Effects of feeding fat-coated butyrate on mucosal morphology and function in the small intestine of the pig

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1 DOI: /j x ORIGINAL ARTICLE Effects of feeding fat-coated butyrate on mucosal morphology and function in the small intestine of the pig Fachgebiet Tierhaltung und Leistungsphysiologie, Institut für Tierhaltung und Tierzüchtung, Universität Hohenheim, Stuttgart, Germany Keywords pig, butyrate, intestinal plicae, sucrase, crypt fission Correspondence Dr. R. Claus, Institut für Tierhaltung und Tierzüchtung, Universität Hohenheim, Garbenstr. 17, Stuttgart, Germany. Tel: ; Fax: ; Received: 24 March 2006; accepted: 5 July 2006 First published online: 7 November 2006 Summary As shown earlier, pig rations with high starch and purine content initiate mucosal hypertrophy by stimulating mitotic activity and DNA formation in the small intestine, whereas in the colon butyrate inhibits apoptosis and thus increases crypt depth. It was the aim of this study to combine these effects by targeting fat-coated butyrate into the small intestine where it usually does not occur, and to investigate effects on mucosal development and function. Three groups of five pigs were fed 3.6 kg/day of either a low-energy ration [deficit group, 6.6 MJ metabolizable energy (ME)/kg] or a high-energy ration (13.7 MJ ME/kg) that was supplemented with brewing yeast as a source of purines. The third ration was of high energy and contained purines and was additionally supplemented with coated butyrate (13.5 MJ ME/kg; 29 g calcium butyrate/kg). Rations were fed for 5 days. After killing, tissue samples were obtained from the proximal, medial and distal parts of jejunum for histology. Chyme samples were obtained from the ileum of all animals and used for sucrase determination. Villus size was not changed by feeding, but butyrate had an effect on plica height and area mainly in the medial jejunum. Plica area in the butyrate group (4.2 mm 2 ) was significantly higher (p 0.01) compared with that of the deficit group (2.3 mm 2 ) and high-energy group (2.7 mm 2 ; p 0.01). For the butyrate group, sucrase activity in the ileum was U/ml and thus significantly higher (0.05) compared with the high-energy group (61.7 U/ml) and the deficit group (28.0 U/ml; p 0.001). Targeting butyrate into the small intestine thus improves digestive and absorptive capacities. The mechanism probably is a specific effect on enterocyte mitosis which in turn leads to an increased plica size by crypt fission. Introduction Pig mucosal cells are renewed every 2 7 days. Mucosal integrity is ensured by an appropriate balance between the mitotic activity of stem cells in the crypt area and apoptosis in the villus tips. In the large intestine, the corresponding compartments are the bottom (mitosis) and the luminal opening (apoptosis) of the crypts (Potten, 1995). Feed has a strong impact on the equilibrium between mitosis and apoptosis and thus the functional status of the gut (Piva et al., 2001). For the pig, we could show that mucosa proliferation in the small intestine is stimulated by an elevated mitotic rate because of a glucose-dependent increase of the insulin-like growth factor 1 (IGF-1) in tissue and peripheral plasma. The additional supply of purines in the feed further increased the mitotic rate because it is used for DNA and RNA synthesis in mitotic enterocytes. An excessive proliferation of villi is avoided by a 312 Journal of Animal Physiology and Animal Nutrition 91 (2007) ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd

2 Butyrate effects on small intestine of pigs counteracting increase of apoptosis, which, however, allows a net gain of villus length (Raab et al., 1998). In the colon, butyrate is the main source of energy for the colonocytes (Bergman, 1990). Transport of butyrate into the cells depends on a monocarboxylate transporter (MCT-I) (Takanaga et al., 1995; Cuff et al., 2002). In the pig colon, butyrate inhibits apoptosis of mucosal cells in vivo through the expression of the antiapoptotic signal Bcl-2. In consequence, crypt depth increased, whereas the mitotic rate was not altered (Mentschel and Claus, 2003). MCT-I is also expressed in the small intestine both in the basolateral membranes of crypt cells and in the microvilli of the villus tip (Cong et al., 2001), suggesting an effect of butyrate also in the small intestine. In this part of the gut, however, microbial formation of butyrate is low or absent, but the addition of butyrate to formulas for total parenteral nutrition (TPN) for neonatal piglets stimulated tissue regeneration after 80% jejunoileal resection (Bartholome et al., 2004). Application of butyrate to normal piglets led either to an increase (Kotunia et al., 2004) or to a decrease (Piva et al., 2002) of villus length. In older pigs, luminal butyrate led to an enlargement of the absorptive surface in the ileum because of an increase of villi size (Gálfi and Bokori, 1990). Such contradictory results are explained by the unknown site of butyrate function within the gut and the fact that only the crypt villus architecture was used to characterize the effects. Because butyrate is a preferred source of energy for enterocytes and additionally has an antiapoptotic effect at least for colonocytes (Hass et al., 1997), we hypothesized that butyrate availability has an effect on small intestinal mucosa in addition to starch energy and purines, which were investigated earlier (Raab et al., 1998). Recently, coated butyrate salts became available which led to normal intake by the animals, whereas uncoated butyrate is usually refused. The fat coating is gradually digested in the small intestine so that butyrate is released. We used such a fat-coated butyrate to study the effects both on the villi and on the plica morphology in the small intestine. The study was based on an earlier investigation, where we demonstrated effects of level of energy (starch) and purine (brewing yeast) on small intestinal mucosa (Raab et al., 1998). In the present study, again energy restriction and high-energy level supplemented with purine served as controls and it was tested whether butyrate has an additional effect. Materials and methods Animals and feeding Fifteen male castrated German Landrace Pietrain pigs from the university herd with a mean weight of 75 kg were kept in individual crates without straw. For adaptation, they were fed 3 kg of a conventional ration per day for a week. It contained 10.9 MJ metabolizable energy (ME) and 418 g crude protein/ kg feed (wet weight). The pigs had free access to water. Thereafter, they were allocated to one of three feeding groups (Table 1). The deficit group was fed a low energy diet with 6.6 MJ ME and 108 g crude protein/kg which contained a low concentration of purines because of the use of casein as the main protein source. In this ration, lignocellulose (Arbocel Ò R; Rettenmaier & Söhne, Rosenberg, Germany) was used because it swells after the addition of water, thus creating a volume of feed similar to the other two groups. Water for volume compensation was added before feeding the pigs. The high-energy group received a ration containing 13.7 MJ ME and 121 g crude protein/kg and was supplemented with purines through the use of brewing yeast instead of casein. The basal ration of the butyrate group was similar to that of the high-energy group (13.5 MJ ME and 117 g crude protein/kg) but was supplemented with 290 g fat-coated calcium butyrate/kg (Sanluc International, Gijzenzele, Belgium). Fat coating ensures that butyrate is primarily directed into the small intestine where the fat coating is gradually removed Table 1 Composition (% weight) of the experimental rations Component Group Deficit High energy Butyrate Wheat Maize Oat Wheat bran Soybean oil 2 2 Brewing yeast Minerals + vitamins CaCO Molasses 2 Casein 5 Lignocellulose 5 Calcium butyrate 2.9 H 2 O* kg feed/day MJ ME/kg feed g crude protein/kg feed *Water was added for volume compensation in this ration. Journal of Animal Physiology and Animal Nutrition. ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd 313

3 Butyrate effects on small intestine of pigs by pancreatic lipases. Each group was adapted to the experimental ration for 3 days. Experimental feeding was then performed for 5 days. Such a time is sufficient to reveal differences in gut mucosa morphology and function (Raab et al., 1998). Thereafter, animals were killed for tissue and chyme sampling by intravenous infusion of Eutha 77 (Essex Pharma, München, Germany). Sampling Tissue samples for histological evaluation were taken within 10 min post-mortem from the proximal, medial and distal parts of the jejunum. Samples were immediately washed with cold physiological saline and fixed in 4% paraformaldehyde (ph 7.2) overnight at 4 C. Where possible, chyme samples were collected from the same intestinal compartments and additionally from the ileum for the determination of the brush border enzyme sucrase. Enterocytes contain brush border enzymes that are still active when apoptotic enterocytes are shedded into the chyme. In consequence, gut mucosa turnover can be continuously followed by measuring enzyme activity in chyme collected through a jejunal fistula (Raab et al., 1998). The chyme samples were shock frozen in liquid nitrogen and then stored at )80 C. Although the pigs were fed prior to killing, only ileal chyme could be collected from all pigs. Chyme from other compartments was evaluated analytically, but the data were excluded from statistical evaluation because of limited number. Sucrase determination Determination of sucrase activity was based on a published method (Dahlqvist, 1968). It determines the enzymatic conversion rate of sucrose to glucose. Endogenous glucose was removed by chromatographing chyme on DEAE 32 cellulose (Serva, Heidelberg, Germany). Hundred milligram portions of DEAE were conditioned with 1 ml 25 mm Na 2 HPO 4 (ph 7.4). Chyme (0.5 ml) was centrifuged (15 min, 3000 g) and 100 ll of the supernatant was added to the conditioned DEAE. Phosphate buffer (900 ll) was added and the mixture shaken for 1 h at 4 C. Samples were then centrifuged (15 min, 2000 g) and washed twice with 5 ml of the phosphate buffer. Sucrase was eluted twice with 0.5 ml buffer (ph 6.0) containing 0.1 m sodium maleate and 0.5 m NaCl by shaking for 30 min at 4 C. In the supernatants, glucose was measured using the glucose oxidase peroxidase system and 2,2-azino-di-(3-ethylbenzthiazolin)-6-sulphonate as substrate. The detection limit of the assay was 0.9 U/ml. Histological and immunocytochemical evaluation Histological samples were taken from the proximal, medial and distal parts of jejunum. They were embedded in paraffin and cut into 4 lm sections for later immunocytochemical staining as described previously (Mentschel et al., 2001). Mitosis was stained with the histoprime monoclonal antibody (Ki67 MIB-1; Linaris, Wertheim-Bettingen, Germany). Cells undergoing apoptosis were identified by the modified TUNEL assay (Gavrieli et al., 1992) with POD Kit (Boehringer, Mannheim, Germany). Evaluation of the stained sections was performed by light microscopy under a constant magnification of 400. For quantification of mitosis, only well-defined crypts were used. The positively stained cells and the total number of cells in the crypt were counted separately. Mitotic activity was expressed as the number of stained cells per 100 total cells. The total number of cells per crypt was used to describe crypt depth. For evaluation of the apoptotic rate at the villus tip, positive cells were referred to as the total of 100 counted cells from the tip (50 cells from both sides). For an objective quantification, plicae were evaluated under a standardized magnification with a 3D Color vision camera module DONPISHA provided by LFT Labortechnik, Wasserburg, Germany and the Color BioCapt version (Vilber Lourmat, Marne la Vallee, France) program. Pictures were printed with a standardized size and evaluated by planimetry (type 11/8470; Ott, Kempten Germany). In addition, size of the villi was characterized by the length in lm. The number of cells per crypt as well as plica height (lm), plica width at the base (lm) and the total amount of villi per plica was determined. Statistical evaluation The results are given as mean values and standard deviations. Group differences were analysed by anova followed by post hoc multiple comparisons using the Bonferroni test. Results The effects of the rations on plicae morphology are summarized in Table 2. Independent of the treatment group, both height and area of plicae decreased towards the distal jejunum as described previously (Tackmann, 1991). A difference of 15% was found 314 Journal of Animal Physiology and Animal Nutrition. ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd

4 Butyrate effects on small intestine of pigs Table 2 Morphological characterization of plicae in three compartments of the jejunum (mean ± SD) Feeding group Proximal jejunum Medial jejunum Distal jejunum Height (mm) Area (mm 2 ) Height (mm) Area (mm 2 ) Height (mm) Area (mm 2 ) Deficit 1.8 ± ± ± ± ± ± 0.8 High energy 2.4 ± ± ± ± ± ± 0.6 Butyrate 2.7 ± ± ± ± ± ± 0.6 But vs. Def p 0.05 n.s. p 0.01 p 0.01 n.s. n.s. But vs. High n.s. n.s. p 0.05 p 0.01 n.s. n.s. Def vs. High n.s. n.s. n.s. n.s. n.s. n.s. But, butyrate; Def, deficit; High, high energy. Significant differences are shown for each compartment at the lower half of the table (n.s., not significant). in favour of the butyrate group for plica height in the proximal jejunum when compared with the deficit group (p 0.05). No significant feeding effect on plica area was found in the proximal jejunum. Maximal effects of butyrate were obvious in the medial jejunum. In this compartment, the deficit group and the high-energy group did not differ in their plica height and area, but butyrate led to a height increase by 50% compared with the deficit (p 0.01) and the high-energy (p 0.05) group. Similarly, the plica area was increased by 83.6% (p 0.01) compared with the deficit group and by 55.6% (p 0.01) compared with the high-energy group. No significant differences between feeding groups were found for height and area in the distal jejunum. The base of the plicae did not change significantly along the entire jejunum and was not significantly influenced by the feeding group (data not shown). The overall mean of the plica base was ± lm. The size of villi was not altered significantly by feeding. The average length was 927 lm in the proximal jejunum, 905 lm in the medial jejunum and 880 lm in the distal jejunum. The number of villi per plica (Table 3) was the highest for the butyrate group throughout. In the proximal jejunum, butyrate and deficit group differed significantly (p 0.01). Effects of butyrate Table 3 Number of villi per plica (mean ± SD) in three compartments of the jejunum Feeding group Proximal jejunum Medial jejunum Distal jejunum Deficit 26.6 ± ± ± 5.1 High energy 34.1 ± ± ± 4.5 Butyrate 39.9 ± ± ± 2.7 But vs. Def p 0.01 p n.s. But vs. High n.s. p 0.01 n.s. Def vs. High n.s. n.s. n.s. But, butyrate; Def, deficit; High, high energy. Levels of significance are shown for each compartment at the lower half of the table (n.s., not significant). were even more pronounced in the medial jejunum when compared with the deficit group (p 0.001) and also when compared with the high-energy group (p 0.01). The number of villi per plica did not differ significantly between the deficit group and the high-energy group. No significant differences were found in the distal part of the jejunum. Altogether, differences and their significance obviously resemble those of the plica height as shown in Table 2. Therefore, an elevated number of villi was associated with an increase in plica height. The mitotic and apoptotic activities are given in Table 4. Values did not differ significantly between feeding groups and compartments in the jejunum. These data, however, are based on individual crypts (mitosis) and villus tips (apoptosis) and do not represent the changes for the whole plicae because of an increase in the number of villi and probably also of crypts per plica. The effects of feeding on the functional status are further characterized by sucrase activity in chyme as shown in Table 5. Because chyme could not be collected from all animals/locations except from the ileum, significance was only calculated for this compartment. The data show that the highest enzyme activity in the ileum was measured for the butyrate group and the lowest activity in the deficit group. Level of significance was p when comparing deficit group and butyrate group or high-energy group and deficit group. Difference between butyrate group and highenergy group was also significant (p 0.05). The same tendency was also found in the other compartments. In consequence, these functional data support the effects on mucosa morphology. The absolute sucrase concentration tended to increase from the proximal jejunum to the distal jejunum. In the ileum, a slight decrease was found compared with the distal jejunum. Compared with the isocaloric high-energy group, butyrate supplementation led to an increase of sucrase activity by a factor of Journal of Animal Physiology and Animal Nutrition. ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd 315

5 Butyrate effects on small intestine of pigs Feeding group Proximal jejunum Medial jejunum Distal jejunum Mitosis Apoptosis Mitosis Apoptosis Mitosis Apoptosis Deficit 23.7 ± ± ± ± ± ± 0.13 High energy 12.5 ± ± ± ± ± ± 0.07 Butyrate 10.6 ± ± ± ± ± ± 0.07 Table 4 Mitotic activity in the crypts (%) and apoptotic activity of the villus tips (positive cells/100 cells) in the three compartments of the jejunum. No significant differences were found Table 5 Sucrase activity (U/ml) in chyme from different compartments of the jejunum and from the ileum Feeding group approximately 5 in the proximal jejunum and a factor of 2 in the distal jejunum and in the ileum. Discussion Proximal jejunum Medial jejunum Distal jejunum Ileum Deficit ± ± ± 8.3 High energy 4.9 ± ± ± ± 11.5 Butyrate 18.8 ± 0.7 No sample 212 ± ± 8.2 But vs. Def p But vs. High p Def vs. High p 0.05 But, butyrate; Def, deficit; High, high energy. Levels of significance for ileum are given at the lower half of the table. Significance was not calculated for the jejunum because chyme could not be sampled from all animals. The study shows that butyrate fed to healthy growing pigs had a remarkable effect on small intestinal morphology and function. In contrast to other studies that investigated feeding effects on crypt villus morphology (Gálfi and Bokori, 1990; Piva et al., 2002; Kotunia et al., 2004), we determined effects on plica morphology but the length of the villi was not altered. Butyrate increased the epithelial surface predominantly in the medial jejunum. In addition, feeding elevated the activity of sucrase so that butyrate is likely to improve digestive capacity of the small intestine. A feeding trial in pigs with a diet containing sodium butyrate led to an improved feed conversion rate, an increased villus length and an increase of brush border enzymes (Gálfi and Bokori, 1990). The reason for the improved performance, however, was not investigated. Other studies with butyrate focussed on the pig as a model for TPN formulas to be used in humans suffering from short bowel syndrome (Bartholome et al., 2004), or to stabilize pig gut morphology and function in neonates (Kotunia et al., 2004) and in the early post-weaning period (Piva et al., 2002). In our study, it was not possible to determine butyrate concentrations in the gut lumen because of the lack of chyme. Additionally, butyrate is rapidly absorbed by the gut mucosa (Schmitt et al., 1976; Ruppin et al., 1980; Bergman, 1990) so that remaining concentrations in the chyme do not characterize a distribution pattern. Compared with other segments of the small intestine, the jejunum is apparently privileged for butyrate absorption. In addition, removal of the lipid coating of calcium butyrate is a gradual process, so that it is reasonable to assume that butyrate in our study was targeted to the medial jejunum, which revealed the maximal morphological response. The evaluation of the mitotic compartment in the crypts did not reveal differences in the mitotic activity between feeding groups. The formation of new villi, however, is dependent on an increase of the mitotic activity, which leads to the formation of new crypts by crypt fission (Totafurno et al., 1988) and thus the enlargement of plicae. Only the number of villi can be counted directly, whereas the number of crypts that serve a single villus is variable. Estimations range in the order of 4 12 crypts per villus (Clarke, 1977; Li et al., 1994). Therefore, the comparatively constant number of mitotic cells between the feeding groups must be multiplied by the number of villi and additionally by the (unknown) number of crypts serving the new villus. Consequently, the overall mitotic activity per plica differs between feeding groups because of the different number of villi. In our earlier study (Raab et al., 1998), the increase of mitotic activity could be explained by the use of starch as the main energy source. Starch is digested to glucose which is absorbed and leads to an IGF-1 release by the liver that initiates mitosis (Claus et al., 1992). In the present study, the starch content was virtually the same in the high-energy and the butyrate groups. For this reason, the effect of butyrate must be mediated by other mechanisms. It is therefore likely that the effect of butyrate on mitosis is mediated by growth factors in the tissue, which are not measurable in peripheral plasma (Potten et al., 1995). The glucagon-like peptide 2 (GLP-2) is a possible candidate to mediate the stimulating effect of 316 Journal of Animal Physiology and Animal Nutrition. ª 2006 The Authors. Journal compilation ª 2006 Blackwell Publishing Ltd

6 Butyrate effects on small intestine of pigs butyrate on mitosis. This factor was shown to increase because butyrate was a component of TPN, and it is known that GLP-2 leads to an increased mitotic activity and decreased apoptosis (Yusta et al., 2000; Boushey et al., 2001). The generally increased apoptotic activity in the butyrate group is also supported by the determination of sucrase in the chyme, which was significantly different between groups in the ileum. This brush border enzyme is still attached to the apoptotic cells that are extruded into the gut lumen and transported with chyme. Consequently, enzyme activity is still maintained in areas of the gut, which are distal to their site of origin (Zhan et al., 2004). Theoretically, enzyme activity might be also dependent on substrate availability, but the rations used were free of sucrase substrate, so that the enzyme activity represents apoptotic activity and thus gut cell formation turnover as a whole. 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