Tarunmeet Gujral

A novel anti-inflammatory role of GPR120 in intestinal epithelial cells

GPR120 (free fatty acid receptor-4) is a G protein-coupled receptor for medium- and long-chain unsaturated fatty acids, including ω-3 fatty acids. Recent studies have shown GPR120 to play cardinal roles in metabolic disorders via modulation of gut hormone secretion and insulin sensitivity and to exert anti-inflammatory effects in macrophages and adipose tissues. However, information on anti-inflammatory role of GPR120 at the level of intestinal epithelium is very limited. Current studies demonstrated differential levels of GPR120 mRNA and protein along the length of the human, mouse, and rat intestine and delineated distinct anti-inflammatory responses following GPR120 activation in model human intestinal epithelial Caco-2 cells, but not in model mouse intestinal epithelial endocrine cell line STC-1. In Caco-2 cells, GPR120 was internalized, bound to β-arrestin-2, and attenuated NF-κB activation in response to 30-min exposure to the agonists GW9508, TUG-891, or docosahexaenoic acid. These effects were abrogated in response to small interfering RNA silencing of β-arrestin-2. Treatment of STC-1 cells with these agonists did not induce receptor internalization and had no effects on NF-κB activation, although treatment with the agonists GW9508 or TUG-891 for 6 h augmented the synthesis and secretion of the gut hormone glucagon-like peptide-1 in this cell line. Our studies for the first time demonstrated a GPR120-mediated novel anti-inflammatory pathway in specific intestinal epithelial cell types that could be of therapeutic relevance to intestinal inflammatory disorders.

Lactobacillus acidophilus Counteracts Inhibition of NHE3 and DRA Expression and Alleviates Diarrheal Phenotype in Mice Infected With Citrobacter rodentium.

Impaired absorption of electrolytes is a hallmark of diarrhea associated with inflammation or enteric infections. Intestinal epithelial luminal membrane NHE3 (Na+/H+ exchanger 3) and DRA (Down-Regulated in Adenoma; Cl-/HCO3- exchanger) play key roles in mediating electroneutral NaCl absorption. We have previously shown decreased NHE3 and DRA function in response to short-term infection with enteropathogenic E coli (EPEC), a diarrheal pathogen. Recent studies have also shown substantial downregulation of DRA expression in a diarrheal model of infection with Citrobacter rodentium, the mouse counterpart of EPEC. Since our previous studies showed that the probiotic Lactobacillus acidophilus (LA) increased DRA and NHE3 function and expression and conferred protective effects in experimental colitis, we sought to evaluate the efficacy of LA in counteracting NHE3 and DRA inhibition and ameliorating diarrhea in a model of C rodentium infection. FVB/N mice challenged with C rodentium [1 × 109 colony-forming units (CFU)] with or without administration of live LA (3 × 109 CFU) were assessed for NHE3 and DRA mRNA and protein expression, mRNA levels of carbonic anhydrase, diarrheal phenotype (assessed by colonic weight-to-length ratio), myeloperoxidase activity, and proinflammatory cytokines. LA counteracted C rodentium-induced inhibition of colonic DRA, NHE3, and carbonic anhydrase I and IV expression and attenuated diarrheal phenotype and MPO activity. Furthermore, LA completely blocked C rodentium induction of IL-1β, IFN-γ, and CXCL1 mRNA and C rodentium-induced STAT3 phosphorylation. In conclusion, our data provide mechanistic insights into antidiarrheal effects of LA in a model of infectious diarrhea and colitis.

All-trans-retinoic Acid Increases SLC26A3 DRA (Down-regulated in Adenoma) Expression in Intestinal Epithelial Cells via HNF-1β

Background: Down-regulation of Cl−/HCO3− exchanger SLC26A3 in gut inflammation results in diarrhea. Does all-trans-retinoic acid (ATRA), an anti-inflammatory agent, affect SLC26A3 (DRA)? Results: ATRA increases DRA expression in enterocytes via transcriptional activation through RAR-β/HNF-1β signaling. Conclusion: ATRA up-regulates DRA expression. Significance: ATRA may act as a therapeutic agent for diarrhea by augmenting DRA expression. All-trans-retinoic acid (ATRA) is an active vitamin A derivative known to modulate a number of physiological processes, including growth and development, differentiation, and gene transcription. The protective effect of ATRA in gut inflammation and diarrheal diseases has been documented. In this regard, down-regulated in adenoma (DRA, a key luminal membrane Cl− transporter involved in NaCl absorption) has been shown to be suppressed in intestinal inflammation. This suppression of DRA is associated with diarrheal phenotype. Therefore, current studies were undertaken to examine the effects of ATRA on DRA expression. DRA mRNA levels were significantly elevated (∼4-fold) in response to ATRA with induction starting as early as 8 h of incubation. Similarly, ATRA increased DRA protein expression by ∼50%. Furthermore, DRA promoter activity was significantly increased in response to ATRA indicating transcriptional activation. ATRA effects on DRA expression appeared to be mediated via the RAR-β receptor subtype, as ATRA remarkably induced RAR-β mRNA levels, whereas RAR-β knockdown substantially attenuated the ability of ATRA to increase DRA expression. Results obtained from agonist (CH-55) and antagonist (LE-135) studies further confirmed that ATRA exerts its effects through RAR-β. Furthermore, ATRA treatment resulted in a significant increase in HNF-1β mRNA levels. The ability of ATRA to induce DRA expression was inhibited in the presence of HNF-1β siRNA indicative of its involvement in ATRA-induced effects on DRA expression. In conclusion, ATRA may act as an antidiarrheal agent by increasing DRA expression via the RAR-β/HNF-1β-dependent pathway.

Identification of Intestinal Ion Transport Defects in Microvillus Inclusion Disease

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.

GLP-1 nanomedicine alleviates gut inflammation

The gut hormone, glucagon like peptide-1 (GLP-1) exerts anti-inflammatory effects. However, its clinical use is limited by its short half-life. Previously, we have shown that GLP-1 as a nanomedicine (GLP-1 in sterically stabilized phospholipid micelles, GLP-1-SSM) has increased in vivo stability. The current study was aimed at testing the efficacy of this GLP-1 nanomedicine in alleviating colonic inflammation and associated diarrhea in dextran sodium sulfate (DSS) induced mouse colitis model. Our results show that GLP-1-SSM treatment markedly alleviated the colitis phenotype by reducing the expression of pro-inflammatory cytokine IL-1β, increasing goblet cells and preserving intestinal epithelial architecture in colitis model. Further, GLP-1-SSM alleviated diarrhea (as assessed by luminal fluid) by increasing protein expression of intestinal chloride transporter DRA (down regulated in adenoma). Our results indicate that GLP-1 nanomedicine may act as a novel therapeutic tool in alleviating gut inflammation and associated diarrhea in inflammatory bowel disease (IBD).

Mechanisms of Intestinal Serotonin Transporter (SERT) Upregulation by TGF-β1 Induced Non-Smad Pathways

TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer’s or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM) attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders.

Mo1757 Trafficking of SLC26A3 (DRA) in Intestinal Epithelial Cells: Effect of EPEC Infection

regulating CIE; however, its role in asymmetric CCH-stimulated signal transduction is not well understood. PURPOSE: Since PLD generates phosphatidic acid (PA) that regulates endocytosis, we (1) investigated whether CCH activates BB PLD, (2) identified which PLD isoform is necessary for CCH-inhibition of NHE3 activity, and (3) determined whether PA or PA-derived diacylglycerol (DAG) is required for this effect. METHODS: 12 day postconfluent Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in Caco-2/BBe cells treated with vehicle, isoform specific PLD inhibitors (EVJ = PLD1; JWJ = PLD2, 5WO = dual), or propranolol (to inhibit conversion of PA to DAG) in the presence or absence of 10μM CCH to elevate [Ca]i. PLD activity was determined in total cell lysates from Caco-2/BBe cells treated with vehicle or CCH in the presence and absence of 10nM (blocks PLD1) or 50nM (blocks PLD2) FIPI. RESULTS: CCH induced rapid (1min) elevation of PLD activity in Caco-2/BBe cells, an effect which was blocked by BB, but not BLM, pretreatment with FIPI. Pretreatment of Caco-2/BBe cells with apical 10μM 5WO abolished CCH-mediated inhibition of NHE3 activity suggesting that BB PLD1 and/or PLD2 are necessary for NHE3 inhibition by CCH. This result was confirmed in studies in which cells were pretreated with both EVJ and JWJ as exposure to each inhibitor alone did not prevent CCH-inhibition of NHE3 activity. Propranolol treatment also prevented CCH-inhibition of NHE3 activity suggesting that PA-derived DAG is required for this effect. CONCLUSIONS: In summary, these data suggest that both PLD1 and PLD2 are activated by CCH signaling and are both necessary to inhibit NHE3 activity, which involves production of PA and DAG. Elevated [Ca]i-mediated signal transduction from the BLMM3 cholingergic receptor activates both BB PLD1 and PLD2 as part of an asymmetric signaling pathway that stimulates production of PA-derived DAG that results in the inhibition of BB NHE3 activity.

Mo1762 A Novel Role of GLP-1 Nanomedicine in Amelioration of Gut Inflammation

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 significant 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; GLP1+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 Crohns disease and ulcerative colitis. (Supported by NIDDK and Dept. of Veteran Affairs)

Mo1792 Inhibition of DRA Expression by NF-κβ Pathway in Human Intestinal Epithelial Cells

Background and aims: Chylomicron retention disease (CRD) is caused by mutations in the SARA2 gene that encodes the SAR1B protein, involved in the vesicular coat protein complex II-dependent transport of proteins/lipoproteins from the endoplasmic reticulum to the Golgi apparatus. Given the large spectrum of CRD phenotypes, it appears important to search for SARA2 polymorphisms and probe functional cause-effects resulting from SAR1B knockdown. Methods: To examine the allelic frequencies of the SARA2 genetic variants, SNPs were assessed in the promoter (2kb, 3.5 and 6 kb regions upstream of the transcription start site), exon 4 and exon 8 in CRD and control subjects. In addition, SAR1B was suppressed in HepG2 and Caco-2/15 cell lines by specific siRNA, and lipid transport was determined. Results: No changes were noted in the polymorphisms in the promoter region (7 variants), exon 4 (2 variants) and exon 8 (1 variant), suggesting that they do not play an essential role in the several biochemical and clinical abnormalities characterizing CRD patients. On the other hand, SAR1B silencing resulted in multiple alterations in lipids, apolipoproteins (apo) and lipoproteins in Caco-2/15 and HepG2 cells. It reduced the output of triglycerides (TG) and TG-rich lipoproteins. Furthermore, SAR1B silencing limited the synthesis of apo B-48 and apo B-100 in Caco-2/15 cells and HepG2 cells, respectively. Conclusions: Our functional experiments show that the suppression of SAR1B has a negative impact on apo B synthesis and lipid/lipoprotein secretion. If previous studies have established only an association between SAR1B defects and lipid transport abnormalities, our findings constitute the first direct demonstration illustrating the SAR1B deficiency cause-effects in enterocytes and hepatocytes. Acknowledgment: This study was supported by the Canadian Institutes of Health Research and the J.A. DeSeve Research Chair in Nutrition