Steven Coon

Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells

Sodium absorption in the mammalian small intestine occurs predominantly by two primary pathways that include Na/H exchange (NHE3) and Na-glucose cotransport (SGLT1) on the brush border membrane (BBM) of villus cells. However, whether NHE3 and SGLT1 function together to regulate intestinal sodium absorption is unknown. Nontransformed small intestinal epithelial cells (IEC-18) were transfected with either NHE3 or SGLT1 small interfering RNAs (siRNAs) and were grown in confluent monolayers on transwell plates to measure the effects on Na absorption. Uptake studies were performed as well as molecular studies to determine the effects on NHE3 and SGLT1 activity. When IEC-18 monolayers were transfected with silencing NHE3 RNA, the cells demonstrated decreased NHE3 activity as well as decreased NHE3 mRNA and protein. However, in NHE3 siRNA-transected cells, SGLT1 activity, mRNA, and protein in the BBM were significantly increased. Thus, inhibition of NHE3 expression regulates the expression and function of SGLT1 in the BBM of intestinal epithelial cells. In addition, IEC-18 cells transected with silencing SGLT1 RNA demonstrated an inhibition of Na-dependent glucose uptake and a decrease in SGLT1 activity, mRNA, and protein levels. However, in these cells, Na/H exchange activity was significantly increased. Furthermore, NHE3 mRNA and protein levels were also increased. Therefore, the inhibition of SGLT1 expression stimulates the transcription and function of NHE3 and vice versa in the BBM of intestinal epithelial cells. Thus this study demonstrates that the major sodium absorptive pathways together function to regulate sodium absorption in epithelial cells.

Glucocorticoids differentially regulate Na-bile acid cotransport in normal and chronically inflamed rabbit ileal villus cells

Previous studies have demonstrated that apical Na-bile acid cotransport (ASBT) is inhibited during chronic ileitis by both a decrease in the affinity as well as a decrease in the number of cotransporters. Methylprednisolone (MP), a commonly used treatment for inflammatory bowel disease (IBD, e.g., Crohns disease), has been shown to reverse the inhibition of several other Na-solute cotransporters during chronic enteritis. However, the effect of MP on ASBT in the chronically inflamed ileum is not known. MP stimulated ASBT in villus cells from the normal rabbit ileum by increasing the cotransporter expression without a change in the affinity of the cotransporter for bile acid. Western blot studies demonstrated an increase in cotransporter expression. MP reversed the inhibition of ASBT in villus cells from the chronically inflamed ileum. Kinetic studies demonstrated that the mechanism of MP-mediated reversal of ASBT inhibition was secondary to a restoration of both affinity as well as cotransporter numbers. Western blot analysis demonstrated restoration of cotransporter numbers after MP treatment of rabbits with chronic ileitis. Thus MP stimulates ASBT in the normal ileum by increasing cotransporter numbers. MP reverses the inhibition of ASBT during chronic ileitis. However, MP restores the diminished affinity as well as cotransporter expression levels during chronic ileitis. Thus MP differentially regulates ASBT in the normal and in the chronically inflamed ileum.

Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells

Previous in vivo studies suggest that constitutive nitric oxide (cNO) can regulate Na- glucose cotransport (SGLT1) and Na-H exchange (NHE3) in rabbit intestinal villus cells. Whether these two primary Na absorbing pathways are directly regulated by cNO and the mechanisms of this regulation in the enterocyte is not known. Thus nontransformed rat small intestinal epithelial cells (IEC-18) were treated with N(G)-nitro-l-arginine methyl ester (l-NAME), which directly decreased cNO in these cells. l-NAME treatment decreased SGLT1 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of inhibition was secondary to a decrease in the affinity of the cotransporter for glucose without a change in the number of cotransporters. In contrast, l-NAME treatment increased NHE3 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of stimulation was by increasing the number of the exchangers without a change in the affinity for Na. Quantitative RT-PCR (RTQ-PCR) and Western blot analysis of SGLT1 demonstrated no change in mRNA and protein, respectively. RTQ-PCR and Western blot analysis of NHE3 indicated that NHE3 was increased by l-NAME treatment by an increase in mRNA and protein, respectively. These results indicate that decreased cNO levels directly mediate the inhibition of SGLT1 and stimulation of NHE3 in intestinal epithelial cells. Thus cNO directly but uniquely regulates the two primary Na-absorptive pathways in the mammalian small intestine.

Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells

We have shown recently that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1) augments H(+) peptide cotransporter (PepT1)-mediated peptide absorption in murine jejunum. While we observed that inhibiting cAMP production decreased this augmentation of PepT1 activity by GIP, it was unclear whether PKA and/or other regulators of cAMP signaling pathway(s) were involved. This study utilized tritiated glycyl-sarcosine [(3)H-glycyl-sarcosine (Gly-Sar), a relatively nonhydrolyzable dipeptide] uptake to measure PepT1 activity in CDX2-transfected IEC-6 (IEC-6/CDX2) cells, an absorptive intestinal epithelial cell model. Similar to our earlier observations with mouse jejunum, GIP but not GLP-1 augmented Gly-Sar uptake (control vs. +GIP: 154 ± 22 vs. 454 ± 39 pmol/mg protein; P < 0.001) in IEC-6/CDX2 cells. Rp-cAMP (a PKA inhibitor) and wortmannin [phosophoinositide-3-kinase (PI3K) inhibitor] pretreatment completely blocked, whereas neither calphostin C (a potent PKC inhibitor) nor BAPTA (an intracellular Ca(2+) chelator) pretreatment affected the GIP-augmented Gly-Sar uptake in IEC-6/CDX2 cells. The downstream metabolites Epac (control vs. Epac agonist: 287 ± 22 vs. 711 ± 80 pmol/mg protein) and AKT (control vs. AKT inhibitor: 720 ± 50 vs. 75 ± 19 pmol/mg protein) were shown to be involved in GIP-augmented PepT1 activity as well. Western blot analyses revealed that both GIP and Epac agonist pretreatment enhance the PepT1 expression on the apical membranes, which is completely blocked by wortmannin in IEC-6/CDX2 cells. These observations demonstrate that both cAMP and PI3K signaling pathways augment GIP-induced peptide uptake through Epac and AKT-mediated pathways in intestinal epithelial cells, respectively. In addition, these observations also indicate that both Epac and AKT-mediated signaling pathways increase apical membrane expression of PepT1 in intestinal absorptive epithelial cells.

Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum

Glucose-dependent insulinotropic polypeptide (GIP) secreted from jejunal mucosal K cells augments insulin secretion and plays a critical role in the pathogenesis of obesity and Type 2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose cotransporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulate other intestinal nutrient absorptive processes. The present study investigated the effect of GIP on proton-peptide cotransporter-1 (PepT1) that mediates di- and tripeptide absorption as well as peptidomimetic drugs. Immunohistochemistry studies localized both GIP receptor (GIPR) and PepT1 proteins on the basolateral and apical membranes of normal mouse jejunum, respectively. Anti-GIPR antibody detected 50-, 55-, 65-, and 70-kDa proteins, whereas anti-PepT1 detected a 70-kDa proteins in mucosal homogenates of mouse jejunum. RT-PCR analyses established the expression of GIPR- and PepT1-specific mRNA in mucosal cells of mouse jejunum. Absorption of Gly-Sar (a nondigestible dipeptide) measured under voltage-clamp conditions revealed that the imposed mucosal H(+) gradient-enhanced Gly-Sar absorption as an evidence for the presence of PepT1-mediated H(+):Gly-Sar cotransport on the apical membranes of mouse jejunum. H(+):Gly-Sar absorption was completely inhibited by cephalexin (a competitive inhibitor of PepT1) and was activated by GIP. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP antagonist). In contrast to GIP, the ileal L cell secreting glucagon-like peptide-1 (GLP-1) did not affect the H(+):Gly-Sar absorption in mouse jejunum. We conclude from these observations that GIP, but not GLP-1, directly activates PepT1 activity by a cAMP-dependent signaling pathway in jejunum.

Su1777 GIP Activation of Signaling Pathways Traffick PepT1 Proteins to the Apical Membrane of Intestinal Epithelial Cells

Background: In our previous study, using lymph fistula rat model, we have shown that fat ingestion stimulates release of mast cell mediators such as histamine, rat mucosal mast cell protease II (RMCP II), and prostaglandin D2 (PGD2) into intestinal lymph; however, it is unclear whether these responses are different depending on the type of fatty acid ingested. Aim: The purpose of this study was to determine the effects of dietary long-chain fatty acids with varying degrees of saturation on intestinal mast cell activation. Methods: We investigated lymphatic mast cell mediator concentrations after administration of three types of triacylglycerols, trilinolein (C18:2 n-6), trilinolenin (C18:3 n-3) or triolein (C18:1 n-9) by using the lymph fistula rat model. After cannulation of the major mesenteric lymph duct and duodenum of male Sprague-Dawley rat, a 3 ml bolus mixture of phosphate-buffered, taurocholatestabilized emulsion containing 120μmol of trilinolein or trilinolenin or triolein was provided to each animal through the intraduodenal feeding tube. The emulsion medium without triacylglycerol was used as control. Lymph was continuously collected for 6 hours and analyzed for RMCPII, histamine, PGD2, triglyceride and protein content. Results: Infusion of trilinolein and trilinolenin significantly increased the lymphatic release of mast cell mediators and influenced lymphatic protein concentration. In contrast, triolein did not induce the release of mast cell mediators. The peak RMCPII concentration after infusion of trilinolein (432.97 ± 77.53 ng/ml) and trilinolenin (317.50 ± 47.59 ng/ml) were significantly greater than triolein (141.46 ± 23.46 ng/ml) ( P,0.01, P,0.05, respectively). Similar to the RMCPII response, the peak values of histamine were significantly greater than triolein (12.97 ± 0.62ng/ml) for trilinolein (34.2 ± 1.7 ng/ml, P,0.01, n=3) and trilinolenin (19.85 ± 1.06ng/ ml, P,0.01, n=3). Infusion of trilinolein induced a significant increase of lymphatic PGD2 concentration peaked at 2 hour compared with the control (1461.23 ± 133.12 vs. 842.30 ± 129.02 pg/ml, P,0.05), but triolein did not induce (978.43 ± 153.74 pg/ml). Conclusion: These observations identify that the intestinal mast cell activation induced by dietary fat is dependent on the degree of unsaturation in fatty acid.

Su1885 Prolonged High-Fat Feeding Decreases SGLT1 and GLUT2 Activity and Blunts Their Upregulation by Glucose-Dependent Insulinotropic Polypeptide (GIP) in Obese Mice

generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.