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* Nestlé Research Center, Nutrition and Health Department, Lausanne, Switzerland and Unité de Nutrition et Métabolisme Protéique, INRA, Theix, France
1 To whom correspondence should be addressed. E-mail: magali.faure{at}rdls.nestle.com.
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mucin • amino acids • protein synthesis • intestine • rats
The nonimmune intestinal barrier relies on epithelial integrity, mucus production, and the presence and equilibrium of commensal bacteria. All of these compartments interact closely with each other and are essential for maintaining gut homeostasis and health. The mucous layer contains large amounts of secreted mucin glycoproteins that form a viscoelastic gel that protects the gastrointestinal epithelium against constant attack from digestive fluids, microorganisms, and toxins (1). The mucus also favors the establishment and maintenance of a balanced commensal microbiota that antagonizes potentially pathogenic bacteria. The final thickness, composition, and protective effect of the mucous layer are determined by the dynamic balance between opposing anabolic (expression, synthesis and secretion from goblet cells) and catabolic (physical and proteolytic degradation) processes.
Many situations of intestinal stress, including inflammatory bowel diseases (IBD)2 such as ulcerative colitis (UC) and Crohn's disease (CD), are associated with intestinal barrier dysfunction (2,3). The composition of the gut microbiota is altered (4–7) and the mucous layer and mucin production are qualitatively and quantitatively impaired [reviewed in (8)]. The ex vivo production of MUC2, the primary mucin secreted in the colon, is reduced in UC patients during the active phase of the disease (9). Because there are no major changes in MUC2 mRNA levels in the colonic tissues of these patients (10), it is likely that the impaired mucin production results from a translational modification. In sum, further investigations are required to better understand the regulatory mechanisms involved.
Under healthy conditions, the gut utilizes threonine more than other essential amino acids (11,12). If dietary threonine is below the requirements of healthy rats, intestinal mucin synthesis is specifically reduced, whereas whole intestinal protein synthesis is unchanged (13). As shown in animal models and humans, inflammatory situations such as those in IBD and sepsis are associated with an overall increased anabolic reaction occurring mainly in the intestine and the liver, respectively (14–17). Consequently, the requirement for threonine, but also serine and cysteine, is increased (18). The synthesis of intestinal mucins for the protection of the epithelium also contributes to a part of this increased requirement (19). Based on all of these data, we hypothesized that in response to inflammation, such an increased utilization of amino acids originating from an overall increase in intestinal protein synthesis may limit the availability of those amino acids (threonine, serine, and cysteine) for the synthesis of intestinal mucins for which they are primarily or likely secondarily limiting (13,20). Similarly, proline, which is highly represented in the composition of mucins [
13%, (20,21) compared with body proteins (4–7%, except collagen)], could also become a secondary limiting amino acid for mucin synthesis in such pathologies. Such limitations in the dietary supply of these specific amino acids could explain the lack of a mucin adaptive response previously observed in IBD models (9,16,17,22,23).
It is not known whether dietary supplementation of these amino acids stimulates colonic mucin synthesis and improves epithelial protection and healing. Although reduced mucin production is often related to an alteration in the gut microbiota, the underlying mechanisms remain unclear. Nevertheless, changes in nutrient substrates or ecological niches of the endogenous bacteria are implicated. The effect of mucin synthesis on the growth and equilibrium of the commensal microbiota is not known.
The main goal of our study was to determine whether dietary supplementation of threonine, serine, cysteine, and proline stimulates mucin synthesis, influences the commensal microbiota, and restores gut barrier homeostasis in a well-recognized animal model of UC.
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MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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During an adaptation period of 8 d, rats from each group were fed their respective diets. Rats in the DSS, DSSM1, and DSSM2 groups were then administered 5% DSS (MW 36–44 kDa, ICN Biomedicals) in their drinking water for 9 d, followed by 2% DSS for a further 19 d (16). The control group received water. During the treatment, body weight, food intake, and stool characteristics (consistency, presence of blood) were recorded. On d 27, before feeding, a blood sample was collected from a lateral tail vein of each rat for assessment of plasma amino acid levels. The day before killing, freshly passed fecal samples from each rat were collected aseptically, suspended in 10% glycerol, frozen in liquid nitrogen, and then stored at –80°C until analysis of the microbiota.
At the end of the experiment, in vivo synthesis rates of total protein and mucins in intestinal tissues were measured using the flooding dose method (21,25), with minor modifications. Briefly, rats in each group received within 30 s a flooding dose injection of L-[1-13C]-valine (150 µmol/100 g body weight, 80 atom% excess, Eurisotop, group CEA) in a lateral tail vein. At 35, 38, 41, 44, 47, 50, 53, and 56 min after the injection, 1 rat/group was killed by exsanguination via the abdominal aorta under sodium pentobarbital (Sanofi) anesthesia. The small intestine and colon were quickly isolated, flushed with cold physiologic buffered saline, and gently dried. The small intestine was divided into the segments of the duodenum (proximal 10 cm), jejunum, and ileum (1/3 distal part). The length and weight of each segment was measured. Aliquots from the proximal parts of each segment were frozen in liquid nitrogen and stored at –80°C until required for analyses of protein synthesis, mucin content, and MUC2 mRNA expression. The mucosa from another aliquot was recovered by scraping, immediately frozen in liquid nitrogen, and stored at –80°C until analyses of mucin synthesis and amino acid composition. Finally, the remaining segments were fixed in Bouin's (Pioneer Research Chemicals) and then stored in 70% ethanol until required for analysis of DSS-induced damages and MUC2 protein expression.
Fecal microbiota. The fecal microbiota was analyzed quantitatively for Enterobacteriaceae, Bacteroides, Enterococci, Lactobacilli, and Bifidobacteria species using plating (26). The counts were expressed as log (base 10) cfu/g feces wet weight with a lower detection limit of 5.50 log cfu/g of feces for Bacteroides and 3.30 log cfu/g for other species.
Amino acid composition in plasma and intestinal mucins. The amino acid composition in plasma and purified intestinal mucins was analyzed as detailed previously (13,21).
Histological examination of DSS-induced damage and MUC2 expression. Fixed tissues were dehydrated, embedded in paraffin blocks, sectioned (5-µm thick), and stained with hematoxylin and eosin for evaluation of epithelial damage. The MUC2 protein expression was analyzed as described previously (27) using a MUC2-specific monoclonal antibody (WE9, kind gift of D. K. Podolsky, Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA).
Mucin content in intestinal mucosa. Mucins were purified from the small and large intestine of healthy rats, as described previously (21). An aliquot was used as a standard in the ELISA assay. The remainder was used to raise rabbit polyclonal antibodies (Eurogentec). The antiserum specificity was assessed by immunoblotting against intestinal homogenates from healthy and DSS-treated rats (data not shown). Pure mucins were used as positive controls, and preimmune antiserum as a negative control.
Intestinal tissues (100 mg) underwent the first steps of mucin purification (21). After the reduction and alkylation steps, the samples were dialyzed against deionized water (21). The protein content was assessed after both the homogenization and the dialysis steps (Bio-Rad dye reagent kit, Bio-Rad Laboratories). Dialyzed samples were diluted to 3 protein concentrations (0.2, 0.3, and 0.4 mg protein/L) in carbonate buffer (15 mmol/L Na2CO3 and 35 mmol/L NaHCO3, pH 9.6). The standard curve (rat mucins) ranged from 0 to 200 mg/L. Microtiter plates (NUNC-immunoplate) were coated with diluted samples (in duplicate) or pure mucins (in triplicate) and incubated overnight at 4°C. After washing with PBS-Tween20 0.05%, pH 7.2, binding sites were saturated with PBS-gelatin 0.5% (1 h at 37°C). Then, microplates were incubated with the anti-rat mucin antiserum (dilution 1:80,000, 2 h, 37°C) and, after washing, with an anti-rabbit peroxidase conjugate (dilution 1:40,000, 2 h, 37°C). The enzymatic reaction was started by adding Tetramethylbenzidine substrate and stopped 10 min later by adding 2 mol/L H2SO4. The optical density was measured at 450 nm against blank wells. A linear standard curve was obtained after logarithmic transformation of the mucin concentrations. The concentration of mucins in samples was calculated from the standard curve by linear regression. Data were expressed as milligrams mucin per liter of dialyzed samples and then correlated with the protein content and weight of the initial tissue. A standard rat intestinal sample was examined in all assays and used as an additional control.
Protein fractional and absolute synthesis rate in tissues and mucins. The synthesis rate of total mucosal proteins and mucins was measured as detailed previously (13,21,25). The fractional synthesis rate (FSR) of tissue proteins or mucins, defined as the percentage of mucosal proteins or mucins synthesized each day (%/d), was calculated as described previously (13). The absolute synthesis rate (ASR) of tissue proteins and mucins, defined as the quantity of proteins or mucins synthesized each day by the whole intestinal tissue (mg/d), was determined by multiplying FSR by total tissue protein or mucin content.
MUC2 gene expression. Total RNA extraction and RT-PCR were performed as detailed previously using specific primers for rat MUC2 and ß-actin as an internal standard (23). PCR products were quantified as described previously (13). The ratio between MUC2 and ß-actin was expressed in arbitrary units (AU).
Statistics. Data are presented as means ± SEM. One-way ANOVA was used to compare groups and repeated-measures ANOVA to compare variables over time. When significantly different at the 0.95 confidence level, a Least-Significant Difference multiple comparison procedure was performed. This classical analysis was then confirmed using a robust analysis [median, robust SD drawn from the "Sn" calculation of P. Rousseeuw et al. (28)].
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Clinical symptoms, food intake, body and tissue weight. The DSS-induced colitis was associated with loose stools that rapidly turned to diarrhea in all rats. Gross rectal bleeding was observed in all DSS-treated rats after d 7. At the macroscopic level, there was a colonic wall thickening with a significant increase in the colonic weight:length ratio (g/cm) in rats in the DSS (0.146 ± 0.06), DSSM1 (0.138 ± 0.007) and DSSM2 (0.146 ± 0.008) groups compared with controls (0.116 ± 0.008).
Compared with d 0, the daily food intake (g/d) of rats at d 9 was significantly reduced by 62% (DSS), 53% (DSSM1), 57% (DSSM2), and then returned to a normal control value 4 d after the switch from 5 to 2% DSS. Throughout the experiment, food intake and body weight evolution did not differ for the 3 DSS-treated groups. Rats in the DSS, DSSM1, and DSSM2 groups lost 11, 13, and 11% of their initial body weight, respectively (not significant), during the first 9 d of the experiment. They recovered poorly during the chronic phase of the colitis to attain 95% (DSS), 91% (DSSM1) and 93% (DSSM2) of their initial weight at the time of killing. In contrast, the body weight of control rats increased by 6% over the study period. Absolute weights of the different intestinal segments were not affected by DSS treatment or by the diets (data not shown). In contrast, relative weights of the segments were significantly increased in the DSS group compared with controls (Table 1). These weights did not differ from each other in the DSSM1 and DSSM2 groups or from the DSS group, and were significantly higher than that of controls.
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Fractional and absolute synthesis rates of intestinal mucins and tissue proteins. The DSS treatment had no effect on the protein fractional synthesis rate (FSR) or absolute synthesis rate (ASR) in the jejunum (data not shown), but it increased protein ASR in the ileum (Fig. 1, B) and both protein FSR and ASR in the colon (Fig. 1, A, C). Amino acid supplementation did not affect protein turnover in either the small or large intestine. The DSS treatment had no significant effect on the mucin FSR or ASR in any of the intestinal segments studied (Fig. 1, D–G, data not shown for jejunum). However, after amino acid supplementation, the colonic mucin FSR and ASR were increased (Fig. 1, E, G), in a dose-dependent manner for ASR, in rats of the DSSM1 and DSSM2 groups.
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Mucin AA composition. In all segments of the intestine, the mucins synthesized in rats of the DSS group had significantly reduced proline, serine, and threonine content compared with the control rats (Table 2). In the jejunum of DSSM1 rats, the mucin content of proline, serine, and threonine was significantly greater than that of DSS rats and did not differ from that of control rats. Mucins synthesized in the ileum and colon of DSSM1 rats had amino acid compositions similar to those of DSS rats. In contrast, colonic mucins of DSSM2 rats, which had significantly greater proline, serine, and threonine content than those of DSS rats, did not differ from controls (Table 2).
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In our experiment, DSS-treated rats showed features similar to those previously reported in the literature, i.e., bloody stools and diarrhea, reduction of food intake and body weight, colonic shortening and wall thickening, and colonic ulcerations with infiltration of inflammatory cells in the lamina propria (16,22,29,30). As expected (16), epithelial mechanisms of defense and tissue repair were initiated. More specifically, protein anabolism in the inflamed colon, and to a lesser extent in the healthy ileum, was observed. In contrast, as previously observed (22), no significant adaptation of the intestinal mucin response was detected, thus suggesting that mucin production could not be increased to promote mucus layer integrity and protection of the epithelium.
We thus hypothesized that after DSS-induced injury, the overall increased intestinal protein synthesis enhances the demand for specific amino acids that may not be adequately supplied by the diet. Because mucins contain a high level of threonine, and lesser amounts of serine, proline, and cysteine (20), their synthesis requires a high availability of threonine (13,19), and also of other amino acids (serine, proline and cysteine) that could be secondarily limiting (18). In DSS-mediated damage, these amino acids may be in limited supply and as such, the adaptive responses to counteract DSS injury may be compromised. In the present study, we tested this hypothesis by administering increasing doses of these specific amino acids to DSS-treated rats. The supplementation levels were clearly reflected in the plasma of rats. Although threonine, serine, and proline gradually accumulated in the plasma of the DSSM1 and DSSM2 groups, the cysteine level remained unchanged, suggesting either its increased utilization or its activated catabolism by DSS-treated rats. Both supplementation doses were well accepted by the rats but did neither appear to induce any improvement in clinical symptoms.
The 2 dietary doses exhibited different beneficial effects at the level of both specific intestinal segments and tissue compartments. In the small intestinal segments exempt from any sign of inflammation, the effect of amino acid supplementation on mucin and protein synthesis was weak. In the inflamed colon, the amino acid supplementation stimulated the mucin synthesis in a dose-dependent manner. The highest supplementation dose was the most efficient at improving the overall mucin status. More specifically, it strongly stimulated mucin synthesis, and globally restored the mucin mucosal content to control values, suggesting a replenishment of mucins in the damaged mucosa. The relative mucin amino acid composition varies from one mucin to another. MUC2 is particularly rich in threonine (>30%) compared with other intestinal mucins (13–20%), in part because of its high threonine content in repeated regions (31). Therefore, significant alterations in the relative expression level of each mucin will be reflected by changes in the measured amino acid composition of overall synthesized mucins. The reduced threonine content in mucins from the DSS group compared with controls likely reflects the synthesis of mucin types less rich in this amino acid, perhaps containing a decreased amount of MUC2. In contrast, the threonine content in mucins synthesized by DSSM2 rats was similar to that of controls, suggesting a qualitative restoration of the mucin, likely MUC2, relative expression profile. Together, these results clearly emphasize that a healthy balanced diet is not adequate in such situations of stress and may not provide optimal amounts of threonine, serine, proline, and cysteine for the establishment of defense and repair processes. Interestingly enough, mucosal tissue compartments were not modified by the amino acid supplementations in the same way. Indeed, the stimulation of colonic protein synthesis was specific to mucins because no such increase in the levels of total colonic protein was observed. There were no regulatory effects of these amino acids at the transcriptional level because mRNA levels of MUC2, the main secreted colonic mucin, were not affected by amino acid supplementation as already suggested in healthy rats (13). However, one cannot exclude differential regulation of the colonic mucins other than MUC2. The amino acids most likely affected mucin production at the translational level, i.e., the synthesis of the mucin protein backbone, which confirms our work hypothesis that the mucin synthesis would be limited by an insufficient dietary supply of specific amino acids during colitis.
In all types of intestinal aggression, innate mechanisms of protection and repair are initiated. Restitution, the initial phase of mucosal repair, is accomplished by rapid migration of the epithelium to reestablish surface epithelial continuity. Goblet cells play an important role in the protection and repair processes because they synthesize and secrete mucins and thus are responsible for the production and maintenance of the protective mucus layer (32). Increased mucin synthesis in inflamed intestinal tissues was reported previously in various animal models (19,23). A normal crypt ultrastructure was not restored in any supplemented group. However, the recovery of numerous MUC2-containing goblet cells in the surface epithelium of ulcerated areas in the DSSM2 rats corroborates the increased measured mucosal mucin synthesis and content and suggests the initiation of recovery/healing processes of the epithelium.
In addition to its protective function due to its chemical and physical properties, the mucus layer also functions as a dynamic defensive barrier. Indeed, it interacts closely with the commensal microbiota and is a key contributor to its maintenance and equilibrium (33). This frail homeostasis between mucus and the microbiota is destabilized in a variety of intestinal disorders, including IBD (4–8). In these patients, mucosal inflammation was shown to be associated with a reduction in the diversity of the microbiota (7). More specifically, a loss of anaerobic bacteria such as Bacteroides, Eubacterium, and Lactobacillus species was reported. Here, we show for the first time that a dietary supplementation of amino acids can promote the overall commensal microbiota growth in an animal model for UC, stimulating the 4 bacterial populations tested including the Bacteroides and Lactobacillus. A direct effect of a small amount of the supplemented amino acids on the microbiota cannot be excluded. Nevertheless, because the amino acids are absorbed mainly in the upper part of the small intestine, their effect on the microbiota was likely mediated via their stimulatory effects on mucin production. Therefore, acting on the host could offer a new approach to support the microbiota and the gut barrier function.
In conclusion, in all pathological situations, initiating intestinal defense and tissue repair processes increases the host's need of specific nutrients. A healthy control diet is clearly not adapted to such situations of stress. Increasing the dietary supply of threonine, serine, proline, and cysteine is a new approach to promote mucin production and a healthy microbiota and to improve epithelial protection and repair.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 5 December 2005. Initial review completed 27 December 2005. Revision accepted 19 March 2006.
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