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*
Ecole Nationale Supérieure Agronomique de Rennes, Rennes Cedex, France and
Institut National de la Recherche Agronomique, Unité Mixte de Recherches sur le Veau et le Porc, Saint Gilles, France
1To whom correspondence should be addressed. E-mail: david{at}roazhon.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • heat shock proteins • weaning • piglet
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). They are classified according to their molecular mass, which ranges from 27 to >100 kDa (3
). They are usually separated into three major families (27, 70 and 90 kDa). Major properties of HSP have been reviewed in recent years for HSP 27 and small HSP (4
), HSP and heat shock cognate 70 (HSC 70) (5
) and HSP 90 family (6
).
Under nonstressful conditions, these proteins maintain cell integrity (7
). They display molecular chaperone properties in the folding of nascent and degraded other proteins, thereby acting as cytoprotectors of cellular constituents (8
). HSP expression also protects cells from various kinds of stress, including hyperthermia, radiation and ischemia (9
). The low-molecular mass HSP family includes several proteins (4
). HSP 27 is associated with the cytoskeleton and is involved in actin filament dynamics (10
). HSP 27 expression is increased in several types of cancers, including breast and prostate carcinomas (11
) as well as uterine and gastric sarcomas (12
). HSP 27 is involved in the protection against stress in general and specifically against toxic stress (4
). The HSP 70 family comprises two forms, the constitutive HSC 70 and the inducible HSP 70, and displays many specific features. HSP 70 confers thermotolerance and protects against apoptosis, endotoxins, reactive oxygen species, radiation and ischemia (9
).
The gastrointestinal tract (GIT) represents a barrier preventing passage of potentially harmful microorganisms or toxic substances to the portal circulation; therefore, its protection is of extreme importance with regard to permanent exposure to such aggressors (13
). A decade ago, expression of HSP 27 (14
) HSC 70, HSP 70 and HSP 90 was detected in the stomach and intestine of mice in the absence of stress (15
). However, most other studies in the GIT have been devoted to HSC 70 and HSP 70. Under normal conditions, HSC 70 and HSP 70 expressions have been observed in gastric mucosal cells (16
) and in the ileum and colon (13
). HSP 70 is induced as a consequence of intestinal aggression by various stressors, including endotoxins (13
) sodium arsenite (17
), ethanol (7
) and ischemia (18
). The observed induction of HSP 70 after this stress results in the protection of the respective parts of the tract (13
,18
–20
). Although found in stomach and intestine (15
), HSP 90 has been studied in these tissues.
Recent evidence has shown that nutrition can modulate the expression of HSP by intestinal epithelial cells or GIT tissues. This expression of HSP, in turn, influences cell and gut protection. For example, the protective effects of glutamine (21
,22
) and short-chain fatty acids (SCFA; especially butyrate) (23
) are mediated specifically through HSP 25 and HSP 70, respectively. By contrast, plant lectins decreased the expression of different HSP and intestinal epithelial cell resistance to heat shock (24
). Glutamine (25
) and butyrate (reviewed in Ref. 26
) have proven to be beneficial to the altered GIT mucosa of young pigs immediately postweaning whereas lectins have antinutritional activity in this species (27
). This prompted us to study HSP along the GIT of pigs and the impact of weaning on their expression, before extending our research to HSP modulation by other nutritional factors. Indeed, virtually nothing is known about gastrointestinal HSP in pigs (28
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All experimental procedures were carried out in accordance with the National Institute of Health Guide and the French Ministry of Agriculture for the Care and Use of Laboratory Animals. All piglets were from the Large White strain.
Piglets were weaned at either 21 or 28 d of age. When weaned, all were transferred from the farrowing unit to the postweaning unit and kept in groups of littermates. Experiments were conducted either over 8 d or 48 h postweaning. Experiments 1 and 2 were performed on piglets weaned at 21 d and complementary experiments 3 and 4 were performed on piglets weaned at 28 d of age. In experiment 1, 15 piglets from five litters were used [weaning body weight (mean ± SEM), 6.22 ± 0.2 kg]. In experiment 2, 24 piglets from three litters were used [weaning body weight (mean ± SEM), 6.19 ± 0.2 kg], In experiment 3, 20 piglets from five litters were used [weaning body weight (mean ± SEM), 6.2 ± 0.2 kg]. In experiment 4, 42 piglets from seven litters were used [weaning body weight (mean ± SEM), 9.3 ± 0.4 kg], with 18 piglets kept with the sows unweaned. Piglets were randomly assigned to slaughter groups of three (unweaned, experiment 4) and four (weaned, experiments 3 and 4; see below) each according to their litter origin, sex and weaning body weight. Experiments were completely balanced block designs in which the block was the litter. The weaned piglets had free access to a solid starter feed (Table 1
) from weaning onward.
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In experiments 1 and 3 piglets were killed at 0, 1, 2, 5 and 8 d postweaning. In experiments 2 and 4 piglets were killed at 0, 6, 12, 24, 36 and 48 h postweaning. Their unweaned littermate controls were killed at the same time points. After chloroform anesthesia (experiment 3) or electronarcose (experiments 1, 2 and 4), pigs were decapitated. Intestinal tissues collected were mid-jejunum (experiment 1 and 3) and stomach, duodenum, mid-jejunum, distal ileon and proximal colon (experiments 2 and 4). For HSP expression, whole thickness tissue samples were placed in extraction buffer [1x TEX buffer, 60 mmol/L Tris-base (pH 6.8), 10% glycerol and 3% sodium dodecyl sulfate (SDS)] with the addition of 5% ß-mercaptoethanol just before use.
Jejunal morphology and enzyme activities
Pieces of mid-jejunum were fixed in buffered formalin and microdissected before villus and crypt morphometry as described by Goodlad et al. (29
). Mucosal scrappings were prepared, snap-frozen in liquid nitrogen and stored in a deep freezer (-80°C) before enzyme activity determination. Lactase-phlorizine hydrolase (EC 3.2.1.23) activity was determined according to Tivey et al. (30
) using lactose as the substrate (L8783; Sigma-Aldrich, St. Louis, MO). The activity of aminopeptidase A (EC 3.4.11.7) was determined using 
-L-glutamic acid-4-nitroanilide as substrate (G6133; Sigma-Aldrich) (31
). The protein content of tissue homogenates was measured using the Bio-Rad protein assay reagent (Bio-Rad, Hemel Hempstead, UK). Enzyme activities were expressed as nanomoles of hydrolyzed substrate per milligram of tissue protein per hour.
Preparation of protein homogenates and Western blotting for HSP
Tissues were immediately homogenized with Poly Fron PT300 (Fisher, Illkirch, France) at maximum speed for 1 min in ice and centrifuged at 26,500 x g for 15 min. Supernatants were collected in series of Eppendorf tubes kept at -20°C for no longer than 1 wk before use. Protein concentrations were measured according to Lowry et al. (32
) using an enzyme-linked immunosorbent assay plate reader (Argus 300; Packard, Rungis, France). Equal amounts of proteins were loaded on a 13% acrylamide gel with a 4% stacking acrylamide gel. Migration was performed in buffer containing 25 mmol/L Tris (pH 7.6), 0.1% SDS and 0.2 mol/L glycine.
After migration, proteins were transferred on Hybond C membranes (Amersham, Saclay, France) for 75 min using buffer containing 25 mmol/L (Tris pH 7.6), 0.1% SDS, 0.2 mol/L glycine and 20% methanol. Blots were washed four times in washing buffer [20 mmol/L Tris-base (pH 7.6), 12.5 mmol/L NaCl and 0.5% Tween 20 (TBST buffer)], soaked in Ponceau red for 1 min to reveal markers and washed again four times in TBST buffer for 5 min each. Molecular weight markers were purchased from Sigma-Aldrich.
Membranes were blocked for 1 h at room temperature in TBST buffer containing 50 g/L milk powder at room temperature and then incubated overnight with the primary antibody. Each sample was tested with an anti-hamster HSP 27 rabbit polyclonal antibody (a generous gift of H. Lambert, University Laval, Quebec, Canada), two human anti-HSC 70 and anti-HSP 70 rabbit polyclonal antibodies (SPA 816 and SPA 812, respectively; Stressgen, Victoria, British Columbia, Canada) and anti-mouse 29 A HSP 90 monoclonal antibody (a generous gift from Dr. A. Wikström, Karolinska Institutet, Huddinge, Sweden). All these antibodies have been shown to respond to the corresponding porcine HSP (3
,33
,34
). Experiments were reproduced four times with separate samples.
After overnight incubation, blots were washed five times for 5 min in TBST buffer and 50 g/L milk powder containing the secondary antibody 1/1000 (IgG peroxidase coupled from Sigma-Aldrich). Dilutions used are shown in Table 2
.
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Standardization of blot densitometry data
To check for equal protein loading of each lane, the same membranes were tested for the expression of ß-actin using a specific antibody (9044; Sigma-Aldrich). An example is given for each figure. ß-Actin expressions were determined for each lane of each membrane. Membranes were classified into two categories for the detection of HSP 90 and HSP 70 (or HSC 70), and HSP 27, respectively.
Detection of HSP 90 and HSP 70
After transfer, membranes were cut into two parts between 70 and 43 kDa. The upper part of the membranes was tested for HSP 90 and HSP 70 and the lower part was tested for ß-actin.
Detection of HSP 27
After transfer, membranes were cut into two parts between 40 and 30 kDa. The upper part was tested for ß-actin and the lower part was tested for HSP 27.
Secondary antibodies were peroxidase-coupled anti-rabbit (HSP 27, HSC 70, HSP 70) or anti-mouse (HSP 90) antibodies from Sigma-Aldrich.
To compare densities, membranes were scanned using a phosphor imager (Quantum Appligene, Illkirch, France). Densitometry measurements were performed using image analysis (ImageQuaNT; Molecular Dynamics, Sunnyvale, CA). The density of each HSP band of interest was expressed as a the percentage of the density of the ß-actin band in the same gel lane.
Statistical analysis
Data are presented as mean ± SEM; n = 4. Data were analyzed using the analysis of variance procedure of the Statistical Analysis System (SAS Institute, Cary, NC) with Dunnett post hoc comparison between means at times postweaning and control means at time zero. Differences between means were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In both groups of piglets weaned at either 21 or 28 d of age, HSP 27, HSP 70 and HSP 90 expressions in the mid-jejunum increased transiently on d 1 (all HSP) and d 2 (HSP 90) after weaning (Fig. 1
); therefore, we investigated possible changes in expression after weaning over a shorter time frame (0–48 h) and at various sites along the GIT.
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Control unweaned pigs, 28 d of age.
Expressions of the four HSP studied were constant in the stomach of control unweaned pigs between 0 and 48 h postweaning (Fig. 2
). Similar observations were made at all other segments of the GIT (data not shown). Thus, these findings exclude any change in HSP expression due to a developmental pattern.
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). In the duodenum and jejunum, an increased expression was observed at 12 and 24 h. In the ileum, expression of HSP 27 was transiently decreased at 12 and 48 h, whereas in the colon an increase was observed from 24 to 48 h.
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), but no changes occurred in the intestines (results not shown).
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). In the duodenum and jejunum, such an increase was seen after 12 and 24 h. In the ileum, expression decreased between 24 and 48 h and in the colon it increased at 36 and 48 h.
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). Decreased expression occurred in the duodenum (at 6, 12 and 36 h), ileum (from 6 to 48 h) and colon (at 24 and 48 h).
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Because the GIT tract is more sensitive to nutritional changes associated with weaning in younger animals, experiments were performed with pigs weaned at 21 d of age. In stomach, increases of HSP 27 expressions were observed 6, 12 and 24 h after weaning (Fig. 7
), as in 28-d-old pigs. For HSP 70, in the same parts of the GIT, increased expressions occurred from 6 to 36 h postweaning. For HSP 90, increased expressions occurred from 24 to 48 h after weaning. These observations are similar to 28-d-old weaned piglets. Comparable results were obtained for duodenum, mid-jejunum, proximal ileum and distal colon (results not shown).
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In experiment 2, jejunal villi of 21-d-old piglets were shorter at 24, 36 and 48 h postweaning, as compared with time 0 (P <0.05) (Table 3
). Although crypt depth did not change over time in this experiment, the villus height to crypt depth ratio was lower at 24, 36 and 48 h postweaning than at time 0 (P < 0.05). As in experiment 1, the activities decreased over time; activities of lactase at 36 and 48 h and of aminopeptidase A at 12, 24, 36 and 48 h were significantly different from corresponding values at time 0.
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). The activity of lactase was dramatically reduced (P < 0.05) between d 0 and 1, and it remained low until d 8. Similar changes over time were recorded for aminopeptidase A activity, but differences from d 0 occurred only at d 5 and 8 (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Biology of HSP in pig GIT
Our first major finding is that in suckling piglets aged 21 or 28 d these four HSP were expressed constantly in every region of the GIT. To our knowledge, little is known about the expression of HSP in the GIT of pigs. In the developmental study carried out by McComb and Spurlock (28
) the gut was not investigated. However, in their experiment with an immunological challenge they reported HSP60, HSC 70 and HSP 70 to be expressed in the jejunum of unchallenged 25-kg male castrates. HSP 27 and HSP 90 were not studied. Our results are in contrast with those of Beck et al. (13
) in rats where HSP 70 did not appear to be expressed in the proximal intestine. In mice, Tanguay et al. (15
) reported that the four HSP they studied (HSP 25, HSP 71, HSC 73 and HSP 84) were expressed in all tissues at variable levels. Both HSP 25 and HSP 71 were present in higher levels in the stomach, intestine and colon. It was also reported that the levels of HSP 25 and HSP 71 in tissues correlated positively. This led to the suggestion that these HSP would have similar roles in the restoration of cellular functions in those tissues exposed to toxic environmental or metabolic products and therefore are subjected to more immediate environmental aggression (15
). In the present study, the linear relationship between HSP 27 and HSP 70 in the GIT was not significant (P < 0.10).
Influence of weaning
The second major finding is that weaning induced profound spatial-temporal changes of expression of most HSP, with the exception of HSC 70. Whereas HSP 27 and HSP 70 expressions increased rapidly after weaning and remained high in the stomach, elevated expression was delayed and shorter in the duodenum, jejunum and colon. In addition, expressions of HSP 27 and HSP 70 decreased transiently in the ileum. Nevertheless, spatial-temporal patterns of expression of HSP 27 and HSP 70 were fairly similar. By contrast, the increase of HSP 90 expression in the stomach and jejunal tissues was delayed by 12–24 h, and the levels in the duodenum, ileum and colon were even transiently reduced. Therefore, we can conclude that the expression of HSP in the GIT was modulated by weaning, the results depending on the HSP, GIT site and time postweaning. However, the actual consequences of such changes on the overall protection of the GIT of pigs are not known at present. Most often, an induction of HSP is considered protective (22
,23
) whereas underexpression of HSP increases the sensitivity of cells or organisms to stress (24
,35
).
Weaning is a complex phenomenon involving dietary, psychological and environmental changes that have serious effects on the physiology of pig GIT (36
). The major features are a transiently reduced feed intake, a growth check and marked changes in the histology and biochemistry of the small intestine, including partial villous atrophy, crypt hyperplasia and depressed activity of most brush border digestive enzymes. Together these factors contribute to decreased digestive and absorptive capacity and to postweaning diarrhea. In the present study, we confirmed postweaning changes in jejunal villus-crypt architecture and disaccharidase and peptidase activities. Such changes were detected as early as 12 h postweaning (aminopeptidase A) and lasted to 8 d at least (most variables). Overall, our observations are in agreement with the literature (36
). Although the older the pigs at weaning are, and the smaller the postweaning intestinal alterations are, published data indicate that significant changes can be observed after weaning at 35 (37
) or even 42 d (38
).
To our knowledge, clear links between expression of HSP and histology or biochemistry of the small intestine have not been established. In the present study, the only linear relationship was between the jejunal villus height to crypt depth ratio and expression of HSP 90 and was negative (n = 24, R2 = 0.3962, P = 0.06). This would support the hypothesis that HSP 90 has a role as a housekeeping protein for cell growth and differentiation (15
).
Factors modulating the expression of HSP
The process of weaning involves major dietary qualitative and quantitative changes, including transient anorexia, in a general context of stress associated with disturbances in social relationships and environment. Whether these factors influence the expression of HSP is largely unknown with regard to the GIT. By contrast, the effect of food restriction on the liver and central nervous system has been studied in rats. Food restriction stimulated HSP 70 but reduced glucose related protein (GRP 78) expression in the liver (39
). Food deprivation induced a twofold increase in mRNA for HSP 70 and HSP 90 (40
). Energy restriction elicited HSP 27, stress protein 34, HSP 70 and HSP 90 in the hypothalamus of both young and adult rats whereas none was found in the hypothalamus of controls that were fed ad libitum (41
). Interestingly, stress protein 34 was induced only by energy restriction and not by heat stress in that study.
The effect of stress has been addressed experimentally in rats subjected to restraint (35
). Although restraint led to an overexpression of HSP 70 in the vasculature and adrenal cortex, no HSP 70 induction was apparent in a wide variety of other tissues, including the small bowel (35
). These observations would suggest that dietary rather than stress factors may be involved in the changes in HSP expression observed in response to weaning in the present study. The specific effect of the transient postweaning anorexia remains to be investigated.
From a hormonal standpoint, weaning is characterized by reduced serum insulin-like growth factor (IGF)-1 and IGF-2, as well as elevated levels of growth hormone (42
) and glucagon 5 d after weaning (43
). Surprisingly, changes in cortisol concentrations were not associated with weaning (42
). In contrast, hormonal regulation of HSP is still poorly understood, especially in the GIT, but it appears to depend strongly on the tissue studied. For example, HSP are selectively induced in the adrenal cortex and vasculature smooth muscle after surgical or restraint stress (35
) and this is dependent on the activation of the hypothalamus-pituitary adrenal axis (35
). Other examples are the expression of HSP 27 in human breast tumor cells, which is modulated by estrogen or the estrogen to progesterone ratio (12
), and HSP 90, which is necessary for proper steroid action in vivo (6
). Thus, hormonal influences on HSP modulation at weaning in pigs cannot be excluded.
HSP and nutrition
The recent ban on in-feed antibiotics in the European Union has renewed the interest in alternative substances or feeding strategies to prevent digestive disorders during critical periods such as weaning in pigs. One promising approach is the dietary manipulation of fermentation in the large intestine to improve the "colonization resistance" exerted by the commensal flora toward enteric pathogens, either directly or indirectly through the fermentation products (26
). Among SCFA, butyrate plays a central role, being preferentially metabolized by colonocytes and exerting cytoprotection on both the small and large intestines (44
). Interestingly, it has been recently shown that butyrate induces a time- and concentration-dependent increase in HSP 25, but not in HSP 72 or HSC 73, expression in rat intestinal epithelial cells (23
). In vivo, increasing dietary fiber also increased colonic, but not proximal ileal, HSP 25, whereas having no effect on HSP 72 or HSC 73 (23
).
Glutamine is a major fuel for rapidly dividing cells and has been shown to prevent gut atrophy in various situations, including the postweaning period in pigs (25
). In vitro, glutamine supplementation reduced heat shock-induced intestinal epithelial cell death (45
). Part of this protective effect has been shown to be mediated by HSP 70 induction (21
,22
).
Contrasting with these favorable effects, antinutritional factors, including lectins, reduce protein digestion (46
) and alter the structure and function of the small intestinal mucosa (27
). Interestingly, recent data demonstrated that kidney bean lectin decreased the levels of HSP in rat gut and Caco2 cells and reduced the resistance of these cells to heat shock (24
).
In conclusion, the present findings clearly showed that weaning modulates the expression of HSP in the GIT of pigs according to various spatial-temporal patterns. The actual modulating factors and mechanisms involved are not fully understood. Thus, further investigations are warranted to examine the respective influences of transient anorexia, nutritional changes and the general stress associated with weaning. Recent data clearly show that HSP are involved in the modulation of cell protection by nutritional factors. The role for HSP as modulators of GIT protection in pigs remains to be demonstrated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 28 February 2002. Initial review completed 11 April 2002. Revision accepted 10 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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