Research Interests: Endocrinology, Chemistry, Life Sciences, Kidney diseases, Internal Medicine, and 15 moreKidney, Animals, Catalase, Glutathione Peroxidase, Alkaline phosphatase, Blood Urea Nitrogen, Brush Border, Enzyme, Lipid peroxidation, Antioxidant enzyme, Carbohydrate metabolism, Body Weight, Bacterial Infection, Biochemistry and cell biology, and Gentamicins
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<jats:title>Abstract</jats:title> <jats:p>Inflammatory bowel diseases (IBDs) are chronic inflammatory multifactorial diseases caused by genetic, immune, and environmental factors. A decrease in intestinal serotonin... more
<jats:title>Abstract</jats:title> <jats:p>Inflammatory bowel diseases (IBDs) are chronic inflammatory multifactorial diseases caused by genetic, immune, and environmental factors. A decrease in intestinal serotonin transporter (SERT), which controls the extracellular availability of serotonin (5-HT) has been implicated in IBD. We previously showed that SERT deletion in mice altered gut bacterial community structure. The gut microbiota-derived metabolites are functional intermediaries between the microbiota and host. Here, we investigated the impact of SERT deficiency on gut metabolites under basal conditions and chronic colitis mimicking human IBD. Methods: Global metabolic profiles were analyzed by "Metabolon" (Durham, NC) on fecal samples from wild type littermates (WT) and SERT KO mice given water or chronic DSS (2.5% DSS for 5 weeks, n=7–9/group). Data were analyzed by ANOVA contrasts (difference between groups) and by two-way ANOVA (P&amp;lt;0.05, q&amp;lt;0.01). Results: SERT KO exhibited more severe colitis vs WT as assessed by histological score, myeloperoxidase activity and colon length. There was more pronounced decrease in the mRNA of tight junction proteins (TJs) occludin-1 and ZO-1 in SERT KO DSS vs WT DSS intestine. Metabolic profiling revealed that SERT deficiency alone resulted in extremely low levels of fecal ectoine, a bacterial derived solute that maintains TJ proteins expression. DSS treatment of WT (but not SERT KO) resulted in a significant increase in microbial derived metabolites including phenylalanine, N-acetylphenylalanine, tyrosine derivatives, glutamate, glutamine and benzoate derivatives. An increase in Trimethylamine N-oxide (TMAO), the short chain fatty acids butyrate/isobutyrate, TCA cycle metabolites; and a decrease in several metabolites including spermidine and various primary and secondary bile acids occurred in DSS treated WT and SERT KO to varying degrees; suggesting that these pathways may contribute to the colitis severity in SERT KO mice. Several secondary bile acids, ketone bodies, the metabolites of pterin, riboflavin pathway (FAD and FMN) and fatty acid metabolism pathways were increased in SERT KO (basal and DSS) suggesting genotype related differences in microbial community. We recently showed an impairment of Aryl hydrocarbon receptor (AhR), an IBD susceptible gene, in SERT KO mice, which could be partly due to altered availability of ligands. Indeed, the bacterial derived AhR ligand tryptamine was extremely low in SERT KO (basal and DSS). DSS increased the host derived AhR ligand, kynurenine in WT, but not in SERT KO.Conclusion:These data highlight the impact of serotonergic machinery and SERT inhibition on host physiology and pathophysiology of IBD. The results provide unique insights into gut bacteria derived metabolites and may aid in the development of novel treatment for disorders with altered SERT and 5-HT availability (Supported by CCFA and NIH).</jats:p>
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☯ These authors contributed equally to this work.
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Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States. While C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile associated diarrhea... more
Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States. While C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile associated diarrhea remains poorly understood. Studies have shown that both NHE3 (Na/H exchanger) and DRA (Down Regulated in Adenoma, Cl/HCO exchanger) resulting in decreased electrolyte absorption are implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and down-regulated in patients with CDI, but the role of DRA in C. difficile infection remains unknown. In the current studies, we examined the effects of C. difficile toxins TcdA and TcdB on DRA protein and mRNA levels in intestinal epithelial cells (IECs). Our data demonstrates that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA,...
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The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or... more
The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used i...
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SLC26A3 or Down-regulated in adenoma (DRA) is the major Cl(-)/HCO3 (-)exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases... more
SLC26A3 or Down-regulated in adenoma (DRA) is the major Cl(-)/HCO3 (-)exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that up-regulate DRA activity may serve as potential anti-diarrheals. In this regard, Sphingosine-1-Phosphate (S1P), a member of bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, current studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-)exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-)exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promo...
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Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus... more
Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus acidophilus (LA) and its culture supernatant (CS) stimulated Cl(-)/HCO3 (-) exchange activity, acutely via an increase in the surface levels of downregulated in adenoma (DRA, SLC26A3) and in long-term treatments via increasing its expression involving transcriptional mechanisms. However, the role of LA in modulating DRA activity under inflammatory conditions is not known. Current in vitro studies using human intestinal epithelial Caco-2 cells examined the efficacy of LA or its CS in counteracting the inhibitory effects of interferon-γ (IFN-γ) on Cl(-)/HCO3 (-) exchange activity. Pretreatment of cells with LA or LA-CS for 1 h followed by coincubation with IFN-γ significantly alleviated the inhibitory effects of IFN-γ on Cl(-)/HCO3 (-) exchange activity. In t...
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Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable toxicity in the kidney. Histological studies have indicated that mitochondria, microsomes, lysosomes and plasma membranes of renal proximal... more
Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable toxicity in the kidney. Histological studies have indicated that mitochondria, microsomes, lysosomes and plasma membranes of renal proximal convoluted tubules in particular are major GM targets. Despite numerous investigations, the biochemical/cellular basis of GM nephrotoxicity is not well understood. Recently reactive oxygen species (ROS) are considered to be important mediators of GM-induced nephrotoxicity. We hypothesize that GM causes damage to intracellular organelles and affects their structural integrity and alters metabolic and other functional capabilities. To address above hypothesis a long-term, time-dependent effect of GM has been studied on blood/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM) and basolateral (BLM), lysosomes and oxidative stress in renal tissues. A nephrotoxic dose of GM (80 mg/kg body weight) was administered to rats daily...
Research Interests: Endocrinology, Chemistry, Life Sciences, Kidney diseases, Internal Medicine, and 15 moreKidney, Animals, Catalase, Glutathione Peroxidase, Alkaline phosphatase, Blood Urea Nitrogen, Brush Border, Enzyme, Lipid peroxidation, Antioxidant enzyme, Carbohydrate metabolism, Body Weight, Bacterial Infection, Biochemistry and cell biology, and Gentamicins
Research Interests: Endocrinology, Biology, Antioxidants, Internal Medicine, Kidney, and 15 moreGreen Tea, Animals, Antioxidant, Glutathione, Catalase, Glutathione Peroxidase, Enzyme, Lipid peroxidation, Human health, Camellia sinensis, Antioxidant enzyme, Digestive System, Carbohydrate metabolism, Glutathione Reductase, and Cell Membrane
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During Ramadan, Muslims the world over abstain from food and water from dawn to sunset for a month. We hypothesised that this unique model of prolonged intermittent fasting would result in specific intestinal and liver metabolic... more
During Ramadan, Muslims the world over abstain from food and water from dawn to sunset for a month. We hypothesised that this unique model of prolonged intermittent fasting would result in specific intestinal and liver metabolic adaptations and hence alter metabolic activities. The effect of Ramadan-type fasting was studied on enzymes of carbohydrate metabolism and the brush border membrane of intestine and liver from rat used as a model. Rats were fasted (12 h) and then refed (12 h) daily for 30 d, as practised by Muslims during Ramadan. Ramadan-type fasting caused a significant decline in serum glucose, cholesterol and lactate dehydrogenase activity, whereas inorganic phosphate increased but blood urea N was not changed. Fasting resulted in increased activities of intestinal lactate (+34 %), isocitrate (+63 %), succinate (+83 %) and malate (+106 %) dehydrogenases, fructose 1,6-bisphosphatase (+17 %) and glucose-6-phosphatase (+22 %). Liver lactate dehydrogenase, malate dehydrogena...
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Research Interests: Chemistry, Acute renal failure, Kidney, Blood Glucose, Green Tea, and 15 moreCholesterol, Animals, Antioxidant, Catalase, Animal Model, Alkaline phosphatase, Blood Urea Nitrogen, Enzyme, Cisplatin, Carbohydrate metabolism, Antineoplastic Agents, Creatinine, Anticancer Drug, Acid Phosphatase, and Kidney cortex
Gentamicin (GM) is an aminoglycoside antibiotic commonly used against life threatening gram negative bacterial infections, however, nephrotoxicity remains the major concern for its long term use. Although its effects on kidney are well... more
Gentamicin (GM) is an aminoglycoside antibiotic commonly used against life threatening gram negative bacterial infections, however, nephrotoxicity remains the major concern for its long term use. Although its effects on kidney are well characterized but there have been no studies regarding its effects on intestine. We hypothesize that GM causes adaptive coordinated effect on enzymes of carbohydrate metabolism and terminal digestion/ absorption in rat intestine. Rats were administerd a nephrotoxic dose of GM (80 mg /kg body weight) daily for 15 days and a time dependent effect was observed on various enzyme activities. Activities of lactate (LDH), malate (MDH) and isocitrate (ICDH) dehydrogenases, significantly increased and peaked at different time intervals of GM treatment. Whereas LDH activity remained higher, MDH and ICDH activity slowly declined from their peak values. Activities of fructose-1,6-bisphosphatase, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase increase...
Research Interests: Biochemistry, Biology, Enzymes, Animals, Digestion, and 15 moreCatalase, Glutathione Peroxidase, Human Experimentation, Alkaline phosphatase, Eating, Enzyme, Intestinal absorption, Digestive System, Carbohydrate metabolism, Body Weight, Enzyme activity, Lactate dehydrogenase, Bacterial Infection, Creatinine, and Gentamicins
Uranium, the heaviest of the naturally occurring elements is widely present as environmental contaminant from natural deposits, industrial emissions and most importantly from modern weapons. Histopathological examinations revealed that... more
Uranium, the heaviest of the naturally occurring elements is widely present as environmental contaminant from natural deposits, industrial emissions and most importantly from modern weapons. Histopathological examinations revealed that uranyl nitrate (UN) exposure caused severe damage to pars recta of renal proximal tubule. However, biochemical events involved in cellular response to renal injury are not completely elucidated. We hypothesized that UN exposure would severely damage kidney tissues and alter their metabolic functions. Rats were treated with a single nephrotoxic dose of UN (0.5mg/kg body weight) i.p. After 5d, effect of UN was studied on the activities of various enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress in different kidney tissues. Activity of lactate dehydrogenase increased whereas activities of isocitrate, succinate and malate dehydrogenases, glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly decreased by UN exposure. Activity of glucose-6-phosphate dehydrogenase decreased whereas that of NADP-malic enzyme increased. The activities of BBM enzymes were significantly lowered and after dissociation from BBM excreted in urine. Lipid peroxidation and the activities of superoxide dismutase and glutathione peroxidase increased whereas catalase activity decreased by UN. UN treatment caused specific alterations in the activities of metabolic and membrane enzymes and perturbed antioxidant defenses.
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Cisplatin (CP) is a major antineoplastic drug for the treatment of solid tumors, however, dose dependent nephrotoxicity remains the major concern for its long term use. Several agents/strategies were attempted to prevent CP nephrotoxicity... more
Cisplatin (CP) is a major antineoplastic drug for the treatment of solid tumors, however, dose dependent nephrotoxicity remains the major concern for its long term use. Several agents/strategies were attempted to prevent CP nephrotoxicity but were not found suitable for clinical practice. Dietary fish oil (FO) enriched in ω-3 fatty acids has been shown to prevent/reduce the progression of certain types of cancers, cardiovascular and renal disorders. The present study was undertaken to see whether FO can prevent CP-induced nephrotoxic and other deleterious effects. Rats were prefed experimental diets for 10days and then received a single dose of CP (6mg/kg body weight) intraperitoneally while still on diet. Serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress in rat kidney were analyzed. CP nephrotoxicity was recorded by increased serum creatinine and blood urea nitrogen. CP decreased the activities of metabolic enzymes, antioxidant defense system and BBM enzymes. In contrast, FO alone increased enzyme activities of carbohydrate metabolism and brush border membrane (BBM). FO feeding to CP treated rats markedly enhanced resistance to CP-elicited deleterious effects. Dietary FO supplementation ameliorated CP induced specific metabolic alterations and oxidative damage due to its intrinsic biochemical antioxidant properties.
Research Interests: Chemistry, Antioxidants, Clinical Practice, Kidney, Animals, and 15 moreAntioxidant, Fish Oil, Dietary Supplements, Blood Urea Nitrogen, Brush Border, Enzyme, Cisplatin, Carbohydrate metabolism, Fatty Acid, Body Weight, Biological markers, Food Sciences, Enzyme activity, Antineoplastic Agents, and Creatinine
DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl− absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as... more
DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl− absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as congenital chloride diarrhea and inflammatory bowel diseases. Previous studies have shown that DRA is subject to regulation by short-term and transcriptional mechanisms. In this regard, we have recently shown that short-term treatment by lysophosphatidic acid (LPA), an important bioactive phospholipid, stimulates Cl−/HCO3−(OH−) exchange activity via an increase in DRA surface levels in human intestinal epithelial cells. However, the long-term effects of LPA on DRA at the level of gene transcription have not been examined. The present studies were aimed at investigating the effects of LPA on DRA function and expression as well as elucidating the mechanisms underlying its transcriptional regulation. Long-term LPA treatment increased the Cl−/HCO3− exchange a...
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Na(+)/H(+) exchanger-3 (NHE3) is crucial for intestinal Na+ absorption and its reduction has been implicated in infectious and Inflammatory Bowel Diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are... more
Na(+)/H(+) exchanger-3 (NHE3) is crucial for intestinal Na+ absorption and its reduction has been implicated in infectious and Inflammatory Bowel Diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HUTU-80 cells were used as models of human intestinal epithelial cells (IECs). Normal C57/BL6, wild type or GADD45b KO mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMinerTM) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a CpG free lucia vector decreased the promoter activity in Caco2 cells. DNA methyltransferase (DNMT) inhibitor, 5-azacytidine (10 M, 24h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco2 cells. Similarly...
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Keratins (K) are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8−/−) mice develop a Th2-type colonic inflammation, epithelial... more
Keratins (K) are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8−/−) mice develop a Th2-type colonic inflammation, epithelial hyperproliferation, and mild diarrhea caused by a keratin level-dependent decrease in short-circuit current and net sodium and chloride absorption in the distal colon. The lack of K8 leads to mistargeting or altered levels of membrane proteins in colonocytes; however, the main transporter responsible for the keratin-related ion transport defect is unknown. We here analyzed protein and mRNA levels of candidate ion transporters CFTR, PAT-1, NHE-3, and DRA in ileum, cecum, and proximal and distal colon. Although no differences were observed for CFTR, PAT-1, or NHE-3, DRA mRNA levels were decreased by three- to fourfold and DRA protein was almost entirely lost in K8−/−cecum and proximal and distal colon compared with K8+/+, whereas the levels in ileum were normal. In K8+...
Research Interests: Physiology, Chemistry, Biology, Medicine, Humans, and 7 moreMice, Animals, Keratin, Medical Physiology, Caco-2 cells, Colon, and Ileum
Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl−/[Formula: see text] exchanger, also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed, Slc26a6-null mice showed defect in... more
Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl−/[Formula: see text] exchanger, also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed, Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore, we investigated the regulation of PAT-1 expression by microRNAs (miRNA), as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3′-untranslated region (UTR) revealed potential binding sites for several miRNAs, suggesting the role of miRNAs in modulating PAT1 expression. miRNAs showing highest context scores (125a-5p, 339-5p, 423-5p, 485-5p, and 501-3p) were selected as candidates for their effects on the activity of a 263-bp PAT-1 3′-untransla...
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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)... more
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 ×...
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Our previous studies showed that L. acidophilus culture supernatant (LA CS) increased P-glycoprotein (P-gp/MDR1) function, expression and promoter activity in Caco2 cells. Current studies were designed to elucidate the molecular... more
Our previous studies showed that L. acidophilus culture supernatant (LA CS) increased P-glycoprotein (P-gp/MDR1) function, expression and promoter activity in Caco2 cells. Current studies were designed to elucidate the molecular mechanisms mediating the stimulatory effects of LA CS on Pgp promoter activity. Deletion analysis indicated that LA CS response element (s) is located between -172/+428 bp region and sequence analysis of this region revealed the presence of 3 potential binding sites for c-Fos or c-Jun, namely proximal AP1a, distal AP1b (-119/-78 bp) & AP1c (+175/+196 bp). LA CS (24h) showed an increase in the protein expression of both c-Fos and c-Jun by ~ 2 fold in Caco2 cells. EMSA showed that LA CS markedly increased the binding of Caco-2 nuclear proteins to AP1a & AP1b but not AP1c. DNA-protein complex was completely eliminated by c-Fos antibody, while c-Jun antibody partially eliminated the complex. ChIP analysis also showed that LA CS enhanced the association of c-Fos ...
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GPR120 (free fatty acid receptor 4, FFAR4) is a G protein-coupled receptor for medium and long-chain unsaturated fatty acids (FA) including ω-3 FA. Recent studies have shown GPR120 to play cardinal roles in metabolic disorders via... more
GPR120 (free fatty acid receptor 4, FFAR4) is a G protein-coupled receptor for medium and long-chain unsaturated fatty acids (FA) including ω-3 FA. 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 (...
Research Interests: Physiology, Inflammation, Cell line, Humans, Intestinal Mucosa, and 13 moreMice, G protein-coupled receptors, Animals, Male, Medical Physiology, Immunoprecipitation, Caco-2 cells, Fluorescent Antibody Technique, Rats, Transfection, Enzyme Linked Immunosorbent Assay, Biochemistry and cell biology, and Immunoblotting
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Enteropathogenic E. coli 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 (DRA) contributing to the... more
Enteropathogenic E. coli 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 (DRA) contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. The current study investigated DRA trafficking under basal conditions and in response to EPEC utilizing Caco-2 cells. 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. Mic...
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Inflammatory bowel diseases (IBDs), including Crohn&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease and ulcerative colitis, are chronic relapsing inflammatory disorders of the... more
Inflammatory bowel diseases (IBDs), including Crohn&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease and ulcerative colitis, are chronic relapsing inflammatory disorders of the gastrointestinal tract. Chronic inflammation of the intestine affects the normal fluid and electrolyte absorption leading to diarrhea, the hallmark symptom of IBD. The management of IBD-associated diarrhea still remains to be a challenge, and extensive studies over the last 2 decades have focused on investigating the molecular mechanisms underlying IBD-associated diarrhea. These studies have shown that the predominant mechanism of diarrhea in IBD involves impairment of electroneutral NaCl absorption, with very little role if any played by anion secretion. The electroneutral NaCl absorption involves coupled operation of Na/H exchanger 3 (NHE3 or SLC9A3) and Cl/HCO3 exchanger DRA (Down Regulated in Adenoma, or SLC26A3). Increasing evidence now supports the critical role of a marked decrease in NHE3 and DRA function and/or expression in IBD-associated diarrhea. This review provides a detailed analysis of the current knowledge related to alterations in NHE3 and DRA function and expression in IBD including the mechanisms underlying these observations and highlights the potential of these transporters as important and novel therapeutic targets.
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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... more
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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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.
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Sodium nitroprusside (SNP) is an antihypertensive drug with proven toxic effects attributed mainly to the production of nitric oxide (NO). Polyunsaturated fatty acids (PUFAs) are widely regarded as functional foods and have been shown to... more
Sodium nitroprusside (SNP) is an antihypertensive drug with proven toxic effects attributed mainly to the production of nitric oxide (NO). Polyunsaturated fatty acids (PUFAs) are widely regarded as functional foods and have been shown to ameliorate the harmful effects of many toxicants. This study examined whether feeding of fish oil (FO)/flaxseed oil (FXO) would have any protective effect against SNP-induced hepatotoxicity and cell death. Male Wistar rats were fed either on normal diet or with 15% FO/FXO for 15 days, following which SNP (1.5 mg/kg body weight) was administered intraperitoneally for 7 days. Animals were killed after treatment, and livers were collected for further analysis. We observed that SNP significantly elevated tissue nitrite levels and lipid peroxidation (LPO) with concomitant perturbation in antioxidant defense systems accompanied with dysregulated glucose metabolism and pronounced cellular death. FO/FXO supplementation to SNP-treated rats caused reversal of tissue injury/cell death and markedly decreased LPO and improved antioxidant defense systems. FO/FXO appear to protect against SNP-induced hepatotoxicity by improving energy metabolism and antioxidant defense mechanism.
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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... more
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.