The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Dietary Sulfur Amino Acid Supplementation Reduces Small Bowel Thiol/Disulfide Redox State and Stimulates Ileal Mucosal Growth after Massive Small Bowel Resection in Rats1,2 Yvonne Shyntum,3,5 Smita S. Iyer,4,5 Junqiang Tian,4,5 Li Hao,5 Yanci O. Mannery,3,5 Dean P. Jones,4–6 and Thomas R. Ziegler4–6* 3 5
Graduate Program in Molecular and Systems Pharmacology, 4Graduate Program in Nutrition and Health Sciences, Department of Medicine, and 6Center for Clinical and Molecular Nutrition, Emory University, Atlanta GA 30322
Abstract Following massive small bowel resection in animal models, the remnant intestine undergoes a dynamic growth response termed intestinal adaptation. Cell growth and proliferation are intimately linked to cellular and extracellular thiol/disulfide redox states, as determined by glutathione (GSH) and GSH disulfide (GSSG) (the major cellular redox system in tissues), and cysteine (Cys) and its disulfide cystine (CySS) (the major redox system in plasma), respectively. The study was designed to determine whether dietary supplementation with sulfur amino acids (SAA) leads to a greater reduction in thiol/ disulfide redox state in plasma and small bowel and colonic mucosa and alters gut mucosal growth in an established rat model of short bowel syndrome (SBS). Adult rats underwent 80% jejunal-ileal resection (RX) or small bowel transection (surgical control) and were pair-fed either isonitrogenous, isocaloric SAA-adequate (control) or SAA-supplemented diets (218% increase vs. control diet). Plasma and gut mucosal samples were obtained after 7 d and analyzed for Cys, CySS, GSH, and GSSG concentrations by HPLC. Redox status (Eh) of the Cys/CySS and GSH/GSSG couples were calculated using the Nernst equation. SAA supplementation led to a greater reduction in Eh GSH/GSSG in jejunal and ileal mucosa of resected rats compared with controls. Resected SAA-supplemented rats showed increased ileal adaptation (increased full-thickness wet weight, DNA, and protein content compared with RX control-fed rats; increased mucosal crypt depth and villus height compared with all other study groups). These data suggest that SAA supplementation has a trophic effect on ileal adaptation after massive small bowel resection in rats. This finding may have translational relevance as a therapeutic strategy in human SBS. J. Nutr. 139: 2272–2278, 2009.
Introduction Short bowel syndrome (SBS)7 is a major cause of intestinal failure and clinical conditions leading to SBS span all age groups (1,2). SBS occurs after massive resection of the small intestine and is characterized by malnutrition and marked loss in body weight due to malabsorption of fluids and nutrients (1,2). Following resection, the remnant intestine in animal models 1
Supported by NIH grants R01 DK55850 and K24 RR023356 (T.R.Z.) and R01 ES011195 (D.P.J.). 2 Author disclosures: Y. Shyntum, S. S. Iyer, J. Tian, L. Hao, Y. O. Mannery, D. P. Jones, and T. R. Ziegler, no conflicts of interest. 7 Abbreviations used: Con, control diet; Cys, cysteine; CySS, cystine; EGF, epidermal growth factor, GSH, glutathione; GSSG, glutathione disulfide; PLSD, protected least significant difference; RX, jejunal-ileal resection; RX-Con, resectioned rats fed the control diet; RX-Suppl, resectioned rats fed the SAA-supplemented diet; NAC, N-acetylcysteine; Eh, redox status; RX, resection; SBS, short bowel syndrome; SAA, sulfur amino acid; Suppl, SAA-supplemented diet; TX, transected; TX-Con, transected rats fed the control diet; TX-Suppl, TX rats fed the SAA-supplemented diet. * To whom correspondence should be addressed. E-mail:
[email protected].
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undergoes a dynamic growth response termed intestinal adaptation, which is characterized by epithelial cell hyperplasia and increased villus diameter, height, and crypt depth (3). Consequently, interventions directed toward improving intestinal adaptation in SBS patients are of immense therapeutic interest. Several lines of evidence show that the biological thiol/ disulfide redox environment functions in the regulation of cell growth (4–6). Cysteine (Cys) and its disulfide cystine (CySS), together with glutathione (GSH) and GSH disulfide (GSSG), comprise the major low-molecular weight thiol/disulfide control systems (6,7). These systems are compartmentalized; GSH/ GSSG comprises the major cellular redox system in tissues, whereas the precursor amino acid Cys and CySS predominate in the plasma (7). In a variety of cell lines, including intestinal epithelial cells, changes in cellular GSH/GSSG redox state (Eh) correlate with the biological status of cells; proliferating cells exhibit a greater reduction in Eh values (i.e. more negative Eh in mV) compared with differentiating or apoptotic cells that have more oxidized
0022-3166/08 $8.00 ã 2009 American Society for Nutrition. Manuscript received February 13, 2009. Initial review completed March 13, 2009. Revision accepted September 29, 2009. First published online October 14, 2009; doi:10.3945/jn.109.105130.
Eh values (i.e. less negative Eh mV) (6,8,9). In contrast, we recently found that more oxidizing Eh is associated with cell proliferation in rat colonic mucosa in vivo (10). In cell lines in vitro, depletion of cellular GSH and associated oxidation of GSH/GSSG Eh inhibits cell proliferation, which is rescued by the supply of exogenous GSH (11). Similar observations have been made with extracellular Eh Cys/CySS. Studies in a human colon carcinoma cell line showed that cell proliferation increased as a function of a greater reduction in Eh Cys/CySS and addition of growth factors, such as keratinocyte growth factor, insulin-like growth factor-I, epidermal growth factor (EGF), or the amino acid glutamine, to the medium led to a greater reduction in extracellular Eh Cys/CySS (4,5). Studies in patients with intestinal pathologies predisposed to SBS have demonstrated compromised redox states. Decreased levels of GSH and increased GSSG levels have been observed in the ileal mucosa of patients with Crohn’s disease (12), with more severe GSH depletion in malnourished patients (13). Patients with inflammatory bowel disease also had compromised mucosal GSH levels and markedly low plasma Cys levels (14). In addition, in a rat model of experimental colitis, a direct association between GSH depletion and mucosal injury was observed and oral treatment with N-acetylcysteine (NAC), a Cys precursor, attenuates acute colitis by increasing mucosal GSH levels (15). In a recent study in intact rats, we showed that inadequate dietary sulfur amino acid (SAA) intake oxidized Cys/ CySS redox in plasma and GSH/GSSG redox in small intestinal and colonic mucosa; SAA supplementation resulted in a greater reduction in plasma Cys/CySS redox potential (16). Taken together, the data support the concept that improving the small intestinal thiol/disulfide redox state may have therapeutic benefits in SBS. The purpose of the present study was to determine in a rat model of SBS following massive small bowel resection if dietary supplementation with SAA leads to a greater reduction in thiol/disulfide redox state in the plasma and jejunal, ileal, and colonic mucosa whether SAA supplementation is associated with improved indices of gut mucosal adaptive growth.
Materials and Methods Rats. Male Sprague-Dawley rats (Charles River Laboratories) weighing 200–250 g were housed in individual cages in the animal care facility under controlled conditions of temperature and humidity with a 12-hlight/-dark cycle. Rats were given free access to water and were initially fed standard pelleted rat food (Laboratory Rodent Chow 5001, PMI Feeds) during a 7-d acclimation period. The study protocol was approved by the Institutional Animal Care and Use Committee of Emory University. Experimental diets. Semipurified diets were custom prepared (HarlanTeklad) to test the specific effects of SAA supplementation. The control and SAA-supplemented diets were isocaloric, isonitrogenous, and contained adequate and identical quantities of energy, nitrogen, carbohydrate, fat, fiber, micronutrients, and essential amino acids (with the exception of methionine), as previously described in detail (16). The SAA and nitrogen content were controlled at the desired experimental levels by varying the amount of the SAA (L-cystine and L-methionine) and the nonessential amino acids, L-alanine, L-aspartic acid, glycine, and L-serine. The SAA-supplemented diet contained 300% of the cystine content and 183% of the methionine content of the control diet (218% of control diet cystine+methionine content) (16). Daily consumption of each of the experimental diets was subsequently confirmed in pilot studies, indicating apparent palatability (data not shown). The experimental diets used were designed in collaboration with scientists at Harlan-Teklad based on prior experience with SAA alterations in
chemically defined diets (16), recognizing that glycine forms part of GSH and L-serine is involved in the synthesis of L-Cys from L-methionine. Experimental design and procedures. A total of 35 rats were divided by body weight into 4 groups: sham-operated, transected (TX) rats fed the control diet (TX-Con; n = 9); TX rats fed the SAA-supplemented diet (TX-Suppl; n = 7); massive small bowel resection (RX) rats fed the control diet (RX-Con; n = 9); and RX rats fed the SAA-supplemented diet (RX-Suppl; n = 10). Rats were food deprived overnight before surgery. The following morning (d 1), rats underwent identical laparotomy procedures and either small bowel TX with reanastomosis or 80% mid-jejuno-ileal RX with jejunoileal in-continuity reanastomosis, as previously described (10,17). Rats were anesthetized with intraperitoneal ketamine (100 g/L) and xylazine (20 g/L). In the RX rats, 80% of the jejunum and ileum was removed using defined landmarks (from 10 cm distal to the ligament of Treitz to 10 cm proximal to the ileocecal junction). In TX rats, the small intestine was TX at a point 10 cm proximal to the ileocecal junction and anastomosed (10). After surgery, rats consumed water ad libitum and the pelleted study diets were provided on the morning of d 2. Because food intake affects blood Cys concentrations (18,19), rats in the TX-Con, TX-Suppl, and RX-Con groups were pair-fed to daily food intake of the RX-Suppl group. Given the expectation that the RX-Suppl group would eat less than the other groups, to optimize pair-feeding, surgery was performed on the RXSuppl group 1 d prior to the other 3 study groups. Food intake was measured daily in the RX-Suppl group and the same amount of food was provided to rats in each of the 3 study groups. Body weight and food intake were measured daily in all 4 treatment groups. Tissue collection. Seven days following the operation, the rats were killed by exsanguination in the morning after overnight food deprivation. Jejunum, ileum, and colon were stripped of mesenteric and vascular connections and sequentially removed from the peritoneum. The lumen was flushed with ice-cold saline to clear intestinal contents and suspended from a ring stand with a constant distal weight. The defined segments used for the endpoints of this study were collected sequentially at the exact anatomical site in each RX or TX rat. The segments used for measurement of mucosal thiols and disulfides were cut longitudinally and the mucosa was obtained by gentle scraping with a glass slide. The mucosa was immediately placed in liquid nitrogen and stored frozen at 2808C until later processing. Blood was drawn by cardiac puncture and processed as previously described (16), after which plasma was obtained by centrifugation and stored at 2808C pending further processing. Histology. The defined segments of jejunum, ileum, and colon were fixed with 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Crypt depth in all tissues and villus height in ileum and jejunum were measured using an Olympus BH-2 light microscope equipped with a calibrated ocular micrometer. Classical morphologic criteria were used to discriminate between the crypt and villus zones (10). At least 10–25 well-oriented crypt-villi units per small intestinal sample and 20 crypts per colon sample were measured and averaged by experienced observers who were unaware of treatment group (L. Hao and J. Tian). Intestinal wet weight, DNA, and protein content. Defined 2-cm segments of full thickness jejunum, ileum, and colon were weighed and homogenized in ice-cold buffer (50 mmol/L PBS, 2 mol/L NaCl, 2 mmol/L EDTA). DNA content per cm was determined by a fluorimetric method (20) and the protein content per cm was determined by the Bradford method (21). GSH, GSSG, Cys, and CySS determination. Mucosal samples were treated with ice-cold 5% (wt:v) perchloric acid containing 0.2 mol/L boric acid and 10 mmol/L g-glutamyl-glutamate (internal standard), and precipitated tissue proteins were separated from the acid-soluble supernatant by centrifugation. The protein pellet was resuspended in 1 mol/L NaOH and protein concentrations were determined using the Bradford method with rabbit g-globulin as the protein standard (Biorad Laboratories). Intracellular GSH and GSSG levels were determined Sulfur amino acids after small bowel resection
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following treatment of the supernatant with iodoacetic acid followed by dansyl chloride. For HPLC analysis, the derivatized samples were separated as previously described (22) on a Supercosil LC-NH2 column (5 mm, 4.6 3 25 cm; Supelco) with the use of Waters 2690 HPLC and autosampler system (Waters) at 228C. Detection was obtained by fluorescence using bandpass filters (305–395 nm excitation, 510–650 nm emission; Gilson Medical Electronics). The thiols were quantified by integration relative to the internal standard and expressed as nmol/mg protein. Conversion to molar values was obtained using 5 mL cell volume/mg protein. To obtain Cys and CySS concentrations in plasma, samples were treated with an equal volume of 10% (wt:v) perchloric acid containing 10 mmol/L g-glutamyl-glutamate. Extracts were centrifuged to remove precipitated protein and the supernatants were processed as described above. Eh calculations. The tissue and plasma Eh of the GSH/GSSG and Cys/ CySS pools were calculated in mV from measured concentrations of GSH, GSSG, Cys, and CySS in molar units with the following terms in the Nernst equation for pH 7.4 in rodent tissues (23): GSH/GSSG, Eh = 2264 + 30 log ([GSSG]/[GSH]2), and a pH 7.4 in plasma Cys/CySS, Eh = 2250 + 30 log [(CySS)/(Cys2)] (22). A greater reduction in Eh values is signified by mV values that are more negative; conversely, more oxidized Eh values are signified by mV values that are less negative (i.e. closer to zero). Statistical analysis. Statistics were conducted using SPSS. Data are presented as means 6 SEM. The study was arranged in a 2 3 2 factorial design with small bowel resection and SAA intake as the main effects. Two-factor ANOVA was used analyze for the main effects and interactions between these. Post-hoc Fishers protected least significant difference (PLSD) tests were used to compare each possible pair of groups analyzed in the ANOVA individually to determine which groups differed significantly from one another. To simplify the presentation of significant differences, the figure symbols denote only significant Fisher PLSD differences between groups; 2-factor comparisons are denoted in the figure legends. We used Pearson correlation coefficients to determine associations between mucosal Eh GSH/GSSG and intestinal markers of adaptation. Significance was set at P , 0.05 for all tests.
FIGURE 1 Effect of dietary SAA supplementation on plasma Eh Cys/CySS in TX and resected rats. Values are the mean 6 SEM, n = 7–10.
Ileum. There was a main effect of both RX and SAA that reduced ileal GSH redox potential (P , 0.05) without interaction. Eh GSH/GSSG responses in the ileal mucosa were similar to those in jejunal mucosa. SAA supplementation in RX animals resulted in a significantly greater reduction in Eh GSH/GSSG in ileal mucosa (~10 mV) compared with the RX-Con and TX groups (Fig. 2B). Ileal mucosal Eh GSH/GSSG did not differ between the TX-Con, TX-Suppl, and RX-Con groups.
Results Postoperative food intake and body weight Food intake was measured daily during the experimental period and averaged ~12 g/d in all study groups. Food intake was similar across the 4 treatments, demonstrating successful pairfeeding (not shown). Mean body weight was similar between the treatment groups at the beginning of the experiment (data not shown). Weight gain did not differ between the 2 TX groups and RX-Con rats (TX-Con, +2.1 6 0.2 g/d; TX-Suppl, +2.2 6 0.2 g/d; RX-Con, +1.7 6 0.1 g/d; and RX-Suppl, +3.1 6 0.3 g/d). Effect of dietary SAA supplementation on plasma Eh Cys/CySS There were no significant main effects of RX and SAA or interactions between these on plasma Eh Cys/CySS (Fig. 1), suggesting that resection or SAA alone did not induce changes in plasma Eh Cys/CySS in this model. Effect of dietary SAA supplementation on gut mucosal Eh GSH/GSSG Jejunum. There was a main effect of both RX and SAA that reduced jejunal GSH redox potential (P , 0.05), without interaction, by 2-factor ANOVA. Dietary SAA supplementation led to a significantly greater reduction in Eh GSH/GSSG (~10 mV) in the jejunal mucosa of RX-Suppl rats compared with the other 3 groups (Fig. 2A). Jejunal mucosal Eh GSH/GSSG did not differ between the TX-Con, TX-Suppl, and RX-Con groups. 2274
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FIGURE 2 Effect of dietary SAA supplementation on gut mucosal Eh GSH/GSSG in TX and resected rats in jejunum (A), ileum (B), and colon (C). Values are the mean 6 SEM, n = 7–10. Significant effects (P , 0.05) were SAA (A) and RX (B). Means with superscripts without a common letter differ, P , 0.05.
Colon. Measurement of colonic mucosal Eh GSH/GSSG revealed no significant main effects of surgery or SAA supplementation on GSH/GSSG redox state (Fig. 2C). Effect of dietary SAA supplementation on full thickness intestinal growth indices Full-thickness wet weight, DNA content, and protein content were determined in defined segments of intestine (Table 1). RX rats in both control and SAA-supplemented dietary groups exhibited a significant adaptive growth response in residual jejunum and ileum and, to a lesser extent, in colon compared with TX rats. Two-factor ANOVA analysis revealed a main effect for RX to increase all 3 of these growth indices in jejunum, ileum, and colon (P , 0.05), but an interaction between RX and SAA supplementation occurred for each index only in ileum (P , 0.05) (Table 1). SAA supplementation in RX animals resulted in a significantly greater increase in ileal wet weight (+20%), DNA (+34%), and protein content (+22%) than in the RX-Con group (Table 1). Regression analysis revealed an association between both DNA (r2 = 20.48; P = 0.012) and protein content (r2 = 20.48; P = 0.012) and Eh GSH/GSSG in the ileum; the greater reduction in (negative mV) Eh GSH/GSSG was positively associated with both DNA and protein content. No effects of SAA supplementation on these markers of intestinal adaptation occurred in either jejunum or colon (Table 1).
FIGURE 3 Effect of dietary SAA supplementation on crypt depth in jejunum, ileum, and colon in TX and resected rats. Values are the mean 6 SEM, n = 7–10. Significant effects P , 0.05 were: in jejunum, RX; in ileum, RX and RX 3 SAA. Means with superscripts without a common letter differ, P , 0.05.
Effect of dietary SAA supplementation on intestinal mucosal crypt depth and villus height We determined indices of gut mucosal adaptive growth in response to 80% small bowel resection, with or without dietary SAA supplementation. Differential and tissue-specific mucosal growth responses occurred in response to resection and dietary SAA content.
main effect for RX to increase ileal crypt depth occurred (P , 0.05), with an interaction between RX and SAA supplementation as evidenced by the marked increase in this mucosal adaptive growth index with SAA supplementation (P , 0.05). Crypt depth in the Tx-Con, TX-Suppl, and Rx-Con groups did not differ, but values in the RX-Suppl group were greater than all of the other groups by post-hoc testing. There were no main effects of resection or SAA diet in colonic crypt depth between groups (Fig. 3).
Crypt depth in jejunum, ileum, and colon There was a significant main effect of RX to increase jejunal crypt depth (P , 0.05) without a significant interaction between RX and SAA supplementation (Fig. 3). Following small bowel resection, with or without SAA, jejunal crypt depth was greater than unresected control and SAA-supplemented groups; values in the RX-Con and RX-Suppl groups were similar. In ileum, a
Villus height in jejunum and ileum Measurement of jejunal villus height demonstrated an adaptive growth response to small bowel resection in control-fed and SAA-supplemented groups compared with the TX rats (Fig. 4). There was a main effect of RX, but not SAA, to increase jejunal villus height without interaction. In the ileum, there was a main effect of both RX and SAA to increase ileal villus height (P ,
TABLE 1
Effects of massive small bowel resection and SAA supplementation on full-thickness intestinal growth indices in rats1
Index n Jejunum, mg/cm Wet weight DNA content Protein content Ileum, mg/cm Wet weight DNA content Protein content Colon, mg/cm Wet weight DNA content Protein content 1 2
Resection main effect
Dietary SAA main effect
Interaction
TX-Con
TX-Suppl
RX-Con
RX-Suppl
7
9
9
10
43.9 6 3.3a 0.43 6 0.03a 12.6 6 1.2a
39.8 6 4.0a 0.30 6 0.02a 9.7 6 1.2a
68.1 6 4.1b 0.65 6 0.05b 22.2 6 1.4b
67.4 6 4.5b 0.67 6 0.03b 25.2 6 0.8b
P , 0.05 P , 0.05 P , 0.05
NS2 NS NS
NS NS NS
36.8 6 2.4a 0.35 6 0.03a 7.8 6 0.5a
30.6 6 2.4a 0.26 6 0.01a 6.1 6 0.2a
61.0 6 4.0b 0.67 6 0.02b 15.0 6 0.9b
73.0 6 3.1c 0.90 6 0.07c 18.6 6 1.52c
P , 0.05 P , 0.05 P , 0.05
NS NS NS
P , 0.05 P , 0.05 P , 0.05
58.0 6 2.0a 0.42 6 0.02a 14.9 6 1.0a
55.5 6 2.1a 0.36 6 0.04a 10.2 6 0.9a
65.6 6 2.9b 0.52 6 0.01b 17.5 6 1.4b
68.2 6 3.6b 0.60 6 0.05b 18.4 6 0.9b
P , 0.05 P , 0.05 P , 0.05
NS NS NS
NS NS NS
Values are mean 6 SEM. Means in a row with superscripts without a common letter differ, P , 0.05 (Fishers PLSD test). NS, Not significant, P $ 0.05.
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FIGURE 4 Effect of dietary SAA supplementation on jejunal and ileal villus height in TX and resected rats. Values are the mean 6 SEM, n = 7–10. Significant effects P , 0.05 were: in jejunum, RX; in ileum, RX and SAA. Means with superscripts without a common letter differ, P , 0.05.
0.05) without interaction. Ileal crypt depth in the RX-Suppl group was greater than in all other groups (P , 0.05).
Discussion Previous studies have demonstrated that nutrition is a critical determinant of thiol/disulfide redox status (Eh). Short-term fasting, protein-energy malnutrition, and protein malnutrition each lead to oxidation of tissue GSH/GSSG redox (the major cellular redox system), and plasma Cys/CySS redox (the major redox system in plasma), respectively (16,18,24,25), while dietary supplementation with Cys precursors such as NAC restores thiol/disulfide homeostasis under these conditions (26). The present study extends these previous findings by examining the effects of SAA supplementation on the principal lowmolecular weight thiol/disulfide redox systems in a rat model of intestinal adaptive growth. The data show that ileal adaptation after small bowel resection is enhanced by SAA supplementation in association with a greater reduction in small bowel mucosal thiol/disulfide redox states. Studies in young, healthy adults show that Cys/CySS redox state is centered at approximately 280 mV in the plasma (27). Remarkably, cultured cells actively regulate extracellular Cys and CySS levels to a value that approximates the Eh Cys/CySS in healthy humans (4,5) and variations in Eh Cys/CySS are associated with major cellular processes, such as proliferation, differentiation, and apoptosis (4,28). In a previous report, we showed that SAA supplementation resulted in a greater reduction in plasma Cys/CySS redox potential in intact rats (16). In healthy humans without SBS, diurnal variation in Cys, CySS, and Eh Cys/CySS occurs as a function of meal timing, suggesting that plasma Eh Cys/CySS can be modulated fairly rapidly by the diet (29). Also, disease states such as diabetes mellitus, infection, malnutrition, and inflammatory bowel disease are associated with oxidized plasma redox states (12,13,18,24,27). In the current study, SAA intake main effects were not significant on plasma Cys/CySS redox state. Thus, data in this model suggest that the plasma Cys pool may not be amenable to modulation by dietary Cys and Met supplementation after massive small bowel resection (possibly due to potential malabsorption of SAA with 80% small bowel resection). 2276
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We have previously shown that in human colonic intestinal cells, a reducing extracellular Eh Cys/CySS increases EGF receptor activation and activation of the mitogen-activated protein kinase pathway, signaling cascades central to cell growth and proliferation (30). Previous studies in rodent models of SBS have defined the critical roles for EGF receptor signaling during resection-induced intestinal adaptation in the ileum (31). Thus, our data suggest the possibility that enhanced ileal adaptive growth with dietary SAA may be due in part to the greater reduction in plasma Eh Cys/CySS and activation of EGF-mitogen activated protein kinase pathways in this gut segment via the epithelial basolateral membrane. SAA supplementation also caused a greater reduction in the GSH/GSSG redox pool in both jejunum and ileum after resection and to a similar extent. The lack of concomitant stimulated mucosal adaptive growth in the jejunum suggests that these distinct proximal and distal small bowel segments exhibit differential growth responses to redox changes systemically in blood (Cys/CySS redox) and locally in mucosa (GSH/GSSG redox) after 80% jejunoileal resection. Such small bowel segment-specific responses to growth stimuli are reminiscent of our previous studies of insulin-like growth factor-I treatment in this identical rat SBS model (32). Studies in intestinal epithelial cells and other cell lines indicate that changes in the cellular GSH/GSSG Eh correlates with the proliferation-apoptosis status of cells; proliferating cells exhibit a greater reduction in Eh values compared with differentiating or apoptotic cells in which more oxidized Eh values occur (6,8,9). In vivo inhibition of GSH synthesis by buthionine sulfoximine, with a consequent depletion of tissue GSH levels, leads to jejunal villus atrophy and epithelial cell damage in mice with an intact intestine (33). Additionally, supplementation with GSH reverses the inhibitory effects of lipid peroxides on intestinal growth (34). Thus, the present observations in ileal mucosa are consistent with the established role of GSH/GSSG redox state in regulating intestinal cell growth in vitro and in vivo. However, in this same rat model, marked depletion of ileal GSH and oxidation of the ileal mucosal GSH/GSSG redox pool by buthionine sulfoximine treatment did not inhibit ileal mucosal adaptive growth (35) and was associated with enhanced cell proliferation in colonic mucosa (10). This suggests the possibility that following small bowel resection, adaptive growth of the remnant ileum is a conserved response that is unaffected by an oxidized mucosal Eh GSH/GSSG but that is able to be stimulated by a greater reduction in plasma Cys/CySS and/or ileal mucosal GSH/GSSG redox. This finding is consistent with the concept that the adaptive growth response in this model may be regulated by multiple, redundant redox systems, as seen in other in vitro and in vivo systems, such that a perturbation in one system does not negatively affect overall cell proliferation and growth (7,36,37). Notwithstanding these possibilities, the novel observation in the present study is that ileal adaptive growth after massive small bowel resection in rats can be augmented by dietary SAA supplementation, which is associated with a greater reduction in plasma Cys/CySS and mucosal GSH/ GSSG redox states. These findings are consistent with our previous studies in a fasting-refeeding rat model, where adequate refeeding after a period of fasting increased duodenal, ileal, and colonic GSH concentrations, leading to a greater reduction in Eh GSH/GSSG in these tissues (24). Dietary SAA supplementation led to a greater reduction in Eh GSH/GSSG in small bowel but not colonic mucosa. Such differential effects on tissue GSH/GSSG redox have also been
observed by Li et al. (38), who demonstrated a greater reduction in GSH/GSSG redox with oral NAC supplementation in the liver and heart but not in the colon of intact mice. Similarly, oral NAC supplementation improved GSH levels in the ileum but not in the colon of aged intact rats (26). Additional studies investigating the distribution of Cys and CySS transport systems and expression of GSH synthesis enzymes in the ileum compared with the colon may aid in understanding the selective effects of dietary SAA supplementation on GSH homeostasis in rat tissues. A limitation of our study is that the results represent a single point in time after SAA supplementation; longitudinal outcomes over a longer period of time with these diets would be of interest to assess the impact of dietary SAA following massive small bowel resection. In conclusion, our data show that dietary SAA supplementation increased ileal adaptive growth following massive small bowel resection in rats, as determined by mucosal wet weight, DNA and protein content, crypt depth, and villus height. Increased adaptive growth in response to SAA supplementation was associated with a greater reduction in Cys/CySS and ileal GSH/GSSG redox states. It remains unclear the degree to which a greater reduction in plasma Eh Cys/CySS or a greater reduction in ileal Eh GSH/GSSG contributes to this improved ileal mucosal adaptive growth response with SAA supplementation and whether such effects are altered by age or other factors affecting these redox systems. These findings have translational importance, because they raise the possibility that SAA-based nutritional therapies may be a strategy to improve overall Eh and potentially enhance adaptive ileal growth in human SBS.
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15.
16.
17.
18.
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Acknowledgments T.R.Z., D.P.J., and Y.S. designed the research (project conception, development of overall research plan, and study oversight). Y.S., J.T., L.H., and Y.O.M. conducted the research. Y.S., J.T., and T.R.Z. analyzed data and performed statistical analysis. Y.S., S.S.I., D.P.J., and T.R.Z. wrote the paper and T.R.Z. had primary responsibility for final content. All authors read and approved the final manuscript.
Literature Cited 1.
2.
3. 4.
5.
6.
7. 8.
Ziegler TR, Evans ME, Fernandez-Estı´variz C, Jones DP. Trophic and cytoprotective nutrition for intestinal adaptation, mucosal repair and barrier function. Annu Rev Nutr. 2003;23:229–61. Ziegler TR, Leader LM. Parenteral nutrition: transient or permanent therapy in intestinal failure? Gastroenterology. 2006;130(2 Suppl 1): S37–42. Bristol JB, Williamson RC. Nutrition, operations, and intestinal adaptation. JPEN J Parenter Enteral Nutr. 1988;12:299–309. Jonas CR, Gu LH, Nkabyo YS, Mannery YO, Avissar NE, Sax HC, Jones DP, Ziegler TR. Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells. Am J Physiol Regul Integr Comp Physiol. 2003;285:R1421–9. Jonas CR, Ziegler TR, Gu LH, Jones DP. Extracellular thiol/disulfide redox state affects proliferation rate in a human colon carcinoma (Caco2) cell line. Free Radic Biol Med. 2002;33:1499–506. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med. 2001;30:1191–212. Jones DP. Redefining oxidative stress. Antioxid Redox Signal. 2006;8: 1865–79. Nkabyo YS, Ziegler TR, Gu LH, Watson WH, Jones DP. Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells. Am J Physiol Gastrointest Liver Physiol. 2002;283: G1352–9.
20. 21.
22. 23.
24.
25.
26.
27.
28.
29.
30.
Kirlin WG, Cai J, Thompson SA, Diaz D, Kavanagh TJ, Jones DP. Glutathione redox potential in response to differentiation and enzyme inducers. Free Radic Biol Med. 1999;27:1208–18. Tian J, Washizawa N, Gu LH, Levin MS, Wang L, Rubin DC, Mwangi S, Srinivasan S, Gao Y, et al. Stimulation of colonic mucosal growth associated with oxidized redox status in rats. Am J Physiol Regul Integr Comp Physiol. 2007;292:R1081–91. Kang YJ. Exogenous glutathione attenuates the antiproliferative effect of buthionine sulfoximine. Toxicology. 1994;88:177–89. Iantomasi T, Marraccini P, Favilli F, Vincenzini MT, Ferretti P, Tonelli F. Glutathione metabolism in Crohn’s disease. Biochem Med Metab Biol. 1994;53:87–91. Miralles-Barrachina O, Savoye G, Belmonte-Zalar L, Hochain P, Ducrotte P, Hecketsweiler B, Lerebours E, De´chelotte P. Low levels of glutathione in endoscopic biopsies of patients with Crohn’s colitis: the role of malnutrition. Clin Nutr. 1999;18:313–7. Sido B, Hack V, Hochlehnert A, Lipps H, Herfarth C, Droge W. Impairment of intestinal glutathione synthesis in patients with inflammatory bowel disease. Gut. 1998;42:485–92. Ardite E, Sans M, Panes J, Romero FJ, Pique JM, Fernandez-Checa JC. Replenishment of glutathione levels improves mucosal function in experimental acute colitis. Lab Invest. 2000;80:735–44. Nkabyo YS, Gu LH, Jones DP, Ziegler TR. Thiol/disulfide redox status is oxidized in plasma and small intestinal and colonic mucosa of rats with inadequate sulfur amino acid intake. J Nutr. 2006;136:1242–8. Washizawa N, Gu LH, Gu L, Openo KP, Jones DP, Ziegler TR. Comparative effects of glucagon-like peptide-2 (GLP-2), growth hormone (GH) and keratinocyte growth factor (KGF) on markers of gut adaptation after massive small bowel resection in rats. J Parent Enteral Nutr. 2004;28:339–409. Iyer SS, Jones DP, Brigham KL, Rojas M. Oxidation of plasma cysteine/ cystine redox state in endotoxin-induced lung injury. Am J Respir Cell Mol Biol. 2009;40:90–8. Iyer SS, Ramirez AM, Ritzenthaler JD, Torres-Gonzalez E, Roser-Page S, Mora AL, Brigham KL, Jones DP, Roman J, et al. Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 2009;296:L37–45. Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980;102:344–52. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. Jones DP. Redox potential of GSH/GSSG couple: assay and biological significance. Methods Enzymol. 2002;348:93–112. Wang X, Wang Q, Andersson R, Bengmark S. Intramucosal pH and oxygen extraction in the gastrointestinal tract after major liver resection in rats. Eur J Surg. 1993;159:81–7. Jonas CR, Estı´variz CF, Jones DP, Gu LH, Wallace TM, Diaz EE, Pascal RR, Cotsonis GA, Ziegler TR. Keratinocyte growth factor enhances glutathione redox state in rat intestinal mucosa during nutritional repletion. J Nutr. 1999;129:1278–84. Badaloo A, Reid M, Forrester T, Heird WC, Jahoor F. Cysteine supplementation improves the erythrocyte glutathione synthesis rate in children with severe edematous malnutrition. Am J Clin Nutr. 2002;76: 646–52. Grattagliano I, Portincasa P, Cocco T, Moschetta A, Di Paola M, Palmieri VO, Palasciano G. Effect of dietary restriction and N-acetylcysteine supplementation on intestinal mucosa and liver mitochondrial redox status and function in aged rats. Exp Gerontol. 2004;39:1323–32. Jones DP, Carlson JL, Mody VC, Cai J, Lynn MJ, Sternberg P. Redox state of glutathione in human plasma. Free Radic Biol Med. 2000;28: 625–35. Jiang S, Moriarty-Craige SE, Orr M, Cai J, Sternberg P Jr, Jones DP. Oxidant-induced apoptosis in human retinal pigment epithelial cells: dependence on extracellular redox state. Invest Ophthalmol Vis Sci. 2005;46:1054–61. Blanco RA, Ziegler TR, Carlson BA, Cheng PY, Park Y, Cotsonis GA, Accardi CJ, Jones DP. Diurnal variation in glutathione and cysteine redox states in human plasma. Am J Clin Nutr. 2007;86:1016–23. Nkabyo YS, Go YM, Ziegler TR, Jones DP. Extracellular cysteine/ cystine redox regulates the p44/p42 MAPK pathway by metalloproteinase-dependent epidermal growth factor receptor signaling. Am J Physiol Gastrointest Liver Physiol. 2005;289:G70–8.
Sulfur amino acids after small bowel resection
2277
31. Warner BW, Erwin CR. Critical roles for EGF receptor signaling during resection-induced intestinal adaptation. J Pediatr Gastroenterol Nutr. 2006;43 Suppl 1:S68–73. 32. Ziegler TR, Mantell MP, Rombeau JL, Smith RJ. Intestinal adaptation after extensive small bowel resection: differential changes in growth and IGF-I system messenger ribonucleic acids in jejunum and ileum. Endocrinology. 1998;139:3119–26. 33. Martensson J, Jain A, Meister A. Glutathione is required for intestinal function. Proc Natl Acad Sci USA. 1990;87:1715–9. 34. Tsunada S, Iwakiri R, Noda T, Fujimoto K, Fuseler J, Rhoads CA, Aw TY. Chronic exposure to subtoxic levels of peroxidized lipids suppresses mucosal cell turnover in rat small intestine and reversal by glutathione. Dig Dis Sci. 2003;48:210–22.
2278
Shyntum et al.
35. Tian J, Washizawa N, Gu LH, Levin MS, Wang L, Rubin DC, Mwangi S, Srinivasan S, Jones DP, et al. Local glutathione redox status does not regulate ileal mucosal growth after massive small bowel resection in rats. J Nutr. 2007;137:320–5. 36. Jones DP, Go YM, Anderson CL, Ziegler TR, Kinkade JM Jr, Kirlin WG. Cysteine/cystine couple is a newly recognized node in the circuitry for biologic redox signaling and control. FASEB J. 2004;18: 1246–8. 37. Jones DP. Radical-free biology of oxidative stress. Am J Physiol Cell Physiol. 2008;295:C849–68. 38. Li J, Wang H, Stoner GD, Bray TM. Dietary supplementation with cysteine prodrugs selectively restores tissue glutathione levels and redox status in protein-malnourished mice. J Nutr Biochem. 2002;13:625–33.
