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1103 Dietary Protein Influences Growth Through a Complex Mechanism Coupling Proteolysis and Intestinal Peptide Transporter Expression
disrupts the complex, releasing FABP1. The released FABP1 binds to the ER membrane and generates PCTV. In humans, genetic defects in Sar1b result in chylomicron retention in the ER.
1103
Adequate intestinal nutrient absorption is required for growth and development. Dietary proteins must be digested to amino acids or peptides that can be absorbed via heteromeric amino acid carriers or intestinal peptide transporters. We hypothesized that intestinal peptide transport impacts growth more than amino acid absorption. Caenorhabditis elegans was used to assay the roles of the intestinal peptide transporter Opt-2 (homologous to PepT1) and amino acid carrier regulatory protein ATGP-1 (neutral amino acid carrier SLC3A1 homologue) on growth. Animals were raised in polymeric (NGM) or elemental (CeHR) diet and harvested daily for 4-days to assay age-related body length. Additionally, RNA was isolated to determine Opt-2 and ATGP-1 transcript levels and dietary green fluorescent protein (GFP) was used as a marker of intestinal proteolysis. Data were analyzed for variance (ANOVA) and expressed as mean±SEM. A significant effect of diet on daily body length was found at days 1 and 2 where animals fed a polymeric diet demonstrated 46% increased growth (d1=508±11, d2=1007±22μm, n=36) compared to those fed an elemental diet (CeHR, d1=370±9, d2=649±11, p<0.05). NGM animals demonstrated almost 100% increased Opt2 mRNA expression compared to CeHR, with no effect on ATGP-1 transcription. NGM animals also demonstrated significantly decreased (80%) intestinal proteolytic activity as measured by increased dietary GFP activity along the entire gut length compared to CeHR. Interestingly, animals transferred to NGM after 1 day in CeHR demonstrated an immediate improvement in growth (d2=822±16, p<0.05) with no increase in Opt-2 or ATGP-1 mRNA compared to CeHR. These transferred animals also showed a marked decrease in dietary GFP proteolytic activity mimicking that of those grown solely in NGM. These findings demonstrate that intestinal absorption of peptides plays a more influential role over growth than amino acid absorption, however the effect is not simply related to intestinal peptide transporter levels. Sole reliance on intestinal amino acid absorption through the provision of elemental diet increases intestinal proteolytic activity with no corresponding increase in amino acid transporter expression or improvement in growth. Animals transferred from elemental to polymeric diet demonstrated significantly improved growth despite decreased proteolytic activity despite expressing far fewer intestinal peptide transporters as animals fed solely polymeric diet. The metabolic cost of complete digestion can apparently be offset by incomplete digestion to permit more intestinal peptide absorption, potentially releasing energy for improved growth. In conclusion, the careful design of enteral formulae to efficiently balance proteolytic activity with intestinal peptide absorption could improve outcomes for short bowel syndrome or pediatric intestinal failure.
Phosphorylation of Sar1b disassembles the FABP1 containing, 75 kDa complex. A) The complex in native cytosol. B) Phosphorylation of Sar1b Thr by PKCζ. C) Phosphorylation of Sar1b completely disassembles the complex. D) The freed FABP1 binds to the ER. Not shown: On FABP1 binding to the ER, PCTV are generated. FABP1 cannot bind to the ER when a member of the 75 kDa complex.
1104 Molecular Aspects of Nutrient Sensing in the Human Gastrointestinal Tract Madusha Peiris, Michael A. Atiba, Andy Hubball, David C. Bulmer, Charles H. Knowles, L. Ashley Blackshaw
1102 Pharmacologic Blockage of Cholesterol Absorption With Ezetimibe Reveals a Novel Homeostatic Network in Enterocytes Luke Engelking, Guosheng Liang, Matthew R. McFarlane, Christina Li
Background: Nutrient sensing within the gastrointestinal (GI) tract occurs via specific receptors that detect a variety of luminal stimuli. Activation of these receptors leads directly to humoral and neural signals that modulate absorption, motility and satiety. However, detailed knowledge of mechanisms involved in nutrient/receptor interaction and nutrient activation of enteroendocrine cells (EEC) is currently lacking. Aim: To characterise taste receptor expression profile and co-localisation with specific gut hormones, as well as, elucidate the effect of nutrient exposure on EEC. Methods: Healthy colon, ileum and antrum samples were obtained from surgical resection specimens. Fluorescent immunohistochemistry was performed on sections cut from Zamboni's fixed tissue. Nutrient exposure studies were carried out on freshly dissected mucosa using an Ussing Chamber. Results: Human antral mucosa showed immunoreactivity (IR) for the amino acid sensing receptors T1R1 and calcium sensing receptor (CaSR), and the long chain fatty acid receptors GPR40 and GPR120. Only CaSR was IR in the ileum and the ascending colon. Gut hormones GLP-1 and PYY were found throughout the GI tract and co-localisation studies in the ascending colon showed 68.7 ± 9.2% of GLP-1 positive cells co-localised with PYY. Both GLP-1 and PYY also co-localised with CaSR (54.5 ± 9.1% and 45.6 ± 8.6%, respectively) in the ascending colon. In order to identify nutrients that would activate EEC, we stimulated mucosa of ascending colon with 10 mmolL-1 glutamic (Glu)/aspartic (Asp) acids (which activate T1R1 and CaSR) and 50 mmolL-1 phenylalanine (Phe)/tryptophan (Trp) (selective activators of CaSR). Using pERK as a marker for cell activation, more EEC were activated in Glu/Asp stimulated mucosa compared to buffer controls. However, in mucosa stimulated with Phe/ Trp, pERK activation was not observed. Since recombinant CaSR activation is associated with pCAMKII activation (Rey et al., Cell Physiol 2010), we localised pCAMKII and found Phe/Trp stimulation markedly increased pCAMKII expression in EECs compared to buffer controls. Conclusions: While short-chain fatty acid receptors are found mainly in the stomach, amino acid receptors, along with associated hormones are expressed until the ascending colon. We have also shown that human colonic EEC respond to specific nutrient stimulation via distinct pathways. Taken together, these results suggest that the colon is an important site for nutrient sensing and is therefore a potential target for appetite regulation. Supported by Wellcome Trust.
BACKGROUND: Ezetimibe is a widely-used drug that lowers plasma cholesterol by blocking NPC1L1-mediated sterol absorption in small intestine. Enterocyte cholesterol homeostasis is controlled by the aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Endogenous cholesterol synthesis and low density lipoprotein (LDL) uptake are coordinately regulated by transcription factors termed Sterol Regulatory Element-Binding Proteins (SREBPs). Liver-X-receptors (LXRs), another family of transcription factors, are activated by sterols and control other aspects of sterol metabolism, especially cholesterol efflux. How blockade of cholesterol absorption by ezetimibe (EZE) affects intestinal SREBPs, LXRs, and key effector molecules, such as LDL Receptor (LDLR) and HMG-CoA Reductase (HMGR) is unknown. AIM: To study the effect of EZE on regulatory molecules that control sterol synthesis and LDL uptake in small intestine. METHODS: C57BL/6 mice were fed a chow diet with indicated drugs. Liver and small intestine were harvested. Jejunal enterocytes were isolated, and tissues fractionated for immunoblotting, which were quantified by densitometry. mRNAs were quantified by qPCR and microarray analysis. RESULTS: Feeding EZE increased active nuclear SREBP-2 by 8-fold in intestine, leading to increases in mRNAs for HMGR and LDLR by 1.3-fold +/- 0.03 (P<0.01) and 2.0-fold +/- 0.13 (P<0.01), respectively. HMGR and LDLR protein levels increased much more profoundly (24-fold and 6-fold, respectively). Microarray analysis revealed that in jejunum, EZE increased the expression of 20 out of 21 SREBP-2 target genes encoding enzymes required for sterol biosynthesis. The mRNA of a LXR target gene, Inducible Degrader Of the LDLR (IDOL), was reduced by 46% +/- 6% (P<0.01) in EZE-treated intestine. Coadministration of EZE with an LXR agonist (T0901317) abolished EZE-mediated reduction in IDOL mRNA and prevented its induction of LDLR protein in the intestine. Hepatic LDLR was unaffected in these studies. CONCLUSION: To maintain sterol homeostasis in the face of EZE-induced blockade of cholesterol uptake, enterocytes boost LDL uptake by increasing LDLR levels and boost sterol synthesis by increasing HMGR and other genes needed for sterol synthesis. These changes in gene expression are mediated by an increase in active SREBP-2. For HMGR and LDLR, it is likely that this regulation is mediated by both transcriptional and posttranscriptional mechanisms. The reduced sterol input caused by EZE is associated with a reduction in LXR target genes including IDOL. The concerted increase in LDLR mRNA through SREBP-2 and reduction in IDOL leads to a dramatic increase in LDLR protein levels. These studies reveal a previously unknown homeostatic network in enterocytes engaged by pharmacologic blockage of cholesterol absorption.
1105 Blocking Intestinal Chylomicron Assembly Improves Systemic Glucose Tolerance Through Incretin Mediated Effects on Glucose Absorption and FGF15 Mediated Alterations of Hepatic Glucose Metabolism Hitoshi Matsumoto, Yan Xie, Susan Kennedy, Jianyang Luo, Nicholas O. Davidson Intestinal microsomal triglyceride transfer protein (Mttp) is required for the assembly and secretion of chylomicrons. Conditional intestine-specific Mttp-knockout (Mttp-IKO) mice exhibit defective intestinal lipid absorption and reduced serum lipid levels. We now find that Mttp-IKO mice exhibit improved glucose tolerance, despite no significant differences in body weight. Oral glucose tolerance testing revealed reduced AUC in Mttp-IKO mice
S-199
AGA Abstracts
AGA Abstracts
Dietary Protein Influences Growth Through a Complex Mechanism Coupling Proteolysis and Intestinal Peptide Transporter Expression Stephanie A. Hansen, Aaron Ashley, Debra Titmus, Brian M. Chung
1103
Adequate intestinal nutrient absorption is required for growth and development. Dietary proteins must be digested to amino acids or peptides that can be absorbed via heteromeric amino acid carriers or intestinal peptide transporters. We hypothesized that intestinal peptide transport impacts growth more than amino acid absorption. Caenorhabditis elegans was used to assay the roles of the intestinal peptide transporter Opt-2 (homologous to PepT1) and amino acid carrier regulatory protein ATGP-1 (neutral amino acid carrier SLC3A1 homologue) on growth. Animals were raised in polymeric (NGM) or elemental (CeHR) diet and harvested daily for 4-days to assay age-related body length. Additionally, RNA was isolated to determine Opt-2 and ATGP-1 transcript levels and dietary green fluorescent protein (GFP) was used as a marker of intestinal proteolysis. Data were analyzed for variance (ANOVA) and expressed as mean±SEM. A significant effect of diet on daily body length was found at days 1 and 2 where animals fed a polymeric diet demonstrated 46% increased growth (d1=508±11, d2=1007±22μm, n=36) compared to those fed an elemental diet (CeHR, d1=370±9, d2=649±11, p<0.05). NGM animals demonstrated almost 100% increased Opt2 mRNA expression compared to CeHR, with no effect on ATGP-1 transcription. NGM animals also demonstrated significantly decreased (80%) intestinal proteolytic activity as measured by increased dietary GFP activity along the entire gut length compared to CeHR. Interestingly, animals transferred to NGM after 1 day in CeHR demonstrated an immediate improvement in growth (d2=822±16, p<0.05) with no increase in Opt-2 or ATGP-1 mRNA compared to CeHR. These transferred animals also showed a marked decrease in dietary GFP proteolytic activity mimicking that of those grown solely in NGM. These findings demonstrate that intestinal absorption of peptides plays a more influential role over growth than amino acid absorption, however the effect is not simply related to intestinal peptide transporter levels. Sole reliance on intestinal amino acid absorption through the provision of elemental diet increases intestinal proteolytic activity with no corresponding increase in amino acid transporter expression or improvement in growth. Animals transferred from elemental to polymeric diet demonstrated significantly improved growth despite decreased proteolytic activity despite expressing far fewer intestinal peptide transporters as animals fed solely polymeric diet. The metabolic cost of complete digestion can apparently be offset by incomplete digestion to permit more intestinal peptide absorption, potentially releasing energy for improved growth. In conclusion, the careful design of enteral formulae to efficiently balance proteolytic activity with intestinal peptide absorption could improve outcomes for short bowel syndrome or pediatric intestinal failure.
Phosphorylation of Sar1b disassembles the FABP1 containing, 75 kDa complex. A) The complex in native cytosol. B) Phosphorylation of Sar1b Thr by PKCζ. C) Phosphorylation of Sar1b completely disassembles the complex. D) The freed FABP1 binds to the ER. Not shown: On FABP1 binding to the ER, PCTV are generated. FABP1 cannot bind to the ER when a member of the 75 kDa complex.
1104 Molecular Aspects of Nutrient Sensing in the Human Gastrointestinal Tract Madusha Peiris, Michael A. Atiba, Andy Hubball, David C. Bulmer, Charles H. Knowles, L. Ashley Blackshaw
1102 Pharmacologic Blockage of Cholesterol Absorption With Ezetimibe Reveals a Novel Homeostatic Network in Enterocytes Luke Engelking, Guosheng Liang, Matthew R. McFarlane, Christina Li
Background: Nutrient sensing within the gastrointestinal (GI) tract occurs via specific receptors that detect a variety of luminal stimuli. Activation of these receptors leads directly to humoral and neural signals that modulate absorption, motility and satiety. However, detailed knowledge of mechanisms involved in nutrient/receptor interaction and nutrient activation of enteroendocrine cells (EEC) is currently lacking. Aim: To characterise taste receptor expression profile and co-localisation with specific gut hormones, as well as, elucidate the effect of nutrient exposure on EEC. Methods: Healthy colon, ileum and antrum samples were obtained from surgical resection specimens. Fluorescent immunohistochemistry was performed on sections cut from Zamboni's fixed tissue. Nutrient exposure studies were carried out on freshly dissected mucosa using an Ussing Chamber. Results: Human antral mucosa showed immunoreactivity (IR) for the amino acid sensing receptors T1R1 and calcium sensing receptor (CaSR), and the long chain fatty acid receptors GPR40 and GPR120. Only CaSR was IR in the ileum and the ascending colon. Gut hormones GLP-1 and PYY were found throughout the GI tract and co-localisation studies in the ascending colon showed 68.7 ± 9.2% of GLP-1 positive cells co-localised with PYY. Both GLP-1 and PYY also co-localised with CaSR (54.5 ± 9.1% and 45.6 ± 8.6%, respectively) in the ascending colon. In order to identify nutrients that would activate EEC, we stimulated mucosa of ascending colon with 10 mmolL-1 glutamic (Glu)/aspartic (Asp) acids (which activate T1R1 and CaSR) and 50 mmolL-1 phenylalanine (Phe)/tryptophan (Trp) (selective activators of CaSR). Using pERK as a marker for cell activation, more EEC were activated in Glu/Asp stimulated mucosa compared to buffer controls. However, in mucosa stimulated with Phe/ Trp, pERK activation was not observed. Since recombinant CaSR activation is associated with pCAMKII activation (Rey et al., Cell Physiol 2010), we localised pCAMKII and found Phe/Trp stimulation markedly increased pCAMKII expression in EECs compared to buffer controls. Conclusions: While short-chain fatty acid receptors are found mainly in the stomach, amino acid receptors, along with associated hormones are expressed until the ascending colon. We have also shown that human colonic EEC respond to specific nutrient stimulation via distinct pathways. Taken together, these results suggest that the colon is an important site for nutrient sensing and is therefore a potential target for appetite regulation. Supported by Wellcome Trust.
BACKGROUND: Ezetimibe is a widely-used drug that lowers plasma cholesterol by blocking NPC1L1-mediated sterol absorption in small intestine. Enterocyte cholesterol homeostasis is controlled by the aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Endogenous cholesterol synthesis and low density lipoprotein (LDL) uptake are coordinately regulated by transcription factors termed Sterol Regulatory Element-Binding Proteins (SREBPs). Liver-X-receptors (LXRs), another family of transcription factors, are activated by sterols and control other aspects of sterol metabolism, especially cholesterol efflux. How blockade of cholesterol absorption by ezetimibe (EZE) affects intestinal SREBPs, LXRs, and key effector molecules, such as LDL Receptor (LDLR) and HMG-CoA Reductase (HMGR) is unknown. AIM: To study the effect of EZE on regulatory molecules that control sterol synthesis and LDL uptake in small intestine. METHODS: C57BL/6 mice were fed a chow diet with indicated drugs. Liver and small intestine were harvested. Jejunal enterocytes were isolated, and tissues fractionated for immunoblotting, which were quantified by densitometry. mRNAs were quantified by qPCR and microarray analysis. RESULTS: Feeding EZE increased active nuclear SREBP-2 by 8-fold in intestine, leading to increases in mRNAs for HMGR and LDLR by 1.3-fold +/- 0.03 (P<0.01) and 2.0-fold +/- 0.13 (P<0.01), respectively. HMGR and LDLR protein levels increased much more profoundly (24-fold and 6-fold, respectively). Microarray analysis revealed that in jejunum, EZE increased the expression of 20 out of 21 SREBP-2 target genes encoding enzymes required for sterol biosynthesis. The mRNA of a LXR target gene, Inducible Degrader Of the LDLR (IDOL), was reduced by 46% +/- 6% (P<0.01) in EZE-treated intestine. Coadministration of EZE with an LXR agonist (T0901317) abolished EZE-mediated reduction in IDOL mRNA and prevented its induction of LDLR protein in the intestine. Hepatic LDLR was unaffected in these studies. CONCLUSION: To maintain sterol homeostasis in the face of EZE-induced blockade of cholesterol uptake, enterocytes boost LDL uptake by increasing LDLR levels and boost sterol synthesis by increasing HMGR and other genes needed for sterol synthesis. These changes in gene expression are mediated by an increase in active SREBP-2. For HMGR and LDLR, it is likely that this regulation is mediated by both transcriptional and posttranscriptional mechanisms. The reduced sterol input caused by EZE is associated with a reduction in LXR target genes including IDOL. The concerted increase in LDLR mRNA through SREBP-2 and reduction in IDOL leads to a dramatic increase in LDLR protein levels. These studies reveal a previously unknown homeostatic network in enterocytes engaged by pharmacologic blockage of cholesterol absorption.
1105 Blocking Intestinal Chylomicron Assembly Improves Systemic Glucose Tolerance Through Incretin Mediated Effects on Glucose Absorption and FGF15 Mediated Alterations of Hepatic Glucose Metabolism Hitoshi Matsumoto, Yan Xie, Susan Kennedy, Jianyang Luo, Nicholas O. Davidson Intestinal microsomal triglyceride transfer protein (Mttp) is required for the assembly and secretion of chylomicrons. Conditional intestine-specific Mttp-knockout (Mttp-IKO) mice exhibit defective intestinal lipid absorption and reduced serum lipid levels. We now find that Mttp-IKO mice exhibit improved glucose tolerance, despite no significant differences in body weight. Oral glucose tolerance testing revealed reduced AUC in Mttp-IKO mice
S-199
AGA Abstracts
AGA Abstracts
Dietary Protein Influences Growth Through a Complex Mechanism Coupling Proteolysis and Intestinal Peptide Transporter Expression Stephanie A. Hansen, Aaron Ashley, Debra Titmus, Brian M. Chung