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School of Dietetics and Human Nutrition, McGill University, Montreal, Quebec, Canada, H9X 3V9
* To whom correspondence should be addressed. E-mail: linda.wykes{at}mcgill.ca.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Improved gut epithelial barrier function has been suggested as a possible mechanism for beneficial effects of probiotics, but data from animal models are inconclusive (8–10). An alternate hypothesis suggests a complex series of molecular events initiated by stimulation of toll-like receptor 9 (11). Dotan and Rachmilewitz (12) proposed that modulation of nuclear factor 
B (NF-B) activation, induction of heat shock proteins mediated by proteasome inhibition, reduced proinflammatory cytokine production, and interference with bacterial adherence in the colon are all responsible for amelioration of IBD symptoms by probiotics. Also emerging from this hypothesis is the idea that these cellular and metabolic changes can be brought about even when nonviable bacteria or bacterial DNA or even secreted factors are administered. This raises questions about how probiotics or specific components of probiotic microbes exert extra-gut effects and systemic modulation of inflammatory processes, including the acute phase response.
Although clinical evidence for the use of probiotics in gastrointestinal diseases is strong, there is little information available about how probiotic use affects the tissues and metabolic processes beyond the gastrointestinal tract. Also, the metric used to qualify a complementary or alternative therapy, like probiotics, is usually against a standard pharmaceutical or lack of treatment for a specific condition. Therefore, comparing the effectiveness of probiotic use in IBD against aggressive nutrition support is a useful approach to qualifying any tangible benefits probiotics might offer in disease management.
Cytokine-induced catabolism in IBD is commonly observed and serves to supply the immune system with required amino acids, micronutrients, and energy needed for the acute phase response (13). Unfortunately, this catabolism is usually coupled with increased energy expenditure (14) and concurrent reduced food intake due to anorexia, food avoidance, and pain (15). Previously, we have shown how skeletal muscle protein synthesis is reduced whereas synthesis of hepatic proteins and acute phase response proteins are increased 2-fold in piglets with macronutrient restriction and short-term acute colitis (16). Because fasting for several days without inflammation in itself can reduce gut integrity and mucosal cell metabolism, fasting during IBD would be expected to compromise protein synthesis and exacerbate damage and inflammation (17). Chronic protein deficiency suppresses gut protein synthesis (18) and when inflammation is superimposed, gut synthesis of the crucial antioxidant glutathione is also decreased (18,19). Enteral nutrition support has emerged as an important component of clinical therapy in pediatric IBD (20,21); therefore, determining the impact of aggressive nutrition support during IBD on visceral protein kinetics and inflammation compared with healthy and diseased controls would provide a metabolic accounting of its benefit as part of the treatment of the disease.
The emergence of complementary therapies for IBD like probiotic supplementation are gaining prominence despite a lack of consensus regarding their mechanism of action and efficacy, as well as their potential effects on nutritional status and metabolism. Approaches that focus on improving nutritional status may be more beneficial for controlling symptoms of the disease while at the same time improving overall nutritional status and health. We hypothesized that aggressive nutritional support is more effective in reducing IBD severity and maintaining protein synthesis rates than probiotic treatment alone. Therefore, our objective, using a piglet model of colitis, was to determine the impact of probiotic supplementation or aggressive nutrition support on synthesis of gut- and liver-derived proteins as well as disease status indicators of colon histopathology and oxidative stress in piglets with moderate macronutrient restriction.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Immediately after piglets were removed from the sow (d 1), catheters were implanted aseptically under isoflurane anesthesia into the femoral vein for infusion of tracers, jugular vein for blood sampling, bladder for urine sampling, and the stomach for diet and DS administration (16,22). On d 2, probiotic supplementation was started. On d 4, DS administration was started and continued for 10 d. Finally, 14 d after surgery, a stable isotope infusion study was conducted to determine protein synthesis rates. Blood was sampled at baseline and throughout the infusion and tissues were sampled immediately after an i.v. injection of Euthansol (sodium pentobarbital; Schering Canada). The study protocol was approved by the McGill University Animal Care Committee in accordance with the Canadian Council on Animal Care Guidelines.
DS-induced colitis model. Our piglet model of DS-induced colitis, originally described by Mackenzie et al. (16), was modified by decreasing the dose and increasing the duration of DS administration. Briefly, a DS solution (200 g/L, 40,000 molecular weight; ICN Biomedicals) was administered through the gastric catheter twice daily at a dose of 1 g/(kg·d). Feces were tested after 5 d for occult blood with Hemoccult test packs (Beckman Coulter) to confirm the presence of colitis.
Probiotics. MRP piglets received 450 x 109 colony-forming units (CFU)/d of the bacterial mixture VSL#3 (VSL Pharmaceuticals), equivalent to 1 VSL#3 packet/d suspended in 30 mL of liquid diet and delivered twice daily as a 15-mL bolus.
Diet. All piglets received a liquid diet designed to meet the following requirements. Piglets in both MR and MRP groups were supplied (Table 1) with a diet providing 50% of the required macronutrients for a growing piglet, while piglets in the WNC group received a nutritionally adequate diet for growing piglets (23,24). Diets were infused over a 16-h period (Compat Enteral Feeding Pump; Novartis Nutrition) via the gastric catheter to achieve the metabolizable energy intake of 925 kJ/(kg·d) for WNC piglets and 461 kJ/(kg·d) for both macronutrient-restricted groups, based on each piglet's daily weight.
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Protein analysis. Total protein and albumin concentrations were determined by automated biochemistry analyzer (Hitachi 911, Hitachi America). Plasma fibrinogen concentration was measured using photometric assay at the clinical laboratory of McGill University Health Centre (25).
Total protein in plasma was isolated by precipitation with ice-cold 0.6 mol/L trichloroacetic acid and processed as reported previously (16). To isolate fibrinogen, an ethanol/saline (1:8) solution was added to plasma (4:1) and placed in an ice bath for 15–30 min. Samples were then centrifuged at 1500 x g; 10 min at 4°C. The resulting fibrinogen pellet was dissolved in SDS-PAGE sample buffer without β-2-mecaptoethanol. To isolate albumin, proteins were precipitated from the fibrinogen-free supernatant with trichloroacetic acid and centrifuged. The supernatant was discarded and albumin resolubilized by vortexing in 95% ethanol. The final supernatant containing the dissolved albumin was mixed 1:1 with SDS-PAGE reducing sample buffer. Both fibrinogen and albumin were resolved separately by SDS-PAGE on a MINI-PROTEAN II System (Bio-Rad Laboratories).
Tissue-free and protein-bound amino acids were isolated and converted to their n-propyl ester heptafluorobutyramide derivatives (16). Tracer:tracee ratios were determined using raw ion abundances and analysis of the tracer and natural abundance of phenylalanine. Isotopic steady state was confirmed between h 3 and 6. The fractional synthesis rate (FSR) for tissue and plasma proteins was calculated using the precursor:product relationship and the intravascular absolute synthesis rate (ASR) for plasma proteins was calculated using the equations previously reported (16).
Oxidative stress markers.
Myeloperoxidase (MPO) activity, a common indicator of neutrophil infiltration and oxidative stress, was assayed on colon tissue samples using the method of Bradley et al. (26). Ferric-reducing ability of plasma (FRAP), a measure of total reducing or antioxidant capacity, was determined based on Friel et al. (27) with modifications for use with a microplate spectrophotometer. Urinary 15-isoprostane F2t concentrations were measured on urine directly sampled from the bladder through the catheter and assayed by competitive ELISA (Oxford Biomedical Research) specific to 15-isoprostane F2t (also known as 8-epi-PGF2
or 8-iso-PGF2). Concentrations were expressed per mmol creatinine, which was determined spectrophotometrically by a creatinine assay kit (Oxford Biomedical Research). Copper and zinc concentrations were determined after appropriate dilution with 0.1 mol/L HNO3 by atomic absorption spectrophotometry (Perkin-Elmer 3100 AAS; Perkin-Elmer Life Sciences) using the method of Makino and Takahara (28).
Histology and immunohistology. Segments of colon were fixed immediately in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin using standard slide preparation techniques. Each section was then examined by 2 investigators unaware of the treatments and graded for histological damage using the scale published by Geboes et al. (29). KI-67 expression, a measure of cell proliferation, was assessed using the KI-67 antigen kit from Vector Laboratories and expressed as percent of crypt and surface epithelial cells which stained positive for KI-67 antigen. Cellular apoptosis was quantified using the in situ death detection kit from Roche (Roche Life Science).
Statistical analysis.
Values in the text and tables are expressed as means ± pooled SEM. Statistical analysis was conducted using SPSS version 11.0 and differences were considered significant at P 
0.05. Hematocrit and protein and oxidative stress variables were analyzed between WNC, MR, and MRP groups only by general linear model univariate ANOVA with CI adjustments using Bonferroni correction and Bonferroni post hoc analysis. All histological outcomes were assessed by Kruskal-Wallis H and Mann-Whitney U tests for post hoc analysis. Oxidative stress and histological outcomes of the colitic piglets were compared independently to the REF piglet by independent Student's t test and Mann-Whitney U tests, respectively. The proportional distribution of the histological values was assessed by chi-square analysis.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Protein synthesis and plasma protein concentrations.
Concentrations of total plasma protein, albumin, and fibrinogen in plasma of REF piglets were 36, 21, and 1.3 g/L, respectively. Neither concentration nor synthesis rate of any plasma protein differed between the REF and WNC piglets. Liver FSR was 100% higher in well-nourished piglets and MRP piglets compared with MR piglets (Table 2). However, whereas the ANOVA met the significance criteria of P < 0.05, the Bonferroni post hoc comparisons did not (WNC vs. MR, P = 0.08; MRP vs. MR, P = 0.06). Colon (mucosa and underlying tissue) FSR was 
50% higher in well-nourished piglets compared with both MR and MRP piglets. Ileal mucosa FSR did not differ among the groups.
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Fibrinogen. The positive acute phase protein fibrinogen demonstrated a similar, but not significant (P = 0.07), pattern for FSR compared with that of the total plasma protein pool (Table 3). Nonetheless, ASR was higher for MRP (P = 0.03) piglets than MR piglets. ASR for WNC piglets was intermediate and did not differ from the other groups.
MPO activity. MPO activity did not differ between REF and WNC piglets but was higher in both MR and MRP piglets (P < 0.05) than in the REF group (Table 4). Neither adequate nutrition nor VSL#3 administration decreased MPO activity compared with the MR piglets.
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50% compared with both MR and MRP piglets. Interestingly, 2 of the 8 piglets from the MRP group had histological scores <2, which were in the range of the REF group, whereas none from the MR group had scores <2.2. However, the groups did not significantly differ when assessed by 
2 analysis (P = 0.29). Cell numbers followed a reciprocal pattern to that of the damage scores, where the REF group had more cells per intact crypt than the piglets with colitis (Table 4; P < 0.001). Despite having fewer cells per intact crypt than the REF piglets, WNC piglets had crypts with more cells than either the MR (P = 0.01) or MRP group (P = 0.05). Goblet cells, as a percentage of total crypt cells, did not differ among the groups. Apoptosis rates were higher for both MR (P = 0.01) and MRP (P = 0.006) than for the REF piglets, whereas the WNC group was intermediate and did not differ from either group (Table 4). The rate of cell proliferation for the WNC group (Table 4; P = 0.04) was lower than the combined macronutrient-restricted colitis groups.
Systemic oxidative stress indicators. At d 14, FRAP and urinary F2-isoprostane concentrations did not differ among the groups. However, the plasma copper:zinc molar ratio was higher in the MR piglets than in the WNC (P = 0.005) and REF piglets (P = 0.006), indicating higher systemic oxidative stress. Interestingly, the copper:zinc ratio for the MRP group was intermediate and did not differ from any other group.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Probiotics did not reduce disease severity during macronutrient restriction in our piglet colitis model. Neither colon protein synthesis rates nor histological damage differed from MR piglets despite others reporting a probiotic-induced reduction in MPO activity and histological damage (8,32). Using a DS colitis model in mice, Rachmilewitz et al. (11) observed reductions in both MPO activity and histological damage in response to administration of both live and nonviable preparations of bacteria in VSL#3. Furthermore, Di Giacinto et al. (33) demonstrated the effectiveness of VSL#3 in ameliorating reoccurring histological damage in rats with trinitrobenzenesulfonic acid-induced colitis. Likewise, Madsen et al. (9) showed a decrease in histological damage in interleukin (IL)-10–deficient mice administered VSL#3. To our knowledge, this is the first study to use probiotics in piglets, a mongastric with 90% gut homology to humans (34). It is possible that our dose of probiotics was too low to be effective in reducing histological damage or inflammation. Our dose of 1 packet of VSL#3 or 450 x 109 CFU in a 5-kg piglet was based on a typical dose for the IL-10–deficient mouse model (0.28 x 109 CFU) (9) and the high doses used in human trials (1800 x 109 CFU) (4) and calculated to be relatively consistent when based on metabolic body weight (kg0.73). Alternatively, the robust nature of the DS-induced colitis used in our model coupled with the malnutrition could have overwhelmed any protective effect of the probiotic treatment. We chose to study probiotic therapy in the macronutrient-restricted state, because this tends to represent the clinical course; however, their use in combination with aggressive nutrition support might yield a more effective reduction in disease severity than even nutrition alone.
Unlike many studies of probiotic use in IBD management, we have explored effects beyond the gastrointestinal tract. Changes in hepatic protein synthesis have been reported in both animal and human studies of gastrointestinal inflammation (16,35,36), yet to our knowledge, the relationship among probiotics, IBD, and hepatic protein metabolism has not been studied. The piglets receiving probiotics demonstrated a 1-fold increase compared with the malnourished colitic piglets in liver protein synthesis as well as synthesis of hepatically derived total plasma protein, albumin, and fibrinogen. This dramatic increase in plasma protein FSR was accompanied by a relatively modest change in concentration of these proteins in plasma. Because concentration in the plasma or intravascular pool represents a balance among entry and removal, increased synthesis without a change in concentration suggests increased losses. Whether these losses are terminal due to proteolysis or to loss through the damaged colon (37) or whether they represent increased pool size due to increased capillary permeability and escape into the interstitial space cannot be determined with this tracer approach. Using plasma protein concentration and plasma volume to calculate ASR likely underestimates actual ASR, especially if losses are high. Low molecular weight proteins, like albumin, are particularly vulnerable to this sort of extravascular loss (38) and consequent underestimation of total ASR. These kinetic data illustrate how stable isotope approaches can identify changes in metabolic processes that may not be evident using only static endpoints. For example, albumin is generally regarded as a negative acute phase protein, because its plasma concentration declines in response to inflammation. Yet the use of stable isotope techniques to measure the FSR of albumin has shown that synthesis actually increases dramatically during this and other inflammatory states (16,39). Stable isotope techniques would complement other experimental outcomes currently used for studying nutrition and metabolism in IBD as they have in surgical research (40–42), helping to describe changes in metabolism of macronutrients caused by both catabolic stress and nutritional interventions.
Several studies reporting the use of probiotics to treat liver disease support our current findings. Loguercio et al. (43) examined how probiotics (VSL#3) affect oxidative stress markers, select plasma cytokine levels, and standard clinical outcomes in patients with nonalcoholic fatty liver disease, alcoholic liver cirrhosis, or hepatitis C virus (with and without cirrhosis). Long-term treatment with probiotics (90 or 120 d) decreased circulating liver enzymes by 50%, illustrating a direct beneficial effect on liver function (43). Similar to our findings, they observed a probiotic-induced increase in total plasma protein concentrations of alcohol-induced cirrhosis patients. The similarities between liver and liver-derived plasma protein synthesis rates in our study and increases in circulating plasma protein concentrations in the cirrhotic patients may be related to changes in cytokine profiles, which stimulate the acute phase response. Loguercio et al. (43) also reported that probiotic treatment decreased circulating levels of tumor necrosis factor- (TNF) and IL-6 while concurrently increasing circulating levels of IL-10. Although the authors offered no explanation for the mechanism of the hepatic effects, their findings of a stimulation of liver protein metabolism are supported by our data and confirm data from others showing a reduction of proinflammatory markers (9,33,44).
Furthermore, in a nonalcoholic steatohepatitis model in ob/ob mice, VSL#3 administration was as effective as anti-TNF antibodies in reducing serum liver enzymes, hepatic Jun N-terminal kinase activity (TNF-stimulated pathway), and NF-B activity (45). This study also supports our findings of extraintestinal effects of probiotic administration, specifically targeting the liver and acute phase proteins. Although a mechanism has yet to be determined, there appears to be a link between probiotic administration and circulating cytokines (decreased JNK activity, decreased circulating levels of TNF and IL-6 with increased circulating IL-10) and decreased cellular proinflammatory response as measured by a decreased NF-B activity and increased hepatic protein production (constitutive and secretory). Stimulation of toll-like receptor 9 by probiotics in the gut may be the initiating factor, although this requires further study (11,12).
In summary, these data illustrate how maintaining adequate nutrition during DS-induced colitis improves protein synthesis rates and histological outcomes. Despite the clear differences between this experimental model and the clinical disease, our findings do point to favorable outcomes associated with attention to maintaining adequate nutritional status during IBD as opposed to macronutrient restriction through anorexia and food avoidance. Moreover, although probiotics did not have a substantial effect on weight gain, colon protein synthesis rates, or histological indices, their use in combination with other therapies needs more study. The most novel aspect of this data was identifying a clear link between probiotic administration during colitis and increased liver and plasma protein synthesis rates in a malnourished state. This increase in hepatic and plasma protein synthesis with only a modest change in plasma concentrations poses a new question: How and why does probiotic administration in a macronutrient-restricted state stimulate hepatic protein synthesis? Furthermore, this increase in protein synthesis could be interpreted as being both advantageous, if the increase in protein synthesis was in some way directed toward controlling the underlying inflammation, and equally disadvantageous if the sustained higher rates of protein synthesis result in increased catabolism of other protein pools to supply the synthetic precursors.
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FOOTNOTES |
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2 Author disclosures: S. Harding, K. Fraser, and L. Wykes, no conflicts of interest.
3 Present address: Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Manitoba, Canada R3T 6C5.
4 Present address: Centre de Recherche du le Centre Hospitalier de l'Université de Montréal, Montreal, Quebec, Canada H2W 1V1.
5 Abbreviations used: ASR, absolute synthesis rate; CFU, colony-forming unit; DS, dextran sulfate; FRAP, ferric-reducing ability of plasma; FSR, fractional synthesis rate; IBD, inflammatory bowel disease; IL, interleukin; MPO, myeloperoxidase; MR, macronutrient-restricted piglets; MRP, macronutrient-restricted piglets with probiotics; NF-B, nuclear factor B; REF, healthy reference piglets; TNF, tumor necrosis factor-; WNC, well-nourished with colitis.
Manuscript received 13 March 2008. Initial review completed 5 May 2008. Revision accepted 24 August 2008.
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LITERATURE CITED |
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