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Uncoupling protein-2 modulates crypt cell apoptosis rate in the colon of mice fed with high-fat diet
also demonstrated. However, the intestinal epithelial cell lines, Caco-2 and HT-29, were unresponsive to CpG motifs under a variety of conditions. LF does not regulate reporter gene transcription, nor did it bind specifically to putative DNA response elements. No LF Iocalisationinto enterocyte and lymphocyte nuclei was detected. Conclusions: We conclude that bacterial DNA binding by LF is an important mechanism of immune gene regulation, with Peyer's patch B-cefls of the breast-feeding infant a likely target. Lack of reporter gene activation, non-specific DNA binding and absence of nuclear localisation argue strongly against LF transcription factor activity.
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The Measurement of Mucosal Protein Turnover in Humans during Feeding and Fasting Stephen J. O'Keefe, Ronzo B. Lee, Jing Li Earlier studies showed that mucosal turnover (MTO) can be measured in vivo in humans by IV infusion of stable isotope-labeled amino acids (13C leucine) and mucosal biopsy (OKeefe AJP 1994). However, the precursor pool for protein synthesis was assumed to be represented by the plasma ketoisocaproic acid (KIC), a product of intraceflufar [eucine, and there is concern that during enteral feeding dietary amino acids may be used directly for mucosal synthesis without equilibration with the systemic circulation. Consequently, we performed studies in healthy volunteers during fasting (n = 5), enteral (n = 12) and parenteral feeding (n = 5), and compared MTO calculated using enteral versus parenteral isotope labeled amino acid administration, and plasma vs mucosal free amino acid pool measurements. Methods: Studies were conducted after an overnight fast. Diets were given for 6h at a constant rate (1.5g protein/kg/d, energy 40kcal/kg/d) by vein or feeding tube, together with a prime/continuous 1Vinfusion of 13C leucine (lmg/kg/h) and continuous duodenal infusion of deuterium (D) labeled leucine (2mg/kg/h). At 6h, endoscopic mucosal biopsies were taken from the distal duodenum. Enrichment of plasma KIC, mucosal free amino acids, and mucosal protein wnh 13C and D was measured after derivatization with HFBA by +CI GC-MS. Results demonstrated that use of the mucosal free amino acid pool as precursor resulted in higher mucosal turnover rates (mean (SE): 240(76)%/d) than use of the plasma KIC pool (40(3.8)%/d) when the isotope was given IV, but that the difference was diminished when isotope was given enterally (255(32) vs 155(20)%/d). Calculations using IV isotope labeling were similar during enteral and parenteral feeding, but higher during parenteral feeding (151(14) vs 108(9)%/d, Mann Whitney: p<0.02) when enteral isotope labeling was used. In contrast, rates during fasting were lower with IV isotope labeling and higher with enteral labeling (33.6(2.9) vs 163(21)%/d, p<0.0001) In conclusion, completely different conclusions can be drawn from the effects of feeding on mucosal turnover when a) the route of isotope administration is changed from IV to enteral, and b) the systemic or mucosal pool of free amino acids is used as the precursor pool for mucosal protein synthesis. Our results suggest that during feeding studies, the isotope label should be co-administrated with the dietary source, and that during fasting conditions, the IV route should be used as the amino acid supply is chiefly supported by endogenous (systemic) sources.
S1754
Enteral L-Arginine Attenuates the Effects of Diabetic Hyperglycemia on Colonic Epithelial Cell Function in Rats Sunny K, Whiteman, Trammel Tillman, Karla Auyeung, Kathleen Madden, Alan Chap, Justin Elfrey, Aiping Zhao, Terez Shea-Donnhue Background: Nitric oxide (NO) plays a major role in colonic function, and diabetics exhibit impaired nitric oxide syuthase (NOS) activity that may contribute to abnormal GI function associated with autonomic neuropathies. We showed previously that L-arginine (ARG) is able to maintain neural NOS activity and attenuate intestinal mucosal damage during periods of oxidative stress (Ward et al, 2000). Aim: To determine the effects enteral L-arginine supplementation on diabetes-induced alterations in colonic epithelial cell function. Methods: Sprague-Dawley rats received vehicle (control) or streptozotocin (65mg/kg ip) to induce diabetes. ARG (0.67-1 gin/day) was added to drinking water for both groups 2 weeks prior to study At 8 weeks of hyperglycemia, sections of stripped mucosae were mounted in Ussing chambers to construct concentration-dependent responses to acetylcholine (ACH), serotouin (5-HT), and histamine (HIST). To evaluate the contribution of enteric nerves, responses were compared in the absence and presence of the nerve toxin, tetrodotoxin (TTX). Results: Diabetic rats weighed less than controls (304 -+ 16 vs 407 -+ 11 gm) and had siginftcantly elevated blood glucose levels (549 -+ 33 vs 110 _+ 10 mg%). These differences were not altered by ARG administration. Diabetes significantly suppressed responses to ACH and 5-HT, but increased responses to HIST. Responses to ACH and 5HI', but not to HIST. were reduced by TTX (p
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Chronic Intake of Subtoxic Peroxidized Lipids Suppresses Mucosal Turnover in Rat Small Intestine and the Reversal by Glutathione Seiji Tsunada, Ryuichi lwakiri, Takahiro Noda, Kazuma Fujimoto, John Fuseler, Carol A. Rhoads, Tak Yee Aw
Diabetic ARG 101 20 59 9* 61:20
Dietary intake of highly polyunsaturated fats is an important source of lipid peroxides in the intestinal lumen. Because oxidized lipids have the ability to generate oxyradicals, they can compromise intestinal cell growth and cell death responses that ultimately could lead to disorders of the digestive systems, such as inflammation and cancer. The objective of this study was to determine the effect of chronic consumption of subtoxic levels of peroxidized lipids on intestinal redox balance and cell turnover responses. Because mucosal glutathione (GSH) governs the elimination of luminal lipid peroxides, we also evaluated the effect of GSH supplementation on intestinal cell proliferation and apoptosis. Male Sprague-Dawley rats were fed standard rat chow or 4% peroxidized Menhaden oil chow (1.2 mmol/g) for 2-8 weeks. Intestinal redox status was determined by GSH and glntathione disulfide (GSSG), lipid peroxide contents, and GSH synthetic and redox enzyme activities. Cell proliferation and cell death were evaluated by omithine decarboxyfase (ODC) activity and apoptosis, respectively. Chronic intake of peroxidized lipids did not affect overall animal growth, but decreased intestinal GSH/GSSG ratio that correlated with increased GSSG content and decreased tissue GSH and glucose 6-phosphate dehydrogenase activity at week 2, consistent with enhanced oxidative stress. Peak circadian duodenal, jejunal and ileal ODC were attenuated and postprandial induction of jejunal and ileal apoptosis was suppressed, consistent with induction of a cytostatic state associated with persistent peroxide challenge. Supplementation with 1% GSH abrogated the peroxide-induced suppression of intestinal cell turnover in association with decreased tissue peroxide accumulation and restoration of mucosal GSH/ GSSG ratio. These data show that chronic exposure of the small intestine to low dose lipid peroxides causes a highly oxidized state that induces mucosa[ GSH redox imbalance and interferes with regulation of cell death and proliferation in vivo. These disruptive effects of lipid peroxides were reversed by GSH supplementation in accordance with the restoration of cellular GSH/GSSG redox balance. Supported by NIH grant DK 44510.
S1755
Uncoupling Protein-2 Modulates Crypt Cell Apoptosis Rate in the Colon of Mice Fed with High-Fat Diet Nofa A. Mouti, Peter Fulop, Murray Resinck, Jack R. Wands, Gyorgy Baffy BACKGROUND. Dietary fat intake has been implicated in the development of colon cancer. While n-3 polyunsaturated fatty acids (PUFA-3) may be protective, other types of fatty acids appear to promote colon cancer. The mechanisms by which fatty acids affect colon tumor formation remain poorly understood, but likely involve effects on crypt cell apoptosis. Uncoupling protein-2 (UCP2) is an inner mitochondnal membrane constituent that mediates proton leak, negatively regulates mitochondrial reactive oxygen species (ROS) formation, and shows a notable expression in the GI tract. Recent evidence links UCP2 to apoptosis m various experimental settings. Transcriptional activity of the UCP2 gene is stimulated by fatty acids. AiMS To study whether diets rich in various fatty acids modulate the rate of apoptosis in colon and assess the influence of absent UCP2 on this effect. METHODS. Groups (n = 4 to 5) of UCP24- and wild-type mice were fed with diets rich in PUFA-3, n6 PUFA (PUFA-6), saturated fatty acids (SFA) or with regular chow (control). After 8 weeks on diet, the mice were sacriheed and proximal, mid, and distal colon segments were removed. Crypt cell apoptosis rate was assessed by morphological criteria on H&E stains and by TUNELassay (expressed as apoptotic cells/24 crypt areas). RESULTS. In wild-type mice fed vath PUFA-3, cumulative crypt cell apoptosis rate was unchanged compared to chow-fed controls (3 08 +/-0.43 vs 3 0 +/-0.48, respectively), while it appeared less in the SFA group (2.18 +/-0.44, NS). In UCP2-/- mice, there were higher rates of crypt cell apoptosis in both the PUFA-3 and chow groups (3.67+/-0.57 and 4.0+/-0.66, respectively), while the rate was significantly lower in the SFA group (2.0+/-0.35, p=0.02 vs. PUFA-3 and p=0.014 vs chow). Results of the PUFA-6 group were intermediate between those of PUFA-3 and SFA (not shown). When apoptosis was assessed in vanous colon segments, these changes were most prominent in the proximal colon. CONCLUSION. Chronic feeding of mice with a diet rich in PUFA-3 and SFA results in subtle but opposing changes in rates of crypt cell apoptosis Absence of UCP2 augments the differential effects of PUFA-3 and SFA suggesting that diminished crypt cell apoptosis and potential colon tumor formation in relation to SFAnch diet is modulated by mitochondrial ROS formation. (Supported in part by NIH grant RR-17(~95)
S1758
The Beneficial Effects of Supplementary Glutamine on Experimental Irradiated Colonic Anastomosis; Histological and Morphological Evaluation Mnhamed M. E1 Malt, Pieter De Metter, Wim Ceelen, Caroline Van Den Broecke, Claude Cuvelier, Wilfried De Neve, Bernard De Hemptinne, Ptet Pattyn Purpose: To investigate the effects of adding glutamme to total partenteral nutrition on the histology and morphology of irradiated colonic anastomosis. Methods: The rectosigmoid colon in male Wistar rats was irradiated up to a total dose of 25 Gy (5 Gy daily for 5 consecutive days). Five days after the end of RT, side-to-side anastomosis was constructed between the irradiated rectosigmoid and the non-irradiated caecum. Postoperatively, animals were divided randomly into 3 groups; group I was fed orally (lab chow), group II received TPN and group fir received TPN enriched with 2% glutamine (GIn-TPN). One week after surgery animals were sacrificed. Results: Mucosa[ regeneration at the anastomotic site was improved with the addition of glutamine to TPN. The depth of the crypts was significantly more in group IIl in comparison to group I1 ( p - 0 04) while it was comparable to that in group I. Mucosal ulceration was less in group llI in comparison to group I (p = 0.005) and group If (p=O.05). The mucosal ulcers that penetrate deeply till the serosa were also less in group Ill (54%) compared to group i (91%) and group lI (70%). The radiation effects
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AGA Abstracts
$1756
The Measurement of Mucosal Protein Turnover in Humans during Feeding and Fasting Stephen J. O'Keefe, Ronzo B. Lee, Jing Li Earlier studies showed that mucosal turnover (MTO) can be measured in vivo in humans by IV infusion of stable isotope-labeled amino acids (13C leucine) and mucosal biopsy (OKeefe AJP 1994). However, the precursor pool for protein synthesis was assumed to be represented by the plasma ketoisocaproic acid (KIC), a product of intraceflufar [eucine, and there is concern that during enteral feeding dietary amino acids may be used directly for mucosal synthesis without equilibration with the systemic circulation. Consequently, we performed studies in healthy volunteers during fasting (n = 5), enteral (n = 12) and parenteral feeding (n = 5), and compared MTO calculated using enteral versus parenteral isotope labeled amino acid administration, and plasma vs mucosal free amino acid pool measurements. Methods: Studies were conducted after an overnight fast. Diets were given for 6h at a constant rate (1.5g protein/kg/d, energy 40kcal/kg/d) by vein or feeding tube, together with a prime/continuous 1Vinfusion of 13C leucine (lmg/kg/h) and continuous duodenal infusion of deuterium (D) labeled leucine (2mg/kg/h). At 6h, endoscopic mucosal biopsies were taken from the distal duodenum. Enrichment of plasma KIC, mucosal free amino acids, and mucosal protein wnh 13C and D was measured after derivatization with HFBA by +CI GC-MS. Results demonstrated that use of the mucosal free amino acid pool as precursor resulted in higher mucosal turnover rates (mean (SE): 240(76)%/d) than use of the plasma KIC pool (40(3.8)%/d) when the isotope was given IV, but that the difference was diminished when isotope was given enterally (255(32) vs 155(20)%/d). Calculations using IV isotope labeling were similar during enteral and parenteral feeding, but higher during parenteral feeding (151(14) vs 108(9)%/d, Mann Whitney: p<0.02) when enteral isotope labeling was used. In contrast, rates during fasting were lower with IV isotope labeling and higher with enteral labeling (33.6(2.9) vs 163(21)%/d, p<0.0001) In conclusion, completely different conclusions can be drawn from the effects of feeding on mucosal turnover when a) the route of isotope administration is changed from IV to enteral, and b) the systemic or mucosal pool of free amino acids is used as the precursor pool for mucosal protein synthesis. Our results suggest that during feeding studies, the isotope label should be co-administrated with the dietary source, and that during fasting conditions, the IV route should be used as the amino acid supply is chiefly supported by endogenous (systemic) sources.
S1754
Enteral L-Arginine Attenuates the Effects of Diabetic Hyperglycemia on Colonic Epithelial Cell Function in Rats Sunny K, Whiteman, Trammel Tillman, Karla Auyeung, Kathleen Madden, Alan Chap, Justin Elfrey, Aiping Zhao, Terez Shea-Donnhue Background: Nitric oxide (NO) plays a major role in colonic function, and diabetics exhibit impaired nitric oxide syuthase (NOS) activity that may contribute to abnormal GI function associated with autonomic neuropathies. We showed previously that L-arginine (ARG) is able to maintain neural NOS activity and attenuate intestinal mucosal damage during periods of oxidative stress (Ward et al, 2000). Aim: To determine the effects enteral L-arginine supplementation on diabetes-induced alterations in colonic epithelial cell function. Methods: Sprague-Dawley rats received vehicle (control) or streptozotocin (65mg/kg ip) to induce diabetes. ARG (0.67-1 gin/day) was added to drinking water for both groups 2 weeks prior to study At 8 weeks of hyperglycemia, sections of stripped mucosae were mounted in Ussing chambers to construct concentration-dependent responses to acetylcholine (ACH), serotouin (5-HT), and histamine (HIST). To evaluate the contribution of enteric nerves, responses were compared in the absence and presence of the nerve toxin, tetrodotoxin (TTX). Results: Diabetic rats weighed less than controls (304 -+ 16 vs 407 -+ 11 gm) and had siginftcantly elevated blood glucose levels (549 -+ 33 vs 110 _+ 10 mg%). These differences were not altered by ARG administration. Diabetes significantly suppressed responses to ACH and 5-HT, but increased responses to HIST. Responses to ACH and 5HI', but not to HIST. were reduced by TTX (p
$1757
Chronic Intake of Subtoxic Peroxidized Lipids Suppresses Mucosal Turnover in Rat Small Intestine and the Reversal by Glutathione Seiji Tsunada, Ryuichi lwakiri, Takahiro Noda, Kazuma Fujimoto, John Fuseler, Carol A. Rhoads, Tak Yee Aw
Diabetic ARG 101 20 59 9* 61:20
Dietary intake of highly polyunsaturated fats is an important source of lipid peroxides in the intestinal lumen. Because oxidized lipids have the ability to generate oxyradicals, they can compromise intestinal cell growth and cell death responses that ultimately could lead to disorders of the digestive systems, such as inflammation and cancer. The objective of this study was to determine the effect of chronic consumption of subtoxic levels of peroxidized lipids on intestinal redox balance and cell turnover responses. Because mucosal glutathione (GSH) governs the elimination of luminal lipid peroxides, we also evaluated the effect of GSH supplementation on intestinal cell proliferation and apoptosis. Male Sprague-Dawley rats were fed standard rat chow or 4% peroxidized Menhaden oil chow (1.2 mmol/g) for 2-8 weeks. Intestinal redox status was determined by GSH and glntathione disulfide (GSSG), lipid peroxide contents, and GSH synthetic and redox enzyme activities. Cell proliferation and cell death were evaluated by omithine decarboxyfase (ODC) activity and apoptosis, respectively. Chronic intake of peroxidized lipids did not affect overall animal growth, but decreased intestinal GSH/GSSG ratio that correlated with increased GSSG content and decreased tissue GSH and glucose 6-phosphate dehydrogenase activity at week 2, consistent with enhanced oxidative stress. Peak circadian duodenal, jejunal and ileal ODC were attenuated and postprandial induction of jejunal and ileal apoptosis was suppressed, consistent with induction of a cytostatic state associated with persistent peroxide challenge. Supplementation with 1% GSH abrogated the peroxide-induced suppression of intestinal cell turnover in association with decreased tissue peroxide accumulation and restoration of mucosal GSH/ GSSG ratio. These data show that chronic exposure of the small intestine to low dose lipid peroxides causes a highly oxidized state that induces mucosa[ GSH redox imbalance and interferes with regulation of cell death and proliferation in vivo. These disruptive effects of lipid peroxides were reversed by GSH supplementation in accordance with the restoration of cellular GSH/GSSG redox balance. Supported by NIH grant DK 44510.
S1755
Uncoupling Protein-2 Modulates Crypt Cell Apoptosis Rate in the Colon of Mice Fed with High-Fat Diet Nofa A. Mouti, Peter Fulop, Murray Resinck, Jack R. Wands, Gyorgy Baffy BACKGROUND. Dietary fat intake has been implicated in the development of colon cancer. While n-3 polyunsaturated fatty acids (PUFA-3) may be protective, other types of fatty acids appear to promote colon cancer. The mechanisms by which fatty acids affect colon tumor formation remain poorly understood, but likely involve effects on crypt cell apoptosis. Uncoupling protein-2 (UCP2) is an inner mitochondnal membrane constituent that mediates proton leak, negatively regulates mitochondrial reactive oxygen species (ROS) formation, and shows a notable expression in the GI tract. Recent evidence links UCP2 to apoptosis m various experimental settings. Transcriptional activity of the UCP2 gene is stimulated by fatty acids. AiMS To study whether diets rich in various fatty acids modulate the rate of apoptosis in colon and assess the influence of absent UCP2 on this effect. METHODS. Groups (n = 4 to 5) of UCP24- and wild-type mice were fed with diets rich in PUFA-3, n6 PUFA (PUFA-6), saturated fatty acids (SFA) or with regular chow (control). After 8 weeks on diet, the mice were sacriheed and proximal, mid, and distal colon segments were removed. Crypt cell apoptosis rate was assessed by morphological criteria on H&E stains and by TUNELassay (expressed as apoptotic cells/24 crypt areas). RESULTS. In wild-type mice fed vath PUFA-3, cumulative crypt cell apoptosis rate was unchanged compared to chow-fed controls (3 08 +/-0.43 vs 3 0 +/-0.48, respectively), while it appeared less in the SFA group (2.18 +/-0.44, NS). In UCP2-/- mice, there were higher rates of crypt cell apoptosis in both the PUFA-3 and chow groups (3.67+/-0.57 and 4.0+/-0.66, respectively), while the rate was significantly lower in the SFA group (2.0+/-0.35, p=0.02 vs. PUFA-3 and p=0.014 vs chow). Results of the PUFA-6 group were intermediate between those of PUFA-3 and SFA (not shown). When apoptosis was assessed in vanous colon segments, these changes were most prominent in the proximal colon. CONCLUSION. Chronic feeding of mice with a diet rich in PUFA-3 and SFA results in subtle but opposing changes in rates of crypt cell apoptosis Absence of UCP2 augments the differential effects of PUFA-3 and SFA suggesting that diminished crypt cell apoptosis and potential colon tumor formation in relation to SFAnch diet is modulated by mitochondrial ROS formation. (Supported in part by NIH grant RR-17(~95)
S1758
The Beneficial Effects of Supplementary Glutamine on Experimental Irradiated Colonic Anastomosis; Histological and Morphological Evaluation Mnhamed M. E1 Malt, Pieter De Metter, Wim Ceelen, Caroline Van Den Broecke, Claude Cuvelier, Wilfried De Neve, Bernard De Hemptinne, Ptet Pattyn Purpose: To investigate the effects of adding glutamme to total partenteral nutrition on the histology and morphology of irradiated colonic anastomosis. Methods: The rectosigmoid colon in male Wistar rats was irradiated up to a total dose of 25 Gy (5 Gy daily for 5 consecutive days). Five days after the end of RT, side-to-side anastomosis was constructed between the irradiated rectosigmoid and the non-irradiated caecum. Postoperatively, animals were divided randomly into 3 groups; group I was fed orally (lab chow), group II received TPN and group fir received TPN enriched with 2% glutamine (GIn-TPN). One week after surgery animals were sacrificed. Results: Mucosa[ regeneration at the anastomotic site was improved with the addition of glutamine to TPN. The depth of the crypts was significantly more in group IIl in comparison to group I1 ( p - 0 04) while it was comparable to that in group I. Mucosal ulceration was less in group llI in comparison to group I (p = 0.005) and group If (p=O.05). The mucosal ulcers that penetrate deeply till the serosa were also less in group Ill (54%) compared to group i (91%) and group lI (70%). The radiation effects
A-261
AGA Abstracts