Search

Background & aimsNormal gestation involves a reprogramming of the maternal gut microbiome (GM) that contributes to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of the GM-maternal metabolism interaction.MethodsThe GM and plasma metabolome of CD1, NIH-Swiss, and C57 mice were analyzed with the use of 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry throughout gestation. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM was studied with the use of fecal microbial transplants (FMTs).ResultsSignificant variation in GM alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time point specific, with the species enriched at gestation day 15/19 (G15/19), a point of heightened IR, being distinct from those enriched before or after pregnancy. Metabolomics revealed elevated plasma kynurenine at G15/19 in all 3 mouse strains. IDO1, the rate-limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine level effects on IR derive from the GM in both mouse and human pregnancy.ConclusionsGM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation, and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1. (Gestational Gut Microbiome-IDO1 Axis Mediates Pregnancy Insulin Resistance; EMBL-ENA ID: PRJEB45047. MetaboLights ID: MTBLS3598).

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]

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