCK is potentiated by leptin, clearly shows that the signals involved in energy homeostasis interact with those inducing satiety [7]. Conversely, hormones regulating food and energy intake could also modulate digestive functions in response to a specific diet. For instance, it has been shown that leptin can inhibit gastric emptying [34]. Moreover, CCK regulates gastric emptying as a function of meal content, but not of the energy content of the meal [38], which also suggests that other signals are involved in the regulation of gastric emptying. In conclusion, the present study confirms and extends our previous results [32]. Chronic dietary adaptation can affect

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