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Metabolic acidosis stimulates intestinal glutamine absorption
log-rank test. Multivariate analysis was performed with the Cox regression model. Significance was considered at p
group and preoperatively and two hours postoperatively in the lean group. All serum samples were collected in the fasting sate. RESULTS: The mean preoperative BM1 was 49.1(range 40-60). The mean preoperative ghrelin level was 1139 -+ 208 pg/ml. At two hours postoperatively the mean was 9 3 7 +- 168 pg/ml. Using Dunnetts ANOVA to compare the two post operative groups to the preoperative values revealed a significant fall in both groups with p = 0.013. At ten days postoperatively the mean was 973 -+ 142 pg/ml. A t-test was performed between the preoperative level and both postoperative samples p =0.012 (recovery room) and p = 0.026 (10 days p/o). There was no statistical difference between both postoperative samples (p=0.053). When the obese group was compared the lean group there was a sigmficant difference between obese preoperative levels and controls (control 1577 -+487 pg/ml p = 0.01). At two hours postoperatively there was not a significant difference between controls and GBP patients (control 1103 -+ 256 pg/ml p = 0.088). Conclusion: Ghrefin levels are significantly different between obese and lean patients preoperatively. Ghrelin fell in all patients postoperatively. This finding refutes the assumption that ghrelin levels are affected only after bypass surgery.
M1327 Metabolic Acidosis Stimulates Intestinal Glutamine Absorption Mark ]. Epler, Qinghe Meng, Wiley W. Souba Jr., Anne M. Karinch, Chengmao Lin, Thomas C. Vary, Ming Pan
M1324
Background: Glutamine is an essential nutrient for cell integrity during acidotic states such as shock but the effect of extracellufar pH on intestinal mucosal cell glutamine uptake is poorly understood. The purpose of this in vitro study was to investigate the intracelhilar signaling pathways involved in controlling intestinal glutamine transport during acidosis. Methods: 3H-ghitamine (50 p,M) transport activity, cellular mitogen-activated protein kinase (MAPK) levels and mRNA levels for the intestinal glutamine transporter ATB~ were measured in cultured intestinal epithelial Caco-2 cells. Cells were incubated with growth medium at various extracellular pHs (pH 6 to pH 9), inhihitors of MAPK MEK1 (PD 98059, 0 - 50 ~M), the phosphatidylinositide-3 kinase (PI-3) (Wortmannin, 0 - 10 ~M), and actinomycinD (0 - 1 ~M) and cycloheximide (0 - 20 ~M). Data were analyzed by ANOVA. Results: Lowing the pH in the cell culture media resulted in an increase of glntamine transport activity in a time and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a 2-fold increase in glutamine transport activity (1.63 _+ 0.25 nmole/mg/minute in acidosis vs. 0.78 _+ 0.11 nmole/mg/minute control, n = 6, p<0.01) and a 3-fold increase of glutamine transport gene ATB~ mRNA levels (p<0.01). This acidosis-induced increase in ghitamine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 13.6 -+ 0.73 nmoldmg/minute in acidosis vs. 6.3 _+ 0.46 nmoledmg/minute in control, n = 6 , p<0.01) rather than a change in transporter affinity (Kin = 0.23 _+ 0.02 mM glutamine in acidosis vs. 0.19 -+ 0.02 mM ghitamine in controls, n = 6 , p=NS). This acidosis-stimulated glutamine transport activity was blocked by actinomycin-D or cycloheximide. Cellular MAPK MEK1/2 and p42/44 levels were elevated in acidotic cells and the acidosis-induced glutaminc transport activity was blocked individually PD 98059 and Wortmanniu. Glutamine transport activity in cells treated with PD 98059, or Wortmannin alone did not differ from control cells treated with vehicle. Conclusions: Acidosis stimulates ghitamine transport in Caco-2 cells via signaling pathways that lead to transcription of the glutamine transporter gene and translation of functional transporters. Mitogen-activated protein kinases and phosphatidylinositide-3 kinase are key intracelMar regulators involved in this signal transduction cascade. An increased availability of glutamine to cells subjected to redox stress may help in maintaining cellular integrity.
Realization of the Esophageal Cancer Model in Mice Martin Fein, Jeffrey H. Peters, Para Chandrasoma, Tom R. Demeester Purpose: There is well established esophageal cancer model in rats. Cancer is induced by surgical induction of duodenogastric reflux (DGR) into the esophagus. Application of this model in transgenic mice may allow to study the relevance of genomic alterations in esophageal carcinogenesis. Therefore, a study was designed to realize this cancer model in mice. Methods: C57B1/6 mice (8 weeks) were treated in four groups: 1. control with sham operation (n= 10), 2. DGR only (n=36), 3. Sham operation and carcinogen (n=28, 3 x 15 mg/kg methyl-N-amyl-nitrosamin intraperitoneal) and 4. DGR and carcinogen (n = 41). Gastrectomy and esophagojejunostomy to induce DGR was done mlcrosurgtcally with interrupted 10-0 stitches. The animals were sacrificed after 24 weeks and examined histologically. Results: DGR (group 2 + 4) induced esophagitis in the distal esophagus with papillomatnsis and hyperceratosis in all animals. Esophageal ulceration was found in 5/41 (12%), columnar lining was detected in one animal of group 4. Esophageal cancer was not induced. Related to carcinogen administration increased hyperceratosis was present in 8/44 (18%) of group 3 + 4. Conclusion: While esophageal adenocarcinoma is induced in 90% of rats following this operation and carcinogen administration, no cancer was induced in mice. This is explained by much less severe esophagitis and possibly by altered effectivity of the carcinogen in mice. Nevertheless, the realization of the reflux induced cancer model in this study led to the application in transgenic mice where cancer development in p53-knockout mice was already documented
M1325 The Effects Of Gastric Division On Systemic Ghrelin Levels In The Morbidly Obese Edward Lin, Nana Gletsu, Kim Fugate, David A. McChisky, Thomas R. Ziegler, Le H. Gu, Dimitris A. Papanicolaou, Bruce J. Ramshaw, C. Daniel Smith BACKGROUND: Circulating ghrelin, produced primarily in the stomach, is a powerful sumulant for food intake. Levels are elevated in states of hunger and rapidly decline postprandially. This study seeks to determine the immediate effects of gastric surgery on ghrehn levels in morbidly obese patients. METHOD: Twenty-eight morbidly obese subjects (BMI>40) were studied prospectively according to IRB approved protocol. Twenty-two underwent roux-eny gastric bypass with divided gastroplasty to create a 15 cc proximal gastric pouch. Six patients underwent other foregnt procedures (3 vertical handed gastroplasty, 2 antireflux surgeries, 1 ulcer operation). Serum samples were obtained preoperatively and immediately post-operatively. In a sub-study, serum was collected after roux-limb formation and after dividing the stomach to determine the point of ghrelin decline. Ghrelni levels were measured by radioimmunoassay and reported in pg/mL. Data were analyzed usmg two-way ANOVA with Neuman-Keuls post-test. RESULTS: BMIs were 46.4 and 42.8, respectively (p=NS). Preoperative and postoperative ghrelin levels in the gastric bypass group were 38.8-+ 2.5 and 26.4 -+ 1.4, respectively (p<0.001). In the non-gastric bypass group, ghrelin levels were 34.5 -+ 5.5 and 29.]~-+ 1.9, respectively (p=NS). The percent change between the gastric bypass group and non-gastric bypass group were 29% and 14%, respectively (p = 0.04). In the gastric bypass group, the percent decline in ghrelin levels was most pronounced < 5 minutes after completely dividing the proximal stomach (p<0.05). CONCLUSIONS: In morbidly obese humans, a divided gastropfasty creating a small proximal gastric pouch results in early declines in circulating ghrelin. This may explain, in part, the rapid weightloss observed following gastric bypass surgery.
M1328 The Role of Cytokines and p21 in the Altered Enterocyte Phenotype Associated with Gut Inflammatory Conditions Mario A. Abedrapo Sr., Madhu S. Malo St., Wenying Zhang, Joseph W. Henderson IV, Aleem Siddique Sr., Moushumi Mozumder, Richard A. Hodin Sr. Introduction:The pro inflammatory cytokines, IL-113 and TNF-cr play a critical role in a variety of gut inflammatory diseases, including Crohn's Disease and Ulcerative Colitis, conditions that are associated with villus atrophy. We have previously identified a molecular phenotype that occurs in the setting of villus atrophy, i.e., silencing of the brush border marker, intestinal alkaline phosphatase (lAD, and hypothesized that ILq3 and/or TNF-c~ might play a role in inducing this enterocyte phenotype by repressing IAP gene transcription. Methods: Sodium Butyrate (NB) treated HT29 cells were used as an in vitro model of enterocyte differentiation. The cytokines were studied at concentrations ranging from 0.01 to 10 ng/ml, and along a range of times prior to and after the differentiation stimulus. Northern analyses were performed using lAP and vilfin probes. A Luciferase reporter construct containing a 2.5 kb segment of the human lAP promoter (IAP-LUC) was transfected with and without a p21 expression plasmid in HT29 and HCT116 (wild-type and p21-deleted) cells. Results: Northem blots confirmed the marked induction in lAP expression after 24 hr of NB treatment (> 20-fold). However, in those cells pre-treated with cytokines there was a dose-dependent decrease in IAP mRNA levels (87% for IL-113, 86% for TNF-c0. Repression of the lAP gene was seen when cytokine treatment occurred before or at the same time, but not 6 hr after NB treatment. These changes were specific for lAP, since other differentiation markers (e.g., viUin) were unaffected by the cytokines. The transfection studies in HT-29 cells revealed that pIAP-LUC was markedly induced by NB (5-fold), but with cytokine pre-treatment the level of lAP activation was significantly diminished (85% and 90%, with IL-I~ and TNF-e~, respectively). Both cytokines decreased lAP gene activation in the wild-type HCTll6 cells, similar to that seen in HT29 cells. In contrast, in the p21deleted cells, whereas IL-l[3 exerted its inhibitory effects, TNF-a did not. The TNF-a inhibitory effect on the lAP gene was rescued by co-transfection of a p21 expression plasmid into the p21-deleted cells. Conclusions: The pro-inflammatory cytokines, 1L-I~ and TNFa, specifically inhibit IAP gene expression, a mechanism that could underlie the altered enterocyte phenotype seen in gut inflammatory conditions. The p21 cell cycle inhibitor is required in the case of TNF-a, but the IL-113 effect is p21-independent.
M1326 The Effects of Ghrelin on post Operative Anorexia: A Comparison Between Gastric Bypass Surgery and Controls on Serum Ghrelin Levels Daniel R. Cottam, Marta Couce, Samer Mattar, Barto Burguera, James Esplen, Jeff Lord, Philip Schauer BACKGROUND: Ghrefin is a stomach hormone believed to regulate appetite. Anorexia following gastric bypass surgery (GBP) is thought to be caused by bypassing the ghrelin producing areas of the stomach. This study was performed to determine the short term differences in serum ghrelin between obese and lean individuals undergoing abdominal surgery. METHODS: The study was comprised of 14 patients who had GBP surgery and 8 lean patients (BMI <30) who underwent other laparotomies. Samples were collected one hour preoperatively, two hours postoperatively, and ten days postoperatively in the obese
SSAT Abstracts
A-796
group and preoperatively and two hours postoperatively in the lean group. All serum samples were collected in the fasting sate. RESULTS: The mean preoperative BM1 was 49.1(range 40-60). The mean preoperative ghrelin level was 1139 -+ 208 pg/ml. At two hours postoperatively the mean was 9 3 7 +- 168 pg/ml. Using Dunnetts ANOVA to compare the two post operative groups to the preoperative values revealed a significant fall in both groups with p = 0.013. At ten days postoperatively the mean was 973 -+ 142 pg/ml. A t-test was performed between the preoperative level and both postoperative samples p =0.012 (recovery room) and p = 0.026 (10 days p/o). There was no statistical difference between both postoperative samples (p=0.053). When the obese group was compared the lean group there was a sigmficant difference between obese preoperative levels and controls (control 1577 -+487 pg/ml p = 0.01). At two hours postoperatively there was not a significant difference between controls and GBP patients (control 1103 -+ 256 pg/ml p = 0.088). Conclusion: Ghrefin levels are significantly different between obese and lean patients preoperatively. Ghrelin fell in all patients postoperatively. This finding refutes the assumption that ghrelin levels are affected only after bypass surgery.
M1327 Metabolic Acidosis Stimulates Intestinal Glutamine Absorption Mark ]. Epler, Qinghe Meng, Wiley W. Souba Jr., Anne M. Karinch, Chengmao Lin, Thomas C. Vary, Ming Pan
M1324
Background: Glutamine is an essential nutrient for cell integrity during acidotic states such as shock but the effect of extracellufar pH on intestinal mucosal cell glutamine uptake is poorly understood. The purpose of this in vitro study was to investigate the intracelhilar signaling pathways involved in controlling intestinal glutamine transport during acidosis. Methods: 3H-ghitamine (50 p,M) transport activity, cellular mitogen-activated protein kinase (MAPK) levels and mRNA levels for the intestinal glutamine transporter ATB~ were measured in cultured intestinal epithelial Caco-2 cells. Cells were incubated with growth medium at various extracellular pHs (pH 6 to pH 9), inhihitors of MAPK MEK1 (PD 98059, 0 - 50 ~M), the phosphatidylinositide-3 kinase (PI-3) (Wortmannin, 0 - 10 ~M), and actinomycinD (0 - 1 ~M) and cycloheximide (0 - 20 ~M). Data were analyzed by ANOVA. Results: Lowing the pH in the cell culture media resulted in an increase of glntamine transport activity in a time and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a 2-fold increase in glutamine transport activity (1.63 _+ 0.25 nmole/mg/minute in acidosis vs. 0.78 _+ 0.11 nmole/mg/minute control, n = 6, p<0.01) and a 3-fold increase of glutamine transport gene ATB~ mRNA levels (p<0.01). This acidosis-induced increase in ghitamine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 13.6 -+ 0.73 nmoldmg/minute in acidosis vs. 6.3 _+ 0.46 nmoledmg/minute in control, n = 6 , p<0.01) rather than a change in transporter affinity (Kin = 0.23 _+ 0.02 mM glutamine in acidosis vs. 0.19 -+ 0.02 mM ghitamine in controls, n = 6 , p=NS). This acidosis-stimulated glutamine transport activity was blocked by actinomycin-D or cycloheximide. Cellular MAPK MEK1/2 and p42/44 levels were elevated in acidotic cells and the acidosis-induced glutaminc transport activity was blocked individually PD 98059 and Wortmanniu. Glutamine transport activity in cells treated with PD 98059, or Wortmannin alone did not differ from control cells treated with vehicle. Conclusions: Acidosis stimulates ghitamine transport in Caco-2 cells via signaling pathways that lead to transcription of the glutamine transporter gene and translation of functional transporters. Mitogen-activated protein kinases and phosphatidylinositide-3 kinase are key intracelMar regulators involved in this signal transduction cascade. An increased availability of glutamine to cells subjected to redox stress may help in maintaining cellular integrity.
Realization of the Esophageal Cancer Model in Mice Martin Fein, Jeffrey H. Peters, Para Chandrasoma, Tom R. Demeester Purpose: There is well established esophageal cancer model in rats. Cancer is induced by surgical induction of duodenogastric reflux (DGR) into the esophagus. Application of this model in transgenic mice may allow to study the relevance of genomic alterations in esophageal carcinogenesis. Therefore, a study was designed to realize this cancer model in mice. Methods: C57B1/6 mice (8 weeks) were treated in four groups: 1. control with sham operation (n= 10), 2. DGR only (n=36), 3. Sham operation and carcinogen (n=28, 3 x 15 mg/kg methyl-N-amyl-nitrosamin intraperitoneal) and 4. DGR and carcinogen (n = 41). Gastrectomy and esophagojejunostomy to induce DGR was done mlcrosurgtcally with interrupted 10-0 stitches. The animals were sacrificed after 24 weeks and examined histologically. Results: DGR (group 2 + 4) induced esophagitis in the distal esophagus with papillomatnsis and hyperceratosis in all animals. Esophageal ulceration was found in 5/41 (12%), columnar lining was detected in one animal of group 4. Esophageal cancer was not induced. Related to carcinogen administration increased hyperceratosis was present in 8/44 (18%) of group 3 + 4. Conclusion: While esophageal adenocarcinoma is induced in 90% of rats following this operation and carcinogen administration, no cancer was induced in mice. This is explained by much less severe esophagitis and possibly by altered effectivity of the carcinogen in mice. Nevertheless, the realization of the reflux induced cancer model in this study led to the application in transgenic mice where cancer development in p53-knockout mice was already documented
M1325 The Effects Of Gastric Division On Systemic Ghrelin Levels In The Morbidly Obese Edward Lin, Nana Gletsu, Kim Fugate, David A. McChisky, Thomas R. Ziegler, Le H. Gu, Dimitris A. Papanicolaou, Bruce J. Ramshaw, C. Daniel Smith BACKGROUND: Circulating ghrelin, produced primarily in the stomach, is a powerful sumulant for food intake. Levels are elevated in states of hunger and rapidly decline postprandially. This study seeks to determine the immediate effects of gastric surgery on ghrehn levels in morbidly obese patients. METHOD: Twenty-eight morbidly obese subjects (BMI>40) were studied prospectively according to IRB approved protocol. Twenty-two underwent roux-eny gastric bypass with divided gastroplasty to create a 15 cc proximal gastric pouch. Six patients underwent other foregnt procedures (3 vertical handed gastroplasty, 2 antireflux surgeries, 1 ulcer operation). Serum samples were obtained preoperatively and immediately post-operatively. In a sub-study, serum was collected after roux-limb formation and after dividing the stomach to determine the point of ghrelin decline. Ghrelni levels were measured by radioimmunoassay and reported in pg/mL. Data were analyzed usmg two-way ANOVA with Neuman-Keuls post-test. RESULTS: BMIs were 46.4 and 42.8, respectively (p=NS). Preoperative and postoperative ghrelin levels in the gastric bypass group were 38.8-+ 2.5 and 26.4 -+ 1.4, respectively (p<0.001). In the non-gastric bypass group, ghrelin levels were 34.5 -+ 5.5 and 29.]~-+ 1.9, respectively (p=NS). The percent change between the gastric bypass group and non-gastric bypass group were 29% and 14%, respectively (p = 0.04). In the gastric bypass group, the percent decline in ghrelin levels was most pronounced < 5 minutes after completely dividing the proximal stomach (p<0.05). CONCLUSIONS: In morbidly obese humans, a divided gastropfasty creating a small proximal gastric pouch results in early declines in circulating ghrelin. This may explain, in part, the rapid weightloss observed following gastric bypass surgery.
M1328 The Role of Cytokines and p21 in the Altered Enterocyte Phenotype Associated with Gut Inflammatory Conditions Mario A. Abedrapo Sr., Madhu S. Malo St., Wenying Zhang, Joseph W. Henderson IV, Aleem Siddique Sr., Moushumi Mozumder, Richard A. Hodin Sr. Introduction:The pro inflammatory cytokines, IL-113 and TNF-cr play a critical role in a variety of gut inflammatory diseases, including Crohn's Disease and Ulcerative Colitis, conditions that are associated with villus atrophy. We have previously identified a molecular phenotype that occurs in the setting of villus atrophy, i.e., silencing of the brush border marker, intestinal alkaline phosphatase (lAD, and hypothesized that ILq3 and/or TNF-c~ might play a role in inducing this enterocyte phenotype by repressing IAP gene transcription. Methods: Sodium Butyrate (NB) treated HT29 cells were used as an in vitro model of enterocyte differentiation. The cytokines were studied at concentrations ranging from 0.01 to 10 ng/ml, and along a range of times prior to and after the differentiation stimulus. Northern analyses were performed using lAP and vilfin probes. A Luciferase reporter construct containing a 2.5 kb segment of the human lAP promoter (IAP-LUC) was transfected with and without a p21 expression plasmid in HT29 and HCT116 (wild-type and p21-deleted) cells. Results: Northem blots confirmed the marked induction in lAP expression after 24 hr of NB treatment (> 20-fold). However, in those cells pre-treated with cytokines there was a dose-dependent decrease in IAP mRNA levels (87% for IL-113, 86% for TNF-c0. Repression of the lAP gene was seen when cytokine treatment occurred before or at the same time, but not 6 hr after NB treatment. These changes were specific for lAP, since other differentiation markers (e.g., viUin) were unaffected by the cytokines. The transfection studies in HT-29 cells revealed that pIAP-LUC was markedly induced by NB (5-fold), but with cytokine pre-treatment the level of lAP activation was significantly diminished (85% and 90%, with IL-I~ and TNF-e~, respectively). Both cytokines decreased lAP gene activation in the wild-type HCTll6 cells, similar to that seen in HT29 cells. In contrast, in the p21deleted cells, whereas IL-l[3 exerted its inhibitory effects, TNF-a did not. The TNF-a inhibitory effect on the lAP gene was rescued by co-transfection of a p21 expression plasmid into the p21-deleted cells. Conclusions: The pro-inflammatory cytokines, 1L-I~ and TNFa, specifically inhibit IAP gene expression, a mechanism that could underlie the altered enterocyte phenotype seen in gut inflammatory conditions. The p21 cell cycle inhibitor is required in the case of TNF-a, but the IL-113 effect is p21-independent.
M1326 The Effects of Ghrelin on post Operative Anorexia: A Comparison Between Gastric Bypass Surgery and Controls on Serum Ghrelin Levels Daniel R. Cottam, Marta Couce, Samer Mattar, Barto Burguera, James Esplen, Jeff Lord, Philip Schauer BACKGROUND: Ghrefin is a stomach hormone believed to regulate appetite. Anorexia following gastric bypass surgery (GBP) is thought to be caused by bypassing the ghrelin producing areas of the stomach. This study was performed to determine the short term differences in serum ghrelin between obese and lean individuals undergoing abdominal surgery. METHODS: The study was comprised of 14 patients who had GBP surgery and 8 lean patients (BMI <30) who underwent other laparotomies. Samples were collected one hour preoperatively, two hours postoperatively, and ten days postoperatively in the obese
SSAT Abstracts
A-796