Search

Serotonin transporter (SERT) deficiency has been implicated in metabolic syndrome, intestinal inflammation, and microbial dysbiosis. Interestingly, changes in microbiome metabolic capacity and several alterations in host gene expression, including lipid metabolism, were previously observed in SERT−/− mice ileal mucosa. However, the precise host or microbial metabolites altered by SERT deficiency that may contribute to the pleiotropic phenotype of SERT KO mice are not yet understood. This study investigated the hypothesis that SERT deficiency impacts lipid and microbial metabolite abundances in the ileal mucosa, where SERT is highly expressed. Ileal mucosal metabolomics was performed by Metabolon on wild-type (WT) and homozygous SERT knockout (KO) mice. Fluorescent-activated cell sorting (FACS) was utilized to measure immune cell populations in ileal lamina propria to assess immunomodulatory effects caused by SERT deficiency. SERT KO mice exhibited a unique ileal mucosal metabolomic signature, with the most differentially altered metabolites being lipids. Such changes included increased diacylglycerols and decreased monoacylglycerols in the ileal mucosa of SERT KO mice compared to WT mice. Further, the ileal mucosa of SERT KO mice exhibited several changes in microbial-related metabolites known to play roles in intestinal inflammation and insulin resistance. SERT KO mice also had a significant reduction in the abundance of ileal group 3 innate lymphoid cells (ILC3). In conclusion, SERT deficiency induces complex alterations in the ileal mucosal environment, indicating potential links between serotonergic signaling, gut microbiota, mucosal immunity, intestinal inflammation, and metabolic syndrome. [ABSTRACT FROM AUTHOR]

Diet–microbiota interactions are emerging as important contributors in the pathogenesis of inflammatory bowel diseases (IBD), characterized by chronic inflammation of the GI tract. The aryl hydrocarbon receptor (AhR) transcription factor regulates xenobiotic metabolism and is activated by exogenous ligands, including indole-3-carbinole (I3C), which is found in cruciferous vegetables. However, studies investigating the impact of dietary I3C and AhR in preclinical models resembling human IBD are lacking. Mice (WT or AhR KO in IECs, 6–8 weeks) or SAMP/YitFC and AKR/J control (4 weeks, m/f) were fed an AhR ligand-depleted or I3C (200 ppm)-supplemented diet. There were increased levels of LPS and exacerbated inflammation, resulting in increased mortality in AhRΔIEC mice fed the AhR ligand-depleted diet in response to chronic DSS. The mechanisms underlying the protective effects of I3C supplementation during colonic colitis involved amelioration of intestinal inflammation and restoration of the altered gut microbiota, particularly the families of clostridicae and lachnospriaceae. Furthermore, the AhR-depleted diet led to the emergence of pathobiont Parvibacter caecicola in WT mice. SAMP/YitFc mice with spontaneous ileitis showed significant recovery in epithelial abnormalities when fed dietary I3C. These data demonstrate the critical role of AhR and the mechanisms of dietary I3C in maintaining epithelial homeostasis and ameliorating inflammation. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBD) involve complex interactions among genetic factors, aberrant immune activation, and gut microbial dysbiosis. While metabolomic studies have focused on feces and serum, fewer investigations have examined the intestinal mucosa despite its crucial role in metabolite absorption and transport. The goals of this study were twofold: to test the hypothesis that gut microbial dysbiosis from chronic intestinal inflammation leads to mucosal metabolic alterations suitable for therapeutic targeting, and to address gaps in metabolomic studies of intestinal inflammation that have overlooked the mucosal metabolome. The chronic DSS colitis was induced for five weeks in 7–9-week-old wild-type C57BL/6J male mice followed by microbial profiling with targeted 16srRNA sequencing service. Mucosal metabolite measurements were performed by Metabolon (Morrisville, NC). The data were analyzed using the bioinformatic tools Pathview, MetOrigin, and Metaboanalyst. The novel findings demonstrated increases in several host- and microbe-derived purine, pyrimidine, endocannabinoid, and ceramide metabolites in colitis. Origin analysis revealed that microbial-related tryptophan metabolites kynurenine, anthranilate, 5-hydroxyindoleacetate, and C-glycosyltryptophan were significantly increased in colon mucosa during chronic inflammation and strongly correlated with disease activity. These findings offer new insights into the pathophysiology of IBD and provide novel potential targets for microbial-based therapeutics. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective [...]

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical [Cl.sup.-]/H[CO.sup.-.sub.3](O[H.sup.-]) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased [Cl.sup.-]/H[CO.sup.-.sub.3](O[H.sup.-]) exchange activity (~2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of [Cl.sup.-]/H[CO.sup.-.sub.3](O[H.sup.-]) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical [Cl.sup.-]/H[CO.sup.-.sub.3](O[H.sup.-]) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-[beta]-cyclodextrin (M[beta]CD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (~45%) by NPY compared with control. M[beta]CD significantly decreased [Cl.sup.-]/H[CO.sup.-.sub.3](O[H.sup.-]) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive [sup.36][Cl.sup.-] uptake (~50%). Our results demonstrate that NPY modulates [Cl.sup.-]/ H[CO.sup.-.sub.3](O[H.sup.-]) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in [Cl.sup.-]/ H[CO.sup.-.sub.3](O[H.sup.-]) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine. chloride absorption; human intestine; downregulated in adenoma; ERK 1/2 MAP kinase doi: 10.1152/ajpgi.00039.2010.

Increased intestinal bile acid absorption and expansion of the bile acid pool has been implicated in the hypercholesterolemia associated with diabetes mellitus. However, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. The ileal apical [Na.sup.+]-dependent bile acid transporter (ASBT) is primarily responsible for active reabsorption of the majority of bile acids. Current studies were designed to investigate the modulation of ASBT function and expression in streptozotocin (STZ)-induced diabetes mellitus in rats and to examine the effect of insulin on rat ASBT promoter by insulin. Diabetes mellitus was induced in Sprague-Dawley rats by intraperitoneal injection of low doses of STZ (20 mg/kg body wt) on five consecutive days. Human insulin (10 U/day) was given to a group of diabetic rats for 3 days before euthanasia. RNA and protein were extracted from mucosa isolated from the small intestine and ASBT expression was assessed by real-time quantitative RT-PCR and Western blotting. Our data showed that ASBT mRNA and protein expression were significantly elevated in diabetic rats. Insulin treatment of diabetic rats reversed the increase in ASBT protein expression to control levels. Consistently, ileal [Na.sup.+]-dependent [[sup.3]H]taurocholic uptake in isolated intestinal epithelial cells was significantly increased in diabetic rats. In vitro studies utilizing intestinal epithelial Caco-2 cells demonstrated that ASBT expression and promoter activity were significantly decreased by insulin. These studies demonstrated that insulin directly influences ASBT expression and promoter activity and that ASBT function and expression are increased in rats with STZ-induced diabetes mellitus. The increase in ASBT expression may contribute to disturbances in cholesterol homeostasis associated with diabetes mellitus. hypercholesterolemia; insulinopenia; enterohepatic circulation doi: 10.1152/ajpgi.00139.2010.

Green tea catechins exhibit hypocholesterolemic effects probably via their inhibitory effects on intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) is responsible for reabsorption of bile acids. The present studies were, therefore, designed to investigate the modulation of ASBT function and membrane expression by green tea catechins in human embryonic kidney HEK-293 cells stably transfected with ASBT-V5 fusion protein and intestinal Caco-2 monolayers. Our data showed that ASBT activity was significantly decreased by (-)-epigallocatechin-3-gallate (EGCG) but not other green tea catechins. Inhibition of PKC, phosphatidylinositol 3-kinase, and MAPK-dependent pathways failed to block the reduction in ASBT activity by EGCG. Kinetics studies showed a significant decrease in the Vmax of the transporter, whereas total ASBT content on the plasma membrane was unaltered by EGCG. Concomitant with the decrease in ASBT function, EGCG significantly reduced ASBT pool in the detergent-insoluble fraction, while increasing its presence in the detergent-soluble fraction of plasma membrane. Furthermore, EGCG decreased the association of ASBT with floating lipid raft fractions of cellular membrane on Optiprep density gradient. In conclusion, our data demonstrate a novel role of lipid rafts in the modulation of ASBT function by the dietary component EGCG, which may underlie the hypocholesterolemic effects of green tea. lipid rafts; (-)-epigallocatechin-3-gallate; hypercholesterolemia doi:10.1152/ajpgi.00360.2009.

Clinical efficacy of probiotics in treating various forms of diarrhea has been clearly established. However, mechanisms underlying antidiarrheal effects of probiotics are not completely defined. Diarrhea is caused either by decreased absorption or increased secretion of electrolytes and solutes in the intestine. In this regard, the electroneutral absorption of two major electrolytes, [Na.sup.+] and [Cl.sup.-], occurs mainly through the coupled operation of [Na.sup.+]/[H.sup.+] exchangers and [Cl.sup.-]/O[H.sup.-] exchangers. Previous studies from our laboratory have shown that Lactobacillus acidophilus (LA) acutely stimulated [Cl.sup.-]/O[H.sup.-] exchange activity via an increase in the surface levels of the apical anion exchanger SLC26A3 (DRA). However, whether probiotics influence SLC26A3 expression and promoter activity has not been examined. The present studies were, therefore, undertaken to investigate the long-term effects of LA on SLC26A3 expression and promoter activity. Treatment of Caco-2 cells with LA for 6-24 h resulted in a significant increase in [Cl.sup.-]/O[H.sup.-] exchange activity. DRA mRNA levels were also significantly elevated in response to LA treatment starting as early as 8 h. Additionally, the promoter activity of DRA was increased by more than twofold following 8 h LA treatment of Caco-2 cells. Similar to the in vitro studies, in vivo studies using mice gavaged with LA also showed significantly increased DRA mRNA (~4-fold) and protein expression in the colonic regions as assessed by Western blot analysis and immunofluorescence. In conclusion, increase in DRA promoter activity and expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of LA. probiotics; Caco-2; DRA (downregulated in adenoma); diarrhea doi:10.1152/ajpgi.00465.2009.

Singla A, Dwivedi A, Saksena S, Gill RK, Alrefai WA, Ramaswamy K, Dudeja PK. Mechanisms of lysophosphatidic acid (LPA) mediated stimulation of intestinal apical [C1.sup.-]/[OH.sup.-] exchange. Am J Physiol Gastrointest Liver Physio1298: G 182-G189, 2010. First published November 12, 2009; doi: 10.1152/ajpgi.00345.2009.--Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical [Cl.sup.-]/[OH.sup.-] exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and [C1.sup.-]/[OH.sup.-] exchange activity was measured as DIDS-sensitive [sup.36][Cl.sup.-] uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical [Cl.sup.-]/[OH.sup.-] exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 [micro]M) significantly stimulated [Cl.sup.-]/[OH.sup.-] exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of [C1.sup.-]/[OH.sup.-] exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical [Cl.sup.-]/[OH.sup.-] exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in [Cl.sup.-]/[OH.sup.-] exchange may contribute to the antidiarrheal effects of LPA. downregulated in adenoma; chloride absorption; human intestine; LPA receptor 2; phosphatidylinositol 3-kinase/Akt doi: 10.1152/ajpgi.00345.2009

Saksena S, Singla A, Goyal S, Katyal S, Bansal N, Gill RK, Alrefai WA, Ramaswamy K, Dudeja PK. Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN-[gamma]. Am J Physiol Gastrointest Liver Physiol 298: G159-G166, 2010. First published November 25, 2009; doi:10.1152/ajpgi.00374.2009.--Two members of the SLC26 gene family, SLC26A3 or DRA (downregulated in adenoma) and SLC26A6 (putative anion transporter 1, PAT1), are known to play a major role in apical [Cl.sup.-]/[OH.sup.-] ([HCO.sub.3]) exchange process in the human intestine. We have previously shown the inhibitory effects of IFN-[gamma] (30 ng/ml, 24 h) on both SLC26A3 and A6 expression and promoter activity. We also demonstrated that the effects of IFN-[gamma] on SLC26A6 gene expression were mediated via IRF-1 transcription factor. However, the molecular mechanisms underlying the transcriptional modulation of SLC26A3 gene expression by IFN-[gamma] in the intestine are not known. The present studies were, therefore, designed to elucidate the signaling mechanisms and transcription factor(s) involved in mediating the inhibitory effects of IFN-[gamma] on DRA promoter (p--1183/+114) activity. Deletion analysis indicated that the IFN-[gamma] response element is located within the - 1183 to -790 region, and sequence analysis of this region revealed the presence of potential [gamma]-activated site (GAS), a binding site (-933/-925 bp) for signal transducer and activator of transcription factor 1 (STAT1). Mutations in the potential GAS element abrogated the inhibitory effects of IFN-[gamma]. These studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3 gene expression by IFN-[gamma] in the human intestine. DRA (downregulated in adenoma) promoter; STAT1 (signal transducer and activator of transcription 1); JAK (Janus kinases); chloride absorption doi: 10.1152/ajpgi.00374.2009

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30-60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM (~60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity ([V.sub.max]) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant ([K.sub.m]). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD 147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake. butyrate absorption; human intestine; CD147; p38 MAPK doi: 10.1152/ajpgi.00283.2009.

Ileal apical [Na.sup.+]-dependent bile acid transporter (ASBT) is responsible for reabsorbing the majority of bile acids from the intestinal lumen. Rapid adaptation of ASBT function in response to physiological and pathophysiological stimuli is essential for the maintenance of bile acid homeostasis. However, not much is known about molecular mechanisms responsible for acute posttranscriptional regulation of ileal ASBT. The protein kinase C (PKC)-dependent pathway represents a major cell signaling mechanism influencing intestinal epithelial functions. The present studies were, therefore, undertaken to investigate ASBT regulation in intestinal Caco-2 monolayers by the well-known PKC activator phorbol 12-myristate 13-acetate (PMA). Our results showed that [Na.sup.+]-dependent [[sup.3]H]taurocholic acid uptake in Caco-2 cells was significantly inhibited in response to 2 h incubation with 100 nM PMA compared with incubation with 4[alpha]-PMA (inactive form). The inhibitory effect of PMA was blocked in the presence of 5 [micro]M bisindolylmaleimide I (PKC inhibitor) but not 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM ([Ca.sup.2+] chelator) or LY-294002 (phosphatidylinositol 3-kinase inhibitor). PMA inhibition of ASBT function was also abrogated in the presence of myristoylated PKC[zeta] pseudosubstrate peptide, indicating involvement of the atypical PKC[zeta] isoform. The inhibition by PMA was associated with a significant decrease in the maximal velocity of the transporter and a reduction in ASBT plasma membrane content, suggesting a modulation by vesicular recycling. Our novel findings demonstrate a posttranscriptional modulation of ileal ASBT function and membrane expression by phorbol ester via a PKC[zeta]-dependent pathway. apical sodium-dependent bile acid transporter; SLC10A2; diacylglycerol; atypical protein kinase C[zeta]

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 [micro]M, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 [micro]M, 24 h) increased the MCT1 promoter activity (-871/+ 91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-[xi] isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine. short-chain fatty acid absorption; transcriptional regulation; human intestine; protein kinase C-[xi]; activator protein 2

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited [Na.sup.+]/[H.sup.+] exchangers (NHE) and [Cl.sup.-]/O[H.sup.-] exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 [micro]M, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular [Ca.sup.2+] and PKC[alpha] inhibition (Go6976). In vitro assays revealed that PKC[alpha] interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause [Ca.sup.2+] and PKC[alpha]-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT. microvilli; serotonin and actin; 5-HT and curvature; stress fibers; ion transporter; cytoskeleton

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-[beta]-cyclodextrin (M[beta]CD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK293 cells treated with M[beta]CD. The inhibition in ASBT activity by M[beta]CD was blocked in the cells treated with M[beta]CD-cholesterol complexes. Kinetic analysis revealed that M[beta]CD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. detergent-insoluble microdomains; floatation on Optiprep density gradient; bile acid absorption

The enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and thus has been implicated in the pathogenesis of various intestinal disorders. To date, SERT expression and function in the human intestine have not been investigated. Current studies were designed to characterize the function, expression, distribution, and membrane localization of SERT in the native human intestine. Real-time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared with colon (ileum [much greater than] duodenum [much greater than] jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9, and 6.8 kb) in the hurhan ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt-villus axis. Immunoblotting studies detected a prominent protein band (~70 kDa) in the ileal apical plasma membrane vesicles (AMVs) isolated from mucosa obtained from organ-donor intestine. Functional studies showed that uptake of [[sup.3]H]serotonin (150 nM) in human ileal AMVs was 1) significantly increased in the presence of both [Na.sup.+] and C[I.sup.-]; 2) inhibited (~50%) by the neuronal SERT inhibitor, fluoxetine (10 [micro]M) and by unlabeled 5-HT; and 3) exhibited saturation kinetics indicating the presence of a carrier-mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotonin in human ileal epithelial cells. serotonin uptake; serotonin transporter expression

Down-regulated in adenoma (DRA), also referred to as SLC26A3, is an intestinal anion transporter essential for intestinal chloride absorption. Mutations in DRA result in congenital chloride diarrhea. DRA expression has been shown to be induced by differentiation and to be modulated by cytokines. However, mechanisms of DRA gene transcription and its tissue-specific targeting have not yet been investigated. In this study, we cloned a 3,765-bp promoter fragment of human DRA gene and characterized its activity in human colonic LS 174T and Caco-2 human colon cell lines. Primer extension identified a single transcriptional initiation site that was identical in both colon cancer cell lines and normal colon. Although hepatic nuclear factor HNF-4 is involved in the basal activity of DRA promoter, sodium butyrate induces its activity in LS 174T cells via the binding of Yin Yang 1 (YY1) and GATA transcription factors to their respective cis-elements in promoter region. We also demonstrated a reduction in DRA promoter activity in Caco-2 cells by IFN-[gamma], suggesting that regulation of DRA promoter by IFN-[gamma] may contribute to the pathophysiology of intestinal inflammation. Furthermore, we showed that the DRA promoter fragment is sufficient to drive human growth hormone transgene expression specifically in villus epithelial cells of the small intestine and in differentiated upper crypt and surface epithelial ceils of the colon. Our studies provide evidence for the involvement of HNF-4, YY1, and GATA transcription factors in DRA expression in intestinal differentiated epithelial cells. SLC26A3; congenital chloride diarrhea; intestinal gene expression

NiemannPick C1-like 1 (NPC1L1) is an essential intestinal component of cholesterol absorption. However, little is known about the molecular regulation of intestinal NPC1L1 expression and promoter activity. We demonstrated that human NPC1L1 mRNA expression was significantly decreased by 25-hydroxycholesterol but increased in response to cellular cholesterol depletion achieved by incubation with Mevinolin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) in human intestinal Caco-2 cells. We also showed that a -1741/+56 fragment of the NPC1L1 gene demonstrated high promoter activity in Caco-2 cells that was reduced by 25-hydroxycholesterol and stimulated by cholesterol depletion. Interestingly, we showed that the NPC1L1 promoter is remarkably transactivated by the overexpression of sterol regulatory element (SRE) binding protein (SREBP)-2, suggesting its involvement in the sterol-induced alteration in NPC1L1 promoter activity. Finally, we identified two putative SREs in the human NPC1L1 promoter and established their essential roles in mediating the effects of cholesterol on promoter activity. Our study demonstrated the modulation of human NPC1L1 expression and promoter activity by cholesterol in a SREBP-2-dependent mechanism. oxysterols; intestinal cholesterol absorption

Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of ~39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells. monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon

Bile acids are efficiently absorbed from the intestinal lumen via the ileal apical sodium-dependent bile acid transporter (ASBT). ASBT function is essential for maintenance of cholesterol homeostasis in the body. The molecular mechanisms of the direct effect of cholesterol on human ASBT function and expression are not entirely understood. The present studies were undertaken to establish a suitable in vitro experimental model to study human ASBT function and its regulation by cholesterol. Luminal membrane bile acid transport was evaluated by the measurement of sodium-dependent [sup.3]H-labeled taurocholic acid ([sup.3]H-TC) uptake in human intestinal Caco-2 cell monolayers. The relative abundance of human ASBT (hASBT) mRNA was determined by real-time PCR. Transient transfection and luciferase assay techniques were employed to assess hASBT promoter activity. Caco-2 cell line was found to represent a suitable model to study hASBT function and regulation. 25-Hydroxycholesterol (25-HCH; 2.5 [micro]g/ml for 24 h) significantly inhibited [Na.sup.+]-dependent [sup.3]H-TC uptake in Caco-2 cells. This inhibition was associated with a 50% decrease in the [V.sub.max] of the transporter with no significant changes in the apparent [K.sub.m]. The inhibition in hASBT activity was associated with reduction in both the level of hASBT mRNA and its promoter activity. Our data show the inhibition of hASBT function and expression by 25-HCH in Caco-2 cells. These data provide novel evidence for the direct regulation of human ASBT function by cholesterol and suggest that this phenomenon may play a central role in cholesterol homeostasis. human apical sodium-dependent bile acid transporter; human intestinal bile acid absorption; oxysterols; transcriptional regulation