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W1746 Cysteine Dioxygenase Expression Is Restricted to the Secretory Lineage in the Small Intestine and Colon
cells relative to absorptive epithelial cells. (Supported by NIH R01 DK061568 and Carle Foundation Hospital) W1747
AGA Abstracts
Hepcidin Produced By Primary Rat Liver Cell Culture Inhibits Iron Transport Across the Basolateral Membrane On CaCo2 Cells Kwo-Yih Yeh, Mary Yeh, Laura Mims, Jonathan Glass Background: Hepcidin, the iron stores regulator produced in the liver, down regulates ferroportin 1 (FPN1) expression by transcriptional and posttranscriptional mechanisms resulting in the reduction of iron export from enterocytes. Recently, using pFPN1-GFP transfected HEK cells, hepcidin has been shown to bind to FPN1 triggering FPN1 phosphorylation, internalization, ubiquitination and subsequently degradation. Similar observations have also been reported in macrophages, but no effect was found on FPN1 in Caco2 cells. Aims: As chemically synthetic or recombinant hepcidin may be less potent than native hepcidin, we hypothesized that primary liver cell cultures may produce native folded hepcidin which would function to inhibit Caco2 cell iron export. Methods: Overnight fasted rats were used to isolate hepatocytes by inferior vena cava perfusion with calcium free Hank's Balance Salt Solution supplemented with 0.05% collagenase and 0.005% trypsin inhibitor. The isolated hepatocytes were cultured in William's E medium and hepcidin synthesis induced by addition of 2 ng/ml of bone morphogenetic protein 4 (BMP4) added 4 h after cell plating and the medium collected 12-24 h later. Separately, Caco2 cells cultured in bicameral chambers until TEERS>700Ω were changed to Opti medium for 12 h and then the hepatocyte conditioned William's E medium ± BMP4 was added to the bottom chamber for 3, 6 and 12 h. Iron transport was assayed by the addition to the upper chamber of 55FeCl3, 2 μM ferric ammonium citrate and 1 mM ascorbate with subsequet monitoring 55Fe in the bottom chamber. Results: After 90 min the transport of 55Fe to the lower chamber was maximally inhibited by conditioned medium produced after 3-6 h of exposure to BMP4 by 20-25%. 55Fe transport to the lower chamber was unaltered by conditioned medium without BMP4 or with BMP4 added to William's E medium. These observations suggest that BMP4 treated hepatocytes secreted hepcidin into the medium to suppress iron transport out of the cells. This hypothesis was verified by treating the 6 h BMP4 condition medium with rat anti-hepcidin antiserum (1:200 dilution) which abolished the inhibition of 55Fe transport into the lower chamber. Western blot analysis of Caco2 cell lysates showed that ferroportin expression was reduced by about 40% by the 6 h after incubation with BMP4 conditioned medium. Northern blot analysis of BMP4 treated hepatocytes is in progress to assess the effects of BMP4 on hepcidin expression. Conclusions: The present data provide the evidence that in Caco2 cells iron transport out of the cells is inhibited by rat hepcidin with posttranscriptional down regulatory mechanism.
p=0.001 for bars not sharing a letter W1745 The Effects of Pluronic F-68 On Cholesterol Absorption John Szymusiak, Qing Yang, Dana Lee, Laura Woollett, James E. Heubi, Patrick Tso Background: The solubilization of cholesterol and fats by bile salts to form micelles is a key step in the intestinal absorption and metabolism of lipids. Pluronic F-68 (Poloxomer 188), a hydrophilic detergent used in artificial blood in humans, has been shown to compete with bile salts for the solubilization of cholesterol, altering the interaction of micelles with the brush border membrane resulting in reduced uptake of cholesterol (but not fatty acids) by enterocytes. Aim: We will determine the effect of Pluronic F-68 on intestinal uptake and lymphatic transport of fatty acids and cholesterol in animals with intact enterohepatic circulation. Methods: Under anesthesia, the lymph duct of adult rats was cannulated and a duodenal infusion tube was installed. Following overnight recovery, the animals were infused intraduodenally with lipid emulsions containing 3H labeled triolein (40 μmol/h) , 14C labeled cholesterol (7.8 μmol/h), and phosphatidylcholine (7.8 μmol/h) . Along with the lipids, an emulsion containing 19mM of either NaTC, F-68, or 2:1 (molar ratio) F-68: NaTC was infused at 3 mL/hr for 6 hr, with lymph collected hourly. The small intestine, cecum, and large intestine were then removed, washed with 10 mM sodium deoxycholate in normal saline and washings were collected. Finally, mucosal lipids were extracted by the Folch method. Aliquots of the lymph, washing, and mucosal lipids were used for radioactivity determination. Results: We observed significantly lower cholesterol levels in the lymph of F-68 treated animals than controls (NaTC) (p<.001, hrs 2-6). There was no significant difference in lumenal cholesterol recoveries between F-68 and control rats, while there were significantly lower levels of radioactive cholesterol in the mucosa of the F-68 group versus control (p<.001). F-68 led to significantly lower levels of triglyceride (TG) in the lymph compared to control rats (p=.023, hrs 4-6). There was no significant difference between lymphatic TG outputs for 2:1 F-68:NaTC and control rats, while the 2:1 F-68:NaTC rats still showed a significant reduction in lymph cholesterol levels (p=.002, hrs 3-6) versus controls. Discussion: F-68 is highly effective in reducing lymphatic cholesterol and TG transport in rats. Partial replacement of F-68 with NaTC rendered F-68 less efficient in reducing lymphatic TG absorption, but not cholesterol absorption. Treatment with a concentrated dose of F-68 lowers cholesterol levels in the lymph and mucosa, without a signifcant effect on luminal cholesterol levels. This suggests that F-68 may influence the relative transport of cholesterol between the lymphatic and the portal route.
W1748 Impaired Intestinal Lipid Metabolism Decreases Mortality and Improves Intestinal Integrity in Pseudomonas Aeruginosa Pneumonia-Induced Sepsis Jessica A. Clark, Yan Xie, Craig M. Coopersmith, Nicholas O. Davidson Sepsis is the leading cause of death in critically ill patients. The intestine plays a crucial role in the pathophysiology of sepsis and has been characterized as the “motor” of the systemic inflammatory response. The proposed mechanism is that certain toxic gut-derived lipid factors, carried in mesenteric lymph, induce systemic injury and distant organ failure. The aim of this study was to determine if blocking intestinal transport of these gut-derived lipid factors confers a survival advantage in pneumonia-induced sepsis. We studied mice with conditional intestine-specific deletion of microsomal triglyceride transfer protein (MTP-IKO), which exhibit virtually complete lipid malabsorption. MTP-IKO mice and control littermates (MTPflox/flox) were intratracheally instilled with 40 μl of 0.1 A600nm Pseudomonas aeruginosa or sterile saline. At 24 hr, intestines were evaluated for apoptosis by caspase-3 staining, proliferation by BrdU staining, and villus length (n=11-18/group). Cytokine and lipid levels in serum at baseline and 24 hr post-sepsis were also measured. Survival studies were performed in a separate cohort of mice (n=14-17/group). At 7 days, septic MTP-IKO mice exhibited significantly decreased mortality compared to septic controls (0% vs. 36% mortality; p<0.01). Compared to shams, septic controls had increased intestinal apoptosis (6 ± 1 vs. 13 ± 2 cells/100 crypts; p<0.05). However, septic MTP-IKO mice exhibited normalized intestinal apoptosis to sham levels (5 ± 1 vs. 5 ± 1 cells/100 crypts; p=ns). Septic controls had decreased proliferation compared to shams (1031 ± 99 vs. 684 ± 49 cells/100 crypts; p<0.05), while proliferation was increased in septic MTP-IKO mice to levels seen in shams (1358 ± 79 vs. 1160 ± 90 cells/100 crypts; p=ns). Compared to shams, septic controls had shorter villi (358 ± 20 vs. 256± 16 μm; p<0.001), while villus atrophy was abrogated in septic MTP-IKO mice (460± 13 vs. 407 ± 13 μm; p=ns). Systemic proinflammatory cytokines IL-6 and MCP-1 were decreased in septic MTP-IKO mice compared to septic controls (2780 ± 942 vs. 36 ± 30 and 5 ± 1 vs. 1 ± 0.2 fold-change over baseline, respectively; p<0.05). Serum levels of cholesterol and phospholipid were increased in septic MTP-IKO mice compared to septic controls (2.4 ± 0.3 vs. 1.2 ± 0.1 and 2.2 ± 0.3 vs. 1.0 ± 0.1 fold-change over baseline, respectively; p<0.05), while there were no differences in triglyceride or free fatty acids between groups. These findings demonstrate that conditional impairment of intestinal lipid transport confers a survival advantage in pneumonia-induced sepsis with improved intestinal integrity and diminished production of systemic cytokines.
W1746 Cysteine Dioxygenase Expression Is Restricted to the Secretory Lineage in the Small Intestine and Colon Jennifer A. Croix, Iori Ueki, Martha H. Stipanuk, Eugene Greenberg, H. Rex Gaskins Background: Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulphinic acid, which is the first and rate limiting step for cysteine utilization. Mammals regulate cysteine metabolism through CDO to maintain sufficient cysteine for protein synthesis and production of other essential molecules such as glutathione, taurine, pyruvate and inorganic sulfate, but below the threshold for cytotoxicity. While CDO is known to be highly expressed in liver, its expression in the intestine remains uncharacterized. Here, we examined CDO expression and localization in mouse small and large intestine and human colon using immunohistochemical and immunofluorescence staining techniques. Methods: Adult mouse small intestine and colon were harvested and immediately fixed in Carnoy's solution. Human colon biopsies were collected from healthy adults undergoing routine screening colonoscopies at Carle Foundation Hospital and immediately fixed in Bouin's solution. Paraffin embedded sections of human colon and mouse duodenum, jejunum, ileum, and proximal and distal colon were stained with an anti-CDO antibody, counterstained with hematoxylin, and analyzed microscopically. Additional sections of mouse tissues were stained using two-color immunofluorescence for CDO and matrix metalloproteinase 7 (MMP7), a marker of Paneth cells, or chromagranin A (ChrA), a marker of enteroendocrine cells. Results: Immunohistochemical analysis of mouse intestine revealed positive CDO staining in goblet cells, Paneth cells (small intestine only), and enteroendocrine cells while CDO staining was notably absent in absorptive eptihelium. In human colon, CDO expression was also observed in goblet cells rather than absorptive coloncytes. Dual immunofluorescent staining for CDO and ChrA or MMP-7 confirmed CDO expression in mouse enteroendocrine cells and Paneth cells, respectively. Conclusions: These results demonstrate that CDO expression in mouse small and large intestine and human colon is restricted to cells of the secretory lineage. This striking difference in CDO expression between the absorptive and secretory cell lineages parallels a high cysteine requirement in goblet, Paneth, and enteroendocrine
AGA Abstracts
A-730
AGA Abstracts
Hepcidin Produced By Primary Rat Liver Cell Culture Inhibits Iron Transport Across the Basolateral Membrane On CaCo2 Cells Kwo-Yih Yeh, Mary Yeh, Laura Mims, Jonathan Glass Background: Hepcidin, the iron stores regulator produced in the liver, down regulates ferroportin 1 (FPN1) expression by transcriptional and posttranscriptional mechanisms resulting in the reduction of iron export from enterocytes. Recently, using pFPN1-GFP transfected HEK cells, hepcidin has been shown to bind to FPN1 triggering FPN1 phosphorylation, internalization, ubiquitination and subsequently degradation. Similar observations have also been reported in macrophages, but no effect was found on FPN1 in Caco2 cells. Aims: As chemically synthetic or recombinant hepcidin may be less potent than native hepcidin, we hypothesized that primary liver cell cultures may produce native folded hepcidin which would function to inhibit Caco2 cell iron export. Methods: Overnight fasted rats were used to isolate hepatocytes by inferior vena cava perfusion with calcium free Hank's Balance Salt Solution supplemented with 0.05% collagenase and 0.005% trypsin inhibitor. The isolated hepatocytes were cultured in William's E medium and hepcidin synthesis induced by addition of 2 ng/ml of bone morphogenetic protein 4 (BMP4) added 4 h after cell plating and the medium collected 12-24 h later. Separately, Caco2 cells cultured in bicameral chambers until TEERS>700Ω were changed to Opti medium for 12 h and then the hepatocyte conditioned William's E medium ± BMP4 was added to the bottom chamber for 3, 6 and 12 h. Iron transport was assayed by the addition to the upper chamber of 55FeCl3, 2 μM ferric ammonium citrate and 1 mM ascorbate with subsequet monitoring 55Fe in the bottom chamber. Results: After 90 min the transport of 55Fe to the lower chamber was maximally inhibited by conditioned medium produced after 3-6 h of exposure to BMP4 by 20-25%. 55Fe transport to the lower chamber was unaltered by conditioned medium without BMP4 or with BMP4 added to William's E medium. These observations suggest that BMP4 treated hepatocytes secreted hepcidin into the medium to suppress iron transport out of the cells. This hypothesis was verified by treating the 6 h BMP4 condition medium with rat anti-hepcidin antiserum (1:200 dilution) which abolished the inhibition of 55Fe transport into the lower chamber. Western blot analysis of Caco2 cell lysates showed that ferroportin expression was reduced by about 40% by the 6 h after incubation with BMP4 conditioned medium. Northern blot analysis of BMP4 treated hepatocytes is in progress to assess the effects of BMP4 on hepcidin expression. Conclusions: The present data provide the evidence that in Caco2 cells iron transport out of the cells is inhibited by rat hepcidin with posttranscriptional down regulatory mechanism.
p=0.001 for bars not sharing a letter W1745 The Effects of Pluronic F-68 On Cholesterol Absorption John Szymusiak, Qing Yang, Dana Lee, Laura Woollett, James E. Heubi, Patrick Tso Background: The solubilization of cholesterol and fats by bile salts to form micelles is a key step in the intestinal absorption and metabolism of lipids. Pluronic F-68 (Poloxomer 188), a hydrophilic detergent used in artificial blood in humans, has been shown to compete with bile salts for the solubilization of cholesterol, altering the interaction of micelles with the brush border membrane resulting in reduced uptake of cholesterol (but not fatty acids) by enterocytes. Aim: We will determine the effect of Pluronic F-68 on intestinal uptake and lymphatic transport of fatty acids and cholesterol in animals with intact enterohepatic circulation. Methods: Under anesthesia, the lymph duct of adult rats was cannulated and a duodenal infusion tube was installed. Following overnight recovery, the animals were infused intraduodenally with lipid emulsions containing 3H labeled triolein (40 μmol/h) , 14C labeled cholesterol (7.8 μmol/h), and phosphatidylcholine (7.8 μmol/h) . Along with the lipids, an emulsion containing 19mM of either NaTC, F-68, or 2:1 (molar ratio) F-68: NaTC was infused at 3 mL/hr for 6 hr, with lymph collected hourly. The small intestine, cecum, and large intestine were then removed, washed with 10 mM sodium deoxycholate in normal saline and washings were collected. Finally, mucosal lipids were extracted by the Folch method. Aliquots of the lymph, washing, and mucosal lipids were used for radioactivity determination. Results: We observed significantly lower cholesterol levels in the lymph of F-68 treated animals than controls (NaTC) (p<.001, hrs 2-6). There was no significant difference in lumenal cholesterol recoveries between F-68 and control rats, while there were significantly lower levels of radioactive cholesterol in the mucosa of the F-68 group versus control (p<.001). F-68 led to significantly lower levels of triglyceride (TG) in the lymph compared to control rats (p=.023, hrs 4-6). There was no significant difference between lymphatic TG outputs for 2:1 F-68:NaTC and control rats, while the 2:1 F-68:NaTC rats still showed a significant reduction in lymph cholesterol levels (p=.002, hrs 3-6) versus controls. Discussion: F-68 is highly effective in reducing lymphatic cholesterol and TG transport in rats. Partial replacement of F-68 with NaTC rendered F-68 less efficient in reducing lymphatic TG absorption, but not cholesterol absorption. Treatment with a concentrated dose of F-68 lowers cholesterol levels in the lymph and mucosa, without a signifcant effect on luminal cholesterol levels. This suggests that F-68 may influence the relative transport of cholesterol between the lymphatic and the portal route.
W1748 Impaired Intestinal Lipid Metabolism Decreases Mortality and Improves Intestinal Integrity in Pseudomonas Aeruginosa Pneumonia-Induced Sepsis Jessica A. Clark, Yan Xie, Craig M. Coopersmith, Nicholas O. Davidson Sepsis is the leading cause of death in critically ill patients. The intestine plays a crucial role in the pathophysiology of sepsis and has been characterized as the “motor” of the systemic inflammatory response. The proposed mechanism is that certain toxic gut-derived lipid factors, carried in mesenteric lymph, induce systemic injury and distant organ failure. The aim of this study was to determine if blocking intestinal transport of these gut-derived lipid factors confers a survival advantage in pneumonia-induced sepsis. We studied mice with conditional intestine-specific deletion of microsomal triglyceride transfer protein (MTP-IKO), which exhibit virtually complete lipid malabsorption. MTP-IKO mice and control littermates (MTPflox/flox) were intratracheally instilled with 40 μl of 0.1 A600nm Pseudomonas aeruginosa or sterile saline. At 24 hr, intestines were evaluated for apoptosis by caspase-3 staining, proliferation by BrdU staining, and villus length (n=11-18/group). Cytokine and lipid levels in serum at baseline and 24 hr post-sepsis were also measured. Survival studies were performed in a separate cohort of mice (n=14-17/group). At 7 days, septic MTP-IKO mice exhibited significantly decreased mortality compared to septic controls (0% vs. 36% mortality; p<0.01). Compared to shams, septic controls had increased intestinal apoptosis (6 ± 1 vs. 13 ± 2 cells/100 crypts; p<0.05). However, septic MTP-IKO mice exhibited normalized intestinal apoptosis to sham levels (5 ± 1 vs. 5 ± 1 cells/100 crypts; p=ns). Septic controls had decreased proliferation compared to shams (1031 ± 99 vs. 684 ± 49 cells/100 crypts; p<0.05), while proliferation was increased in septic MTP-IKO mice to levels seen in shams (1358 ± 79 vs. 1160 ± 90 cells/100 crypts; p=ns). Compared to shams, septic controls had shorter villi (358 ± 20 vs. 256± 16 μm; p<0.001), while villus atrophy was abrogated in septic MTP-IKO mice (460± 13 vs. 407 ± 13 μm; p=ns). Systemic proinflammatory cytokines IL-6 and MCP-1 were decreased in septic MTP-IKO mice compared to septic controls (2780 ± 942 vs. 36 ± 30 and 5 ± 1 vs. 1 ± 0.2 fold-change over baseline, respectively; p<0.05). Serum levels of cholesterol and phospholipid were increased in septic MTP-IKO mice compared to septic controls (2.4 ± 0.3 vs. 1.2 ± 0.1 and 2.2 ± 0.3 vs. 1.0 ± 0.1 fold-change over baseline, respectively; p<0.05), while there were no differences in triglyceride or free fatty acids between groups. These findings demonstrate that conditional impairment of intestinal lipid transport confers a survival advantage in pneumonia-induced sepsis with improved intestinal integrity and diminished production of systemic cytokines.
W1746 Cysteine Dioxygenase Expression Is Restricted to the Secretory Lineage in the Small Intestine and Colon Jennifer A. Croix, Iori Ueki, Martha H. Stipanuk, Eugene Greenberg, H. Rex Gaskins Background: Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulphinic acid, which is the first and rate limiting step for cysteine utilization. Mammals regulate cysteine metabolism through CDO to maintain sufficient cysteine for protein synthesis and production of other essential molecules such as glutathione, taurine, pyruvate and inorganic sulfate, but below the threshold for cytotoxicity. While CDO is known to be highly expressed in liver, its expression in the intestine remains uncharacterized. Here, we examined CDO expression and localization in mouse small and large intestine and human colon using immunohistochemical and immunofluorescence staining techniques. Methods: Adult mouse small intestine and colon were harvested and immediately fixed in Carnoy's solution. Human colon biopsies were collected from healthy adults undergoing routine screening colonoscopies at Carle Foundation Hospital and immediately fixed in Bouin's solution. Paraffin embedded sections of human colon and mouse duodenum, jejunum, ileum, and proximal and distal colon were stained with an anti-CDO antibody, counterstained with hematoxylin, and analyzed microscopically. Additional sections of mouse tissues were stained using two-color immunofluorescence for CDO and matrix metalloproteinase 7 (MMP7), a marker of Paneth cells, or chromagranin A (ChrA), a marker of enteroendocrine cells. Results: Immunohistochemical analysis of mouse intestine revealed positive CDO staining in goblet cells, Paneth cells (small intestine only), and enteroendocrine cells while CDO staining was notably absent in absorptive eptihelium. In human colon, CDO expression was also observed in goblet cells rather than absorptive coloncytes. Dual immunofluorescent staining for CDO and ChrA or MMP-7 confirmed CDO expression in mouse enteroendocrine cells and Paneth cells, respectively. Conclusions: These results demonstrate that CDO expression in mouse small and large intestine and human colon is restricted to cells of the secretory lineage. This striking difference in CDO expression between the absorptive and secretory cell lineages parallels a high cysteine requirement in goblet, Paneth, and enteroendocrine
AGA Abstracts
A-730