AGA Abstracts BBM Na-dependent nutrient co-transporters and Cl-HCO3 exchangers in intestinal epithe- lial cells. Mo1762 A Novel Role of GLP-1 Nanomedicine in Amelioration of Gut Inflammation Arivarasu Natarajan Anbazhagan, Shubha Priyamvada, Anoop Kumar, Tarunmeet Gujral, Dulari Jayawardena, Mentor Thaqi, Edurne Mugarza, Hayat Onyuksel, Pradeep K. Dudeja Glucagon-like peptide-1 (GLP-1) is an important gut hormone, which in intestine is secreted by L-cells. In addition to its beneficial roles in insulin secretion, obesity, cardio and neuro protection, recently GLP-1 has also been shown to act as an anti-inflammatory agent. However, its use in clinical practice has been hampered by its short half-life and by the fact that commercially available analogues have increased risk of immunogenicity. It has recently been shown that novel formulation composed of human GLP-1 self-associated to a sterically stabilized phospholipid micelles (SSM), GLP-1 nanomedicine, increased the stability of circulating GLP-1 in the blood, and the nanomedicine had an anti-inflammatory effect against LPS induced inflammation in lungs. Whether GLP-1 nanomedicine exerts anti-inflammatory effects in intestine is not known. We hypothesized that GLP-1 nanomedicine decreases intestinal inflammation and attenuates the associated diarrhea. To test this hypothesis, we used Dextran Sodium Sulfate (DSS) (3% in drinking water for 7 days) induced colitis mouse as a model for intestinal inflammation. Animals were divided into 4 groups (5 mice/group) and i.p. injections of vehicle (SSMs) or GLP-1 (15 nmole/100μl) were given daily: Group 1(Control): vehicle alone; Group 2 (DSS): vehicle+DSS; Group 3 (GLP-1): GLP-1 alone; Group 4 (GLP-1+DSS). Mucosa was scraped from distal colon on day 8 for qRT-PCR and western blotting. Tissue sections were used for immunostaining. DSS mice showed a signifi- cant decrease in body weight as compared to control. However, GLP-1 treatment to DSS mice partially attenuated the weight loss. When colons were harvested from experimental mice and examined, loose fecal pellets were observed in the colon of DSS colitis mice reflecting diarrheal phenotype, which was partly alleviated in GLP-1 treated mice. Further, treatment with GLP-1 alleviated the increase in expression of the pro-inflammatory cytokines in colonic epithelium of DSS mice e.g. IL-1β (Control: 2±0.6; DSS: 201±43; GLP-1: 2±0.3 GLP-1+DSS: 78±20); and CXCL-1 (Control: 1±0.3; DSS: 18.9±7; GLP-1: 1.3±0.9; GLP- 1+DSS: 12±3). Increased levels of pro-inflammatory cytokines have been implicated in the associated diarrhea by decreased expression of chloride transporter SLC26A3 or down regulated in adenoma (DRA). Western blot results showed that DSS decreased the expression of DRA as compared to control but GLP-1 abrogated this effect of DSS (Control: 0.9±0.1; DSS: 0.3±0.1; GLP-1: 1.2±0.1; GLP-1+DSS: 0.6±0.2). This data was further confirmed by immunostaining. Our data showed that GLP-1 nanomedicine is effective in reducing intestinal inflammation and the associated diarrhea. We speculate that GLP-1 nanomedicine could be used as a novel therapeutic approach to treat patients with Crohn's disease and ulcerative colitis. (Supported by NIDDK and Dept. of Veteran Affairs) Mo1763 Sorting Nexin 27 (SNX27) Is a Member of NHERF (Na + /H + Exchanger Regulatory Factor) Family of PDZ Adaptor Proteins Karen Bannert, Peggy Bodammer, Julia Glamann, Katja Bovensiepen, Georg Lamprecht Introduction: Intestinal electroneutral NaCl absorption is mediated by parallel Na + /H + and Cl - /HCO 3 - exchange in the enterocyte apical membrane. The ion transporters involved are NHE3 (SLC9A3) and DRA (SLC26A3), both of which are regulated by adaptor proteins of the NHERF (Na + /H + exchanger regulatory factor) family via PDZ domain (PSD95, disc large, ZO1) interaction. Sorting Nexin 27 (SNX27) is a protein specifically expressed in early endosomes which contains a PX and a single PDZ domain and binds to phosphatidylinositol 3-phosphate-enriched compartments. SNX27 is involved in PDZ-directed sorting and recy- cling of transmembrane proteins from early endosomes to the plasma membrane and thereby regulates protein function. The PDZ domain of SNX27 shares high homology with those present in NHERF family proteins NHERF and E3KARP. SNX27 has thus been proposed to belong to the NHERF family. We therefore investigated whether there is a specific PDZ domain-mediated interaction of SNX27 with DRA. Methods: FLISA (fluorophore-linked immunosorbent assay): The PDZ domain of SNX27 (PDZSNX27) expressed as a His-tag fusion protein was bound to Nickel-coated microplates. Immobilized PDZSNX27 was incu- bated with increasing amounts of the biotin-labeled C-terminus of DRA and bound protein was detected by fluorescent labeled streptavidin. GST pull down assay: The fusion construct GST-PDZSNX27 was used to pull down EGFP-DRA from HEK/EGFP-DRA cells using mag- netic glutathione beads. Precipitated EGFP-DRA was detected by anti-EGFP. CoIP: The fusion construct mCherry-HA-SNX27 was transiently transfected into HEK/EGFP-DRA cells. SNX27 was immunoprecipitated using anti-HA-agarose. Co-immunoprecipitated EGFP-DRA was detected by anti-EGFP. In all interaction experiments, a mutant of DRA which lacks the C-terminal PDZ interaction motif (ETKFminus), served as a negative control. Results: In the FLISA, the C-terminus of DRA bound to PDZSNX27 with a half-maximal binding of 50 nM. Thus, binding affinity of DRA to SNX27 was similar to NHERF and E3KARP (n= 5). In contrast, C-DRA-ETKFminus did not bind to PDZSNX27 (n=5). GST-PDZSNX27 pulled down EGFP-DRA but not EGFP-DRA-ETKFminus from cell lysates (n=8). EGFP- DRA but not EGFP-DRA-ETKFminus was co-immunoprecipitated with mCherry-HA-SNX27 (n=5). Summary and Conclusion: Three different assays (FLISA, GST pull down and CoIP) indicate that SNX27 binds to DRA via its PDZ domain. We have previously shown that DRA endogenously cycles through the plasma membrane, early and recycling endosomes and that the PDZ interaction motif of DRA is required for this process. Because SNX27 is specifically located in early endosomes, it seems possible that it is involved in the recycling of DRA. Current investigations address the influence of SNX27 on the recycling of DRA and therefore the functional consequences of the interaction of the two proteins. S-654 AGA Abstracts Mo1764 Protein Kinase G Mediated Regulation of Na-Glucose Co-Transport in Na:H Exchange Silenced Intestinal Epithelial Cells Balasubramanian Palaniappan, Subha Arthur, Uma Sundaram Background: Previous studies have demonstrated that direct inhibition of intestinal epithelial cell brush border membrane (BBM) Na:H exchange (NHE3) results in the stimulation of Na-glucose co-transport (SGLT1). Also previous in vitro and in vivo studies has demonstrated that constitutive nitric oxide (cNO) inhibits BBM NHE3 while stimulating SGLT1 in the BBM of rat intestinal epithelial cells (IEC-18) and rabbit villus cells. Also, NHE3 has been shown to regulate SGLT1 through cNO in stable siRNA NHE3 transfected IEC-18 cells (NHE3KO). However, the molecular mechanism of regulation of SGLT1 by NHE3 by cNO in NHE3KO cells is unknown. Hypothesis: Ca++ mediated increase in cNO production stimulates SGLT1 in NHE3KO cells through protein kinase G (PKG) pathway. Aim: Determine the role of Ca++ and PKG pathway in the regulation of SGLT1 in NHE3KO cells. Methods: NHE3KO cells grown to confluent monolayers on 24 well plates were used. BAPTA was used to chelate intracellular Ca++ (20 μM/24 hr), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin- 1-one (ODQ; 500 μM/24 hr) was used to inhibit guanylyl cyclase, Rp-cGMP (50 μM/24 hr) to inhibit PKG while 8-bromo cGMP(8-Bro) was used to activate PKG. Na-dependant 3H- OMG uptake was used to determine SGLT1 activity. Rat specific SGLT1 antibody was used for Western blot analysis. Results: The reversal of stimulation of SGLT1 when Ca++ was inhibited with BAPTA in NHE3KO cells was restored by PKG activation with 8-Bro in these cells (control 1701 ± 92.7; NHE3KO 2297 ± 180.2; NHE3KO + BAPTA + 8-Bro: 2309 ± 31.6 pmol/mg protein; n=3, p<0.05). Specific inhibition of cGMP formation with ODQ or PKG with Rp-cGMP showed a reversal of stimulation of SGLT1 in NHE3KO cells (control 1887 ± 15.4; NHE3KO 2395 ± 55.7; NHE3KO + ODQ: 1842 ± 47.9; NHE3KO + Rp- cGMP: 1810 ± 39.0; pmol/mg protein; n=3, p<0.05). Western blot studies showed that PKG pathway inhibitors, Rp-cGMP or ODQ restored SGLT1 protein levels in NHE3KO cells. Conclusions: Increased intracellular Ca++ likely mediates the stimulation of SGLT1 in NHE3KO cells by up regulating cNO production. Unregulated cNO production activates protein kinase G mediated pathway, which in turn directly mediates the stimulation of SGLT1 in NHE3 silenced intestinal epithelial cells by increasing the number of co-transporters. Mo1765 3D Cell Culture of CaCo2: A Better Model to Study the Functionality and Regulation of Intestinal Ion Transporters Ishita Chatterjee, Anoop Kumar, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja Traditional two-dimensional (2D) cell monolayers have been widely used as an in vitro model of intestinal epithelium. However, 2D system fails to represent the physiological complexity and pose limitations to some extent by forcing the cells to adapt to an artificial, flat and rigid surface. Thus, development of more advanced and reliable in vitro model systems is crucial as an alternative to conventional in vivo studies. In this regard, the three- dimensional (3D) culture almost mimics physiological conditions that exist in vivo. Utilizing Caco-2 cells as a 3D model epithelium, recent studies have demonstrated their suitability for testing drug permeability and epithelial morphogenesis. However, the suitability of 3D models for studies of intestinal epithelial ion transporters has not been addressed. Current studies were performed to validate 3D Caco2 model system for investigating expression of major apical ion transporters and their modulation by key pro-inflammatory agents. Caco- 2 cells (10 4 cells/ml) were grown in 96 well plates on matrigel for up to 12 days. Unlike 2D monolayers, in 3D culture, Caco2 cells started forming spheroid like structures at day 3 and were utilized at 6d or 12d post-plating for examining the expression of ion transporters by RT-PCR, western blotting and microscopy. To evaluate the differentiation status of cells, expression of SLC26A3 (DRA), the major apical Cl - /HCO 3 - exchanger, (highly expressed in differentiated cells) was measured at 6d and 12d post plating. DRA expression was increased by ~100 fold at 12d as compared to 6d. The 3D culture at 12d also exhibited decent expression of other ion transporters such as SLC9A3 (Na + /H + exchanger isoform 3) and SLC6A4 (serotonin transporter). In 2D model, Caco2 cells do not respond well to pro- inflammatory agents e.g. lipopolysaccharide (LPS) and tumor necrosis factor (TNF). To evaluate the importance of the 3D model over 2D for inflammatory responses, we also examined the expression of MyD88, an activator of MAP-kinase essential for the response of epithelial cells to LPS. Unlike 2D culture, in 3D culture Caco-2 showed responsiveness to LPS (20 ng/ml), as it markedly decreased DRA mRNA levels (~70%) at 48h. In parallel, 3D culture also showed significantly higher levels of MyD88 mRNA (~4 fold) and protein (~2 fold) compared to 2D model, where MyD88 was almost undetectable. Similarly, respon- siveness of tumor necrosis factor (TNF, 50 ng/ml) in decreasing DRA mRNA levels of Caco- 2 cells in 3D microenvironment was also considerably higher, ~50% reduction in DRA as compared to a modest decrease of DRA in 2D cells. In conclusion, our studies for the first time demonstrate that 3D culture model of Caco-2 cells may be more physiologically relevant to assess the responsiveness of ion transporters to various inflammatory agents.(Supported by Dept. of Veterans Affairs and NIDDK). Mo1766 Effects of the Dipeptidyl Peptidase-4 Inhibitor, Sitagliptin, on Colorectal Carcinogenesis in a Model of Type 2 Diabetes Takuya Inoue, Naoki Yorifuji, Kaori Fujiwara, Taisuke Sakanaka, Munetaka Iguchi, Ken Narabayashi, Toshihiko Okada, Sadaharu Nouda, Kazuki Kakimoto, Kumi Ishida, Ken Kawakami, Yosuke Abe, Toshihisa Takeuchi, Eiji Umegaki, Kazuhide Higuchi Background: Patients with type 2 diabetes mellitus are known to have an increased risk of colorectal neoplasia compared with those without diabetes. Dipeptidyl peptidase-4 (DPP- 4) inhibitors delay the degradation of the gut incretin, glucagon-like peptide-1 (GLP-1), which in turn results in a reduction in post-prandial glucose. DPP4 inhibitors are therefore regarded as a new therapeutic agent for type 2 diabetes. Because the intestinotrophic hormone, GLP-2, is also rapidly degraded and deactivated by DPP-4, DPP-4 inhibition may increase the risk of colorectal tumors by potentiating the effects of endogenous GLP-2. However,