Effects of dietary whey proteins, their peptides or amino-acids on the ileal mucosa of normally fed and starved rats

CIinical Nurrifion (1991) lo: 4%54 @ Longman GroupUK Ltd 1991

Effects of dietary whey proteins, their peptides or amino-acids on the ileal mucosa of normally fed and starved rats M.G. POULLAIN, J.P. BROYART

J.P. CEZARD, C. MARCHE*,

J. MACRY,

L. ROGERt,

E. GRASSETt

and

d’Anatomopathologie, iNSERM U120, Robert Debr& Hospital, 48 Bd S&wrier, 75019, Paris, *Laboratoire Bichat Hospital, Paris and tsopharga R. U., Paris, France (Reprint requests and correspondence to J. P. Cl

ABSTRACT-The effects of three liquid diets, differing only in the molecular form of the nitrogen source (whole whey proteins, WP; trypsic whey protein hydrolysate, WPH, and amino-acid mixture, AAM) were studied on the mucosa morphology and brush border hydrolase (BBH) activities (disaccharidases, peptidases) of the ileum of normally fed male Wistar rats (controls) and during refeeding of rats starved for 72h. All three diets produced repair of the fasting induced mucosal atrophy; the AAM diet gave the most rapid response and highest villus height (p < 0.01). This was correlated with an increase in crypt mitoses (p < 0.01). Similar results were obtained in controls with AAM. The sucrase (S) and acid amino peptidase (AAP) specific activities of controls were higher (p c 0.01) on the WPH diet; neutral amino peptidase (NAP) was unaffected. Dipeptidyl peptidase IV (DDP) was lowest on AAM while glucoamylase fG) highest on WP. Fasting increased S and DDP activity, and produced no change in the other BBH. Large variations in BBH occurred during refeeding except for NAP which remained stable. Control values were restored at 96 h, except for AAP. The results show that BBH and mucosa morphology of the ileum in the rat can be modified by the molecular form of the nitrogen source and that the nutritional status interferes with this adaptation. These data could have implications for the therapy of small bowel disease.

Introduction

intake (proteins and calories) (14, 20, 21) and, especially for the brush border enzymes. on the quantities of their specific stimulatory substrates (22, 23). We have shown previously that diets having the same nitrogen and caloric content and differing only in the molecular forms of the alimentary proteins lead to a different jejunal mucosal adaptation in normally fed and in severely undernourished rats (24). This study was therefore carried out to determine the changes which occur in the ileum of rats fed the three types of liquid diets. Similar synthetic diets are widely used for therapeutic purposes in human ileal pathologies (1, 25, 26).

Synthetic liquid diets which contain nitrogen in the form of hydrolysed proteins or amino-acids are frequently used in the nutrition support of nutritionally deficient patient with or without intestinal diseases (1, 2). Their nutritional values (3-6) and intestinal absorption rates (7, 8) have been studied extensively but their effects on small intestinal adaptation in normal or pathological states (9-12) have received less attention. Many of these studies on small intestine adaptation used diets that differed from each other in many ways, including their nitrogen or caloric content, protein composition, hydrolysis methods used, or in being confined to the jejunum (9, 10, 12). Nutritional deficiency results in severe impairment of small intestine morphology and functions (13-17). Intestinal function is rapidly restored by refeeding (13, 14, H-20). The extent of the changes (mucosal morphology and brush border enzyme activities) depends on the amount of food

Materials and methods Diets and animals

All three diets used were provided by Sopharga RU (Creully, France). Each diet (80m1, 90kcaV 49

50

ILEAL ADAPTATION

AND ALIMENTARY

PROTEINS

day) had the same protein, carbohydrate (CaloreenR Sopharga RU), lipid (Liporocil,R Sopharga RU), vitamin and mineral mixture content. They differed only in the molecular forms of the whey proteins: purified whey proteins (lactose free) (WP), whey protein peptide hydrolysate (WPH), or amino-acid mixture (AAM). WPH was a mixture of small peptides obtained by tryptic and chymotryptic hydrolysis of purified whey proteins in an enzymatic membrane reactor (27). The mean molecular weight distribution of the peptides (Table 1) was determined by exclusion high-performance liquid chromatography (HPLC) (28). The free amino-acid content was <3g/lOOg N, as determined by reverse-phase (RP)-HPLC (29). The amino-acid mixture composition, equivalent to that of whey proteins, was obtained by mixing pure crystalline L-amino-acids (Ajinomoto Co Jnc, Tokyo, Japan) and was such as to fulfil the needs of growing rats (30). Male Wistar rats (280-3008) were purchased from Elevage Janvier (Laval, France). They were housed individually in wire-bottom cages in an animal room maintained at 22°C with a 12h light period (06:00-18:OOh). They were maintained for 1 wk on a laboratory pelleted diet (UAR 113, Villemoisson/Orge, France) ad libitum before experimentation. Three groups of control (unstarved) rats were used; each group (six rats) were fed one of the diets for 96 h. A total of 92 rats were starved for 72 h with

Table 1 Composition

Volume (ml) Energy (kcal) Carbohydrates (% of energy)** Lipids (% of energy)*** Nitrogen (% of energy)

Vitamin and mineral mixture

free access to water; they were then divided in three groups and fed one of the three liquid diets. Animals were killed at various times (6-8 per time period) from 0-96h after the beginning of the new diet. This was done between 09.00 and 10.00 to minimise the influence of circadian rhythm on brush border enzyme level (31). Seventy-two hours of starvation produced an average body weight loss of 12%. Rats began to recover weight more rapidly on the WPH (+7% of the starvation weight vs +3% on WP and +2% on AAM). Food intake of the three groups was identical and corresponded to the amount eaten after 72h of starvation on a normal pelleted diet. Rats that did not eat the total amount of the diet (90 kcal/d) were excluded from the experiment; they constituted 15% of the rats in each group. The National Research Council guide for the care and use of laboratory animals was followed. Protein and enzyme assays

Rats were killed by decapitation at the end of each time period. The terminal ileum, starting at the ileo-caecal valve was removed from its vascular and peritoneal connections by slight traction. The last 120mm were measured without any traction. This segment was washed with cold 0.9% NaCYL and weighed. The first 20mm were kept for the histomorphometric study. The mucosa from the remaining 1OOmmwas removed by scraping with a

of diets* Diet WP

Diet WPH

Diet AAM

80 90 58

80 90 58

80 90 58

15

15

15

21 purified whey proteins

27 peptides from whey proteins: 10000-5000 MW: 5% 5000-1000 MW: 30% 1000 MW: 65% Adjust to quantity recommended

27 free aminoacids

(27)

*WP, diet with purified whey proteins; WPH, diet with small peptides from whey proteins; AAM, amino-acid mixtures equivalent to whey protein amino-acid composition. **Caloree# - glucose polymers, Gl (glucose, 1 molecule), 1.5%; maltose, 13% and G3-G10 85% ***LiprociF - medium-chain triglycerides, 80% ; long-chain triglycerides, 20% (14.7% Cl*:*; 3.1%Cn3.1).

CLINICAL NUTRITION

glass slide and homogenized in of 0.02 mol NaHzPO~-KZHPO~/L buffer, pH 6.1 (2ml//g). Protein was determined by the Lowry method (32); sucrase and glucoamylase activities were assayed by a modification of the Dahlquist technique (33) and leucyl P-naphthylamide hydrolase activity (neutral brush border aminopeptidase) as previously described (34). Dipeptidyl peptidase IV and acid aminopeptidase were measured as described in (35). Results are expressed per milligram protein (specific activity).

Histomorphometric

study

The histomorphometric study was carried out on the first 20mm of each ileal segment. The tissue was fixed in Bouin’s solution and embedded in ParaplastR (Prolabo Co, Paris). Longitudinal sections (5 km) were stained with hematoxylin and the periodic acid-Schiff (PAS) procedure (14). The variables measured were villus and crypt heights, enterocyte height, and the number of mitoses per crypt. Each variable was determined at least 10 times per rat using a ocular micrometer (Leitz Co, Wetzlar, FRG).

Statistical analysis

Numerical data are expressed as mean + SEM of six or more rats studied. The significance of the differences between paired numerical data in the three groups was calculated by analysis of variance and Student’s t-test (36).

51

Results Effects of the three diets on the hktomorphometry of ileal mucosa in controls and during refeeding after starvation (Table 2)

The control rats fed the AAM diet had the greatest villus height (p < 0.01). This was correlated with a statistically higher number of mitoses. Fasting produced no trophic change from the WP control diet, despite a statistically significant reduction in the number of mitoses per crypt compared to the WPH and AAM diets, there was a mucosal hypoplasia characterised by a diminution of villus height and number of mitoses per crypt. Refeeding produced repair of the mucosa hypoplasia. The most rapid and greatest villus growth was on the AAM diet. The mucosa of animals fed the WPH diet did not return to control values before 96 h of refeeding. The greater villus hyperplasia observed with the AAM diet was associated with a significant increase in the number of mitosis per crypt at all the times studied.

Effects of the three diets on brush border disaccharidases and peptidases in controls and during refeeding after starvation

Sucrase and acid amino peptidase specific activities in controls were higher (p 0.01) with the WPH diet, glucoamylase with the WP diet (Fig. 1). Dipeptidyl peptidase IV was decreased in rats on the AAM diet while for N-amino peptidase activity was unchanged.

Table 2 Effect of WP, WPH and AAM diets on villus and crypt height and number of mitoses per crypt of the ileum in controls and during refeeding Time of refeeding Controls n=6

Oh n=8

24h n-8

48h n=6

72h n=8

96h n=6

Villus height (pm)

WP WPH AAM

251 f 44 271 + 7 302 k 8”

238 + 6

250 * 7 233 + 3* 263 + lo”

254 f 10 234 + 4* 270 f 7”

249 f 6 232 + 6* 269 + 8”

257 + 8 251 + 8 301 + 5”

Crypt height (pm)

WP WPH AAM

98 + 6 111 f 2 109 f 3

107 + 3

113 + 6 106f2 100+3

117 f 2 115 +3 111 * 4

115 f 3 113 f 4 111 f 2

118 ?c 9 123 + 6 111 I2

WP WPH AAM

2.1 + 0.3 1.8 + 0.4 2.6 + 0.06+

1.2 f 0.1

2 + 0.1 2.7 + 0.1” 3.1 k 0.3*

2 f 0.05* 2.2 f 0.1 2.9 + 0.3”;

1.8 + 0.1’ 2.1 + 0.1: 3.3 + 0.2=*

2.1 rt 0.1’ 1.2 + 0.1 2.9 f 0.2x*

Mitoses per crypt (n)

“statistically different (p < 0.05) from the other groups at the same time of refeeding ‘N.S. as compared with the preceding time of refeeding for the same group

52

ILEAL ADAPTATION

AND ALIMENTARY

PROTEINS

Discussion

400

100

g p P

50

2

IO SUCRASE

IP II

GLUCOAMYLASE

NAP

PAP

Fig. Effects of WP (hatched bars), WPH (clear bars) and AAM (stippled bars) diets on ileum brush-border hydrolase adaptation (specific activities) in controls (96h of feeding). XP < 0.01.

Fasting increased sucrase and dipeptidyl peptidase IV activities and decreased that of glucoamylase (Table 3), the other hydrolase activities were not changed. During refeeding (Table 3), sucrase and dipeptidyl peptidase IV were decreased notably with the AAM diet while glucoamylase was increased. Control values were reached at 96 h of refeeding, except for AAP. NAP remained unchanged.

This study confirms previous reports demonstrating that starvation has no effect on ileal morphology (13, 1.5, 37) which remains similar to that of rats on natural diets (such as the WP diet). This was observed despite the fact that there was a significant reduction in the number of mitoses per crypt. These data contrast with the jejunal mucosa atrophy observed after severe malnutrition (13, 15,19,37). Brush border hydrolase activities were either unmodified (N aminopeptidase, acid aminopeptidase), decreased (glucoamylase) or increased (sucrase, dipeptidyl peptidase IV). These data suggest that natural alimentary substrates have little or no direct influence on ileal mucosa adaptation. The maintenance of normal morphology and functions in the ileum after starvation appears to be the consequence of reduced enterocyte turnover rate and brush border enzyme hydrolysis secondary to a decrease in pancreatic protease secretion induced by starvation (38,39,40). The relative hypoplasia in starved rats compared to rats on the WPH and AAM control diets could be the result of a delayed absorption of WPH and free amino-acids in rats on these two diets and to a specific effect of these substrates on the ileal mucosa (41), or to a

Table 3 Brush border hydrolase specific activities after starvation and during refeeding with the WP, WPH and AAM diets Time of refeeding 0

24h n=8

48h n=6

96h n=6

Diet

n=8

WP WPH AAM

57 f 4

43 f 4” 31 f4O 29 f 5O

WP WPH AAM

81 + 7

1.53+ 29O 140 + loo 170 + loo

98 f lox0 140 + 10 175 f 20

150 + 200 125 + 20 85 + gxo

N Amino peptidase

WP WPH AAM

107 f 7

114 f 6 107 + 4 120 f 13

130 f 8 108 + 10 101 + 5

104 f 4 108 + 9 107 + 4

Acid Amino peptidase

WP WPH AAM

227 + 30

288 i lox0 175 + 12 167 + 10

265 + 15 19Of18 233 f 23

246 + 20 230 f 29O 250 + 27

Dipeptidyl peptidase IV

WP WPH AAM

78 + 4

68 f 3 56 f 5O 41 + 5xo

69 + 3 57 + 3 49 f 3x

67 + 3 60 + 3 50 + 6x

Sucrase

Glucoamylase

32 + 4O 29 + 3 40 + 10x0

38 -t 6 45 + 30 26 + 6”

Xstatistically different (p < 0.05) from the other groups at the same time of refeeding Ostatistically different (p < 0.05) as compared to the preceding time of refeeding for the same group

CLINICAL NUTRITION

reduction in pancreatic secretion induced by these diets (42). Neuro-hormonal regulation may also be involved eventually mediated through pancreatic secretion (37,38,43,44). Refeeding rapidly returned ileal morphology to normal. However there were significant differences in the rate, depending on the molecular form of the alimentary nitrogen source. The AAM diet produced the greatest villus height during both the refeeding period and in controls. This was associated with an increase of the number of mitoses per crypt, indicating a growth-promoting effect of amino-acids when they are ingested as free amino acids. This effect is related only to the molecular form of the nitrogen source, as nitrogen and caloric intakes were the same, as were the nature and quantity of carbohydrates and lipids in the three diets. In the amino-acid mixture, there was no glutamine or asparagine as found in native lactalbumin, but glutamic and aspartic acids were present, so the potential growth promoting effect of these two substrates in the AAM could not be suggested. Brush border hydrolase activities were also modified by the diets. However, as previously found in the jejunum (24), each enzyme responded differently depending on the dietary nitrogen source and the nutritional status. Sucrase and acid amino peptidase was increased in controls by the WPH diet; dipeptidyl peptidase IV was decreased by the AAM diet and the N amino peptidase remained unchanged. Sucrase and DDP IV were decreased during refeeding, notably with the AAM diet, while glucoamylase increased. NAP remained unchanged and control values were reached at 96 h except in rats on AAP. These findings strongly suggest that, in contrast to the jejunum, where specific enzyme substrates have an important role in the brush border enzyme adaptation (18,20,22,23,41), other factors, such as nutritional status and the molecular form of the alimentary nitrogen, participate in the regulation of ileal adaptation. They must therefore be taken into account when such regulation is studied at this level. These results demonstrate that, depending on the nutritional status, the molecular form of the dietary proteins may be varied to obtain different growth promoting effects on the ileal mucosa or enterocyte brush border enzyme adaptation. Further studies are necessary to clarify the multiple mechanisms involved in their changes. Nevertheless these data may be significant as synthetic diets are widely used in nutritional

53

support and also for therapeutic purpose such as ‘bowel rest’ in ileal diseases (1, 25, 26) and ileal growth stimulation in the short bowel syndrome (1,37, 38).

Acknowledgements This work was supported by INSERM, Sopharga R.U. Cie and the Facultt Xavier Bichat, University Paris VII.

References 1. Koretz R L, Meyer J H 1980 Elemental diets - facts and fantasies. Gastroenterology 78: 393-410 2. Youne E A. Heuler. N. Russel P. Weser E 1975 Com&ative nutritional analysis of chemically defined diets. Gastroenterology 69: 1338-1345 3. Anderson H L, Heindel M B, Linkwile H 1969 Effects on nitrogen balance of adult man on varying source of nitrogen and level of caloric intake. Journal of Nutrition 99: 82-86 4. Meguid M, Dubonis D, Meguid V. Terz J J 1983 Nutritional support in cancer. Lancet 2: 230-231 5. Meguid M M, Landel A M, Terz J J, Akrabawi S S 1984 Effect of elemental diet on albumin and urea synthesis comparison with partially hydrolyzed diet. Journal of Surgical Research 37: 16-24 6. Smith J L, Arteaga C, Heymsfield S B 1982 Increased urea genesis and impaired nitrogen use during infusion of a synthetic amino-acid formula. New England Journal of Medicine 306:l 1013-1018 7. Keohane P P, Grimble G K, Brown B, Spiller R C, Silk D B A 1985 Influence of protein compostion and hydrolysis method on intestinal absorption of protein in man. Gut 26: 907-913 8. Silk D B A, Clark M L, Marrs T C, et al 1975 Jejunal absorption of an amino-acid mixture simulation casein and an enzymic hydrolysate of casein prepared for oral administration to normal adults. British Journal of Nutrition 33: 95-100 9. Fenyo G, Hallberg 0 1976 The influence of a chemical diet on the intestinal mucosa after jejuno-ileal by pass in the rat. Acta Chirurgica Scandinavia 142: 270-274 10. Kimura T, Seto A, Kato T, Yoshiora A 1977 Effects of dietary amino acids and proteins on jejunal disaccharidase and leucine amino peptidase. Nutr Rep Int 16: 621-630 11. Spector M H, Traylor J, Young E, Weser E 1981 Stimulation of mucosal growth by gastric and heal infusion of single amino-acids in parenterally nourished rats. Digestion 21: 33-40 12. Vanderhoof J A, Grandjean C J, Burkley K T, Antonson D L 1984 Effects of casein versus casein hydrolysate on mucosal adaptation following massive bowel resection in infant rats. Journal of Pediatric Gastroenterology and Nutrition 3: 262-267 13. Altman G G 1972 Influence of starvation and refeeding in mucosal size and epithelial renewal in the rat small intestine. American Journal of Anatomy 133: 391-400 14. Levine G M, Deren J J, Steiger E 1974 Role of oral intake in maintenance of gut mass and disaccharidase activity. Gastroenterology 67: 975982

54

ILEAL ADAPTATION

AND ALIMENTARY

PROTE INS

15. MacManus J P A, Isselbacher K G 1970 Effect of fasting versus feeding on the rat small intestine. Gastroenterology 59: 214-222 16. Newev H, Sanford P A, Smvth D H 1970 Effects of fasting on intestinal transfer-of sugar and amino acids in vitro. Journal of Phvsioloev (Land) 208: 705-710 17. Prosper J, Murry R-L, Keyn F 1968 Protein starvation and the small intestine. Gastroenterology 55: 223-227 18. Gorostiza E, Marche C, Boryart J P, Balmain N, CCzard J P 1984 Influence of starvation on sucrase regulation by dietary sucrose in the rat. American Journal of Clinical Nutrition 40: 1017-1022 19. Gorostiza E, Poullain M G, Marche C, Cezard J P 1985 Effect of fasting and refeeding on small intestinal adaptation in the rat. Gastroenterology Clinique et Biologique 9: 79@-?96 20. Lis T M, Crampton R E, Matthews D M 1972 Effects of dietary changes on intestinal absorption of L-methionine and L-methionyl-methionine in the rats. British Journal of Nutrition 27: 159-162 21. Lifshitz F, Hawkins R L, Oraz-Bensussen S, Wapir R A 1972 Absorption of carbohydrates in malnourished rats. Journal of Nutrition 102: 1303-1307 22. CCzard J P. Brovart J P. Cuisinier-Gleizes P. Mathieu H 1983 Sucrase-isdmaltasd regulation by dietary sucrose in the rat. Gastroenterology 84: 18-25 23. Nicholson M C, Carty D A, Kim Y S 1974 The response of rat intestinal brush border and cytosol peptide hydrolase activities to variation in dietary protein content. Journal of Clinical Investigation 890-898 24. Poullain M G, Cezard J P, Marche C, Roger L, Mendy F, Broyart J P 1989 Dietary whey proteins, their peptides or amino acids: effects on the jejunal mucosa of starved rats. American Journal of Clinical Nutrition 49: 71-76 25. Navarro J, Vargas J, Cezard J P 1982 Prolonged constant rate elementai nutrition in Crohn’s disease. Journal of Pediatric Gastroenterology-. and Nutrition 1: 541-549 26. Greenberg G R, Fleming C R, Jeejeebhoy K N et al 1988 Controlled trial of bowel rest and intestinal support in the management of Crohn’s disease. Gut 29: 13& 1315 27. Maubois J L, Roger L, Brule G, Pior M, inventors 1984 Institut National de Recherches Agronomiques (INRA) Paris, France, assignee. Total enzymatic hydrolysates from whey proteins and process for obtaining the same. US patent 4 427 658. January 24,18 p. Int CI’ A61K37100, A61K31/195, A23C21/100 28. Irvine G B, Shaw C 1986 High-performance gel permeation chromatography of proteins and peptides on columns of TSK-G2000-SW and TSK G3OOOSW: a volatile solvent giving separation based on charge and size of poly peptides. Analytical Biochemistry 155: 141148 29. Bohlen P, Castillo F, Ling N, Guillemin R 1980

30.

31.

32.

33.

34.

35.

36.

37.

38. 39.

40.

41

42

43.

44.

Submission date: 22 May 1990; Accepted after revision: 28 September

Purification of peptides: an efficient procedure for the separation of peptides from amino-acids and salts. International Journal of Peptide and Protein Research 16: 306-310 Committee on Animal Nutrition Nutrient requirements of laboratory animals. Vol 10. Washington, DC: National Academy of Sciences 1978 Saito M, Kato H, Suda M 1980 Circadian rhythm of intestinal disaccharidases of rats fed with a diurnal periodicity. American Journal of Physiology 238: G97101 Lowry 0 H, Rosenbrough N J, Farr A L, Randall R J 1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265275 Cezard J P, Conklin K A, Das B C, Gray G M 1979 Incomplete intracellular forms of intestinal surface membrane sucrase-isomaltase. Journal of Biological Chemistry 254: 8969-8975 Ahnen D J, Santiago N A, Cezard J P. Gray G M 1982 Intestinal aminooligopeptidase. In vivo synthesis on intracellular membranes of rat jejunum. Journal of Biological Chemistry 257: 12129-12135 Triadou N. Bataille J, Schmitz J 1983 Longitudinal study of the human intestinal brush border membrane proteins: distribution of the main disaccharidases and peptidases. Gastroenterology 85: 13261332 Raul F. Noriega R, Duffoel R et al 1982 Modifications of brush border enzyme activities during starvation in the jejunum and ileum of adult rats. Enzyme 28: 328-335 Dowling R G 1982 Small bowel adaptation and its regulation. Scandinavian Journal of Gastroenterology 17: 53-74 Williamson R C N 1978 Intestinal adaptation. Mechanisms of control. N Engl J Med 298: 1444-1450 Weser E, Heller R, Tawil T 1977 Stimulation of mucosal growth in the rat ileum by bile and pancreatic secretions after jejunal resection. Gastroeneterology 73: 524-529 Alpers D H, Tedesco F J 1975 The possible role of pancreatic proteases in the turnover of intestinal brushborder proteins. Biochem Biophys Acta 401: 28-40 Weser E, Babbitt 3, Hoban M. et al 1986 Intestinal adaptation. Different growth responses to disaccharides compared with monosaccharides in rat small bowel. Gastroenterology 91: 1521-1527 Green G M, Miyasaka K 1983 Rat pancreatic response to intestinal infusion of intact and hydrolyzed protein. American Journal of Physiology 245: G39&G397 Howard F. Yudkin J 1963 Effect of dietarv chanae unon the amylase and trypsin activities of the rat pancyeas: British Journal of Nutrition 17: 281-294 Grey V J, Morin C 1988 Role of food in the appearance of growth stimulation activity in rat proximal intestine after small bowel resection. Digestive Diseases and Sciences 33: 78-82

1990